IgA Nephropathy Today

IgA Nephropathy Today

Contributions to Nephrology Vol. 157

Series Editor

Claudio Ronco, Vicenza

IgA Nephropathy Today

Volume Editor

Yasuhiko Tomino, Tokyo

24 figures, 5 in color, and 10 tables, 2007

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Contributions to Nephrology (Founded 1975 by Geoffrey M. Berlyne)

Yasuhiko Tomino Division of Nephrology Department of Internal Medicine Juntendo University School of Medicine 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421 (Japan)

Library of Congress Cataloging-in-Publication Data International Symposium on IgA Nephropathy (11th : 2006 : Tokyo, Japan) IgA nephropathy today / volume editor, Yasuhiko Tomino. p. ; cm. – (Contributions to nephrology, ISSN 0302-5144 ; v. 157) “11th International Symposium on IgA Nephropathy, October 5–7, 2006, Tokyo, Japan”–Foreword. Includes bibliographical references and index. ISBN-13: 978–3–8055–8286–5 (hard cover : alk. paper) 1. IgA glomerulonephritis–Congresses. I. Tomino, Yasuhiko, 1949– II. Title. III. Series. [DNLM: 1. Glomerulonephritis, IGA–etiology–Congresses. 2. Glomerulonephritis, IGA–therapy–Congresses. W1 CO778UN v.157 2007 / WJ 353 I6085i 2007] RC918.I35I585 2006 616.6⬘1–dc22 2007011107

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Contents

IX Foreword Tomino, Y. (Tokyo) Chairman’s Reviews

1 Pathogenesis of IgA Nephropathy Tomino, Y. (Tokyo)

8 Treatment for IgA Nephropathy Tomino, Y. (Tokyo) Clinical Reviews of IgA Nephropathy

13 International IgA Nephropathy Network Clinico-Pathological Classification of IgA Nephropathy Feehally, J.; Barratt, J. (Leicester); Coppo, R. (Turin); Cook, T. (London); Roberts, I. (Oxford); on behalf of the International IgA Nephropathy Network

19 IgA Nephropathy: A Clinical Overview Julian, B.A. (Birmingham, Ala.); Wyatt, R.J. (Memphis, Tenn.); Matousovic, K. (Prague/Pilsen); Moldoveanu, Z.; Mestecky, J.; Novak, J. (Birmingham, Ala.)

27 Angiotensin Antagonists and Fish Oil for Treating IgA Nephropathy Coppo, R.; Amore, A.; Peruzzi, L.; Mancuso, D.; Camilla, R. (Turin)

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37 Treatment of IgA Nephropathy: Corticosteroids, Tonsillectomy, and Mycophenolate Mofetil Kawamura, T. (Tokyo)

44 Interpretation of Renal Biopsies in IgA Nephropathy Cook, H.T. (London) Basic Reviews of IgA Nephropathy

50 Altered Expression of Lymphocyte Homing Chemokines in the Pathogenesis of IgA Nephropathy Buren, M.; Yamashita, M. (Cleveland, Ohio); Suzuki, Y.; Tomino, Y. (Tokyo); Emancipator, S.N. (Cleveland, Ohio)

56 IgA Nephropathy: Current Views of Immune Complex Formation Mestecky, J. (Birmingham, Ala.); Suzuki, H.; Yanagihara, T. (Birmingham, Ala./Tokyo); Moldoveanu, Z.; Tomana, M. (Birmingham, Ala.); Matousovic, K. (Birmingham, Ala./ Prague/Pilsen); Julian, B.A.; Novak, J. (Birmingham, Ala.)

64 Pathogenic Role of IgA Receptors in IgA Nephropathy Monteiro, R.C. (Paris)

70 The Mucosa-Bone-Marrow Axis in IgA Nephropathy Suzuki, Y.; Tomino, Y. (Tokyo)

80 Searching for IgA Nephropathy Candidate Genes: Genetic Studies Combined with High Throughput Innovative Investigations Schena, F.P.; Cerullo, G.; Torres, D.D.; Zaza, G.; Cox, S. (Bari); Bisceglia, L. (San Giovanni Rotondo); Scolari, F. (Brescia); Frascà, G. (Ancona); Ghiggeri, G.M. (Genoa); Amoroso, A. (Turin); on behalf of the European IgA Nephropathy Consortium Clinical Advances (Update)

90 Clinicopathological Influence of Obesity in IgA Nephropathy: Comparative Study of 74 Patients Tanaka, M.; Tsujii, T.; Komiya, T.; Iwasaki, Y.; Sugishita, T.; Yonemoto, S.; Tsukamoto, T.; Fukui, S.; Takasu, A.; Muso, E. (Osaka)

94 A Multicenter Prospective Cohort Study of Tonsillectomy and Steroid Therapy in Japanese Patients with IgA Nephropathy: A 5-Year Report Miyazaki, M.; Hotta, O. (Sendai); Komatsuda, A. (Akita); Nakai, S. (Osaka); Shoji, T. (Nagoya); Yasunaga, C. (Kitakyusyu); Taguma, Y. (Sendai); Japanese Multicenter Study Group on the Treatment of IgA Nephropathy (JST-IgAN)

99 Tonsillectomy and Corticosteroid Therapy with Concomitant Methylprednisolone Pulse Therapy for IgA Nephropathy Suwabe, T.; Ubara, Y.; Sogawa, Y.; Higa, Y.; Nomura, K.; Nakanishi, S.; Hoshino, J.; Sawa, N.; Katori, H.; Takemoto, F.; Hara, S.; Ohashi, K.; Takaichi, K. (Tokyo)

Contents

VI

104 Impact of Annual Urine Health Check-Up System to Obtain Clinical Remission in Patients with IgA Nephropathy Ieiri, N.; Hotta, O.; Taguma, Y. (Miyagi)

109 Sequential Immunosuppressive Therapy in Progressive IgA Nephropathy Rasche, F.M.; Keller, F.; von Müller, L.; Czock, D. (Ulm); Lepper, P.M. (Berne)

114 Prospective Trial of Combined Therapy with Heparin/Warfarin and Renin-Angiotensin System Inhibitors in Progressive IgA Nephropathy Ishii, T.; Kawamura, T.; Tsuboi, N.; Ogura, M.; Utsunomiya, Y.; Hosoya, T. (Tokyo) Basic Advances (Update)

120 Downregulation of the ␤1,3-Galactosyltransferase Gene in Tonsillar B Lymphocytes and Aberrant Lectin Bindings to Tonsillar IgA as a Pathogenesis of IgA Nephropathy Inoue, T.; Sugiyama, H.; Kikumoto, Y.; Fukuoka, N.; Maeshima, Y.; Hattori, H.; Fukushima, K.; Nishizaki, K. (Okayama); Hiki, Y. (Toyoake); Makino, H. (Okayama)

125 Development of IgA Nephropathy-Like Disease with High Serum IgA Levels and Increased Proportion of Polymeric IgA in ␤-1,4-Galactosyltransferase-Deficient Mice Nishie, T. (Kanazawa); Miyaishi, O. (Aichi); Azuma, H. (Osaka); Kameyama, A. (Ibaraki); Naruse, C.; Hashimoto, N.; Yokoyama, H. (Kanazawa); Narimatsu, H. (Ibaraki); Wada, T.; Asano, M. (Kanazawa)

129 IgA Nephropathy: Characterization of IgG Antibodies Specific for Galactose-Deficient IgA1 Suzuki, H. (Birmingham, Ala./Tokyo); Moldoveanu, Z.; Hall, S.; Brown, R.; Julian, B.A. (Birmingham, Ala.); Wyatt, R.J. (Memphis, Tenn.); Tomana, M. (Birmingham, Ala.); Tomino, Y. (Tokyo); Novak, J.; Mestecky, J. (Birmingham, Ala.)

134 IgA Nephropathy and Henoch-Schoenlein Purpura Nephritis: Aberrant Glycosylation of IgA1, Formation of IgA1-Containing Immune Complexes, and Activation of Mesangial Cells Novak, J.; Moldoveanu, Z.; Renfrow, M.B.; Yanagihara, T.; Suzuki, H.; Raska, M.; Hall, S.; Brown, R.; Huang, W.-Q.; Goepfert, A. (Birmingham, Ala.); Kilian, M.; Poulsen, K. (Aarhus); Tomana, M. (Birmingham, Ala.); Wyatt, R.J. (Memphis, Tenn.); Julian, B.A.; Mestecky, J. (Birmingham, Ala.)

139 Patients with IgA Nephropathy Respond Strongly Through Production of IgA with Low Avidity Against Staphylococcus aureus Shimizu, Y.; Seki, M.; Kaneko, S.; Hagiwara, M.; Yoh, K.; Yamagata, K. (Tsukuba); Koyama, A. (Ami)

Contents

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144 Transferrin Receptor Engagement by Polymeric IgA1 Induces Receptor Expression and Mesangial Cell Proliferation: Role in IgA Nephropathy Tamouza, H.; Vende, F.; Tiwari, M.; Arcos-Fajardo, M.; Vrtovsnik, F.; Benhamou, M.; Monteiro, R.C.; Moura, I.C. (Paris)

148 IgA Fc Receptor I Is a Molecular Switch that Determines IgA Activating or Inhibitory Functions Kanamaru, Y.; Blank, U.; Monteiro, R.C. (Paris)

153 The Role of PDGF-D in Mesangioproliferative Glomerulonephritis Floege, J.; van Roeyen, C.; Boor, P.; Ostendorf, T. (Aachen)

159 Analysis of Innate Immune Responses in a Model of IgA Nephropathy Induced by Sendai Virus Yamashita, M.; Chintalacharuvu, S.R. (Cleveland, Ohio); Kobayashi, N. (Tokyo); Nedrud, J.G.; Lamm, M.E. (Cleveland, Ohio); Tomino, Y. (Tokyo); Emancipator, S.N. (Cleveland, Ohio)

164 Roles of Bone Marrow, Mucosa and Lymphoid Tissues in Pathogenesis of Murine IgA Nephropathy Aizawa, M.; Suzuki, Y.; Suzuki, H.; Pang, H.; Kihara, M.; Yamaji, K.; Horikoshi, S.; Tomino, Y. (Tokyo)

169 Concluding Remarks Glassock, R.J. (Los Angeles, Calif.) Abstracts

174 Clinical Reviews of IgA Nephropathy 180 Basic Reviews of IgA Nephropathy 185 Clinical Advances (Update) 215 Basic Advances (Update) 243 Closing Remarks 244 Author Index 249 Subject Index

Contents

VIII

Foreword

Almost 40 years have passed since Dr. Jean Berger first described primary IgA nephropathy as a new disease entity. This disease may lead to end-stage renal disease (ESRD) with its enormous economic impact on healthcare everywhere. Since the pathogenesis of IgA nephropathy is still obscure, specific treatment is not yet available. However, efforts by many investigators around the world have gradually clarified various aspects of the pathogenesis and treatment of this disease. The objectives of the 11th International Symposium on IgA nephropathy (October 5–7, 2006) are (1) to discuss the most up-to-date findings on pathogenesis and treatment of IgA nephropathy and (2) to build friendship among us. This symposium is truly a small specialized meeting with the participation of international nephrologists and basic scientists involved in the large topic of IgA nephropathy. This article is a summary of the topics presented in the 11th International Symposium on IgA nephropathy. This symposium was made possible by the generosity of our sponsors. Thanks go to the members of the Organizing and Scientific Committees, my colleagues in the Division of Nephrology at Juntendo University and the sponsors. Autumn 2006 Yasuhiko Tomino, MD, PhD President, 11th International Symposium on IgA Nephropathy Professor, Division of Nephrology, Department of Internal Medicine Dean, Juntendo University School of Medicine, Tokyo, Japan

IX

Acknowledgments

We sincerely thank the people mentioned below for their collaboration with the 11th International Symposium of IgA Nephropathy in Tokyo, and with the editing of this book. In addition, we especially appreciate the excellent and dedicated assistance by Mr. Makishima and Dr. Hidaka. Medical Toyu Co. Ltd. Nobukazu Kai Syunta Aramaki Shigehiro Makishima Nana Harada Hiroyuki Miyazawa Michiko Kai

Juntendo University School of Medicine Satoshi Horikoshi Isao Ohsawa Toshinao Tsuge Noriyoshi Kobayashi Hitoshi Suzuki Teruo Hidaka Masao Kihara Eri Izumida Hirofumi Ohba Rei Kuze Terumi Shibata Kazutaka Yoshida

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Chairman’s Reviews Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 1–7

Pathogenesis of IgA Nephropathy Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine, Juntendo University, Tokyo, Japan

Abstract IgA nephropathy is generally considered to be an immune-complex-mediated or aggregated (polymerized) IgA (IgA1)-mediated glomerulonephritis. Since the pathogenesis of IgA nephropathy is still obscure, it is important to determine the initiation and progression of this disease using the spontaneous animal model. The ddY mouse strain can serve as a spontaneous animal model for IgA nephropathy. Genetic factors are considered to be involved in the initiation and progression of IgA nephropathy. It has been hypothesized that susceptibility genes for IgA nephropathy can be detected by a genome-wide scan using this model. The peak marker D10MIT 86 on chromosome 10 is located on the region syntenic to human 6q22–23 with IGAN1, which is responsible for familiar IgA nephropathy. There are several developmental and/or exacerbating factors in this disease. Among them, the loss of glomerular epithelial cells (podocytes) and interstitial mast cell infiltration are important factors for progression of glomerulosclerosis and tubulointerstitial injury in patients with IgA nephropathy. Copyright © 2007 S. Karger AG, Basel

Determination of Pathogenesis of IgA Nephropathy using ddY Mice, a Spontaneous Animal Model for IgA Nephropathy

Although the etiology and pathogenesis of IgA nephropathy are still obscure, much is known about serum IgA and mesangially deposited IgA (fig. 1). IgA is the most common immunoglobulin produced by lymphocytes and plasma cells lining the mucosal membranes, and is the main immunoglobulin directed against bacterial or viral antigens in exogenous secretions. Imai et al. [1] reported that the ddY mouse strain can serve as a spontaneous animal model for IgA nephropathy, since these mice show mesangioproliferative glomerulonephritis with severe glomerular IgA deposition. In these mice, at over 40 weeks of age

Antibody

Antigen Trigger ? Genetic ?

Alteration of molecules in the hinge region of IgA (IgA1)

Virus, fungus, bacteria, food, IgG, IgM, IgA1, fibronectin, laminin Genetic factors

Antigen antibody-dependent

Antigen antibody-independent Aggregated IgA1 (nephritogenic IgA1)

IgA (IgA1)-IC

• Pinocytosis • Charge • Cytokine • Complement

Receptors

• Fc␣R, novel Fc␣R • Fc␣␮R • ASGPR • plgR • Transferrin R

IgA deposition in mesangial areas/cells

Fig. 1. Initiation of IgA nephropathy.

marked deposition of IgA and C3 occurs in the glomerular mesangial areas, in association with an increase in the levels of IgA and macromolecular IgAimmune complex in the serum [1, 2]. IgA nephropathy is generally considered to be an immune-complex-mediated or aggregated (polymerized) IgA (IgA1)mediated glomerulonephritis. However, the antigens or stimulators that produce the aggregated (polymerized) IgA involved in this disease are still obscure. Several antigens originating in the respiratory, intestinal and/or biliary tracts and some dietary antigens have been implicated. Previous studies revealed that murine retroviral gp70 is involved in the pathogenesis of lupus nephritis in systemic lupus erythematosus (SLE)-prone NZB, NZB ⫻ NZWF1, BXSB and MRL/Mp-lpr/lpr mice [3, 4]. Takeuchi et al. [5] reported that the murine retroviral envelope glycoprotein, gp70, is deposited in the glomerular mesangial areas in ddY mice over 24 weeks, in the same way as IgG and IgA. Gp70 is also present in various lymphoid tissues. Thus, they suggested that gp70 derived from lymphoid tissues circulates as immune complexes and is deposited in the glomerular mesangial areas. It may be one of the pathogenic antigens involved in renal disease of ddY mice. We examined the deposition of the major retroviral envelop glycoprotein, gp70, in glomeruli of ddY mice by immunofluorescence [6]. Positive staining of gp70 was not observed in glomeruli of our strain of ddY mice at any age examined using two different anti-gp70 antisera and three

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different staining conditions, whereas deposition of IgA, IgG and IgM was manifest in mice aged over 40 weeks. It appears that gp70 deposition may not be sine qua non for the pathogenesis of IgA nephropathy, and that ddY mice may have a heterogenous genetic background, resembling the situation in humans. Genetic factors are considered to be involved in the initiation and progression of IgA nephropathy on the basis of racial differences in prevalence and familial aggregation. It has been hypothesized that susceptibility genes for IgA nephropathy can be detected by a genome-wide scan using this model [7]. First, serial renal biopsies were performed at 20, 40 and 60 weeks of age in 361 ddY mice. The ddY mice were classified into three groups on the basis of onset of glomerular injury as follows: early onset at 20 weeks (31.9%), late onset at 40 weeks (37.9%) and quiescence at 60 weeks (30.2%). The severity of glomerular lesions in both onset groups correlated with the intensity of glomerular IgA deposition but not with serum IgA levels. A genome-wide scan using 270 microsatellite markers identified three chromosomal regions on chromosomes 1, 9 and 10, which were significantly associated with the glomerular injuries. Surprisingly, the peak marker D10MIT86 on chromosome 10 is located on the region syntenic to human 6q22–23 with IGAN1, which might be responsible for familial IgA nephropathy [7]. In addition, D1MIT16 on chromosome 1 was located very close to the locus of the selectin gene, which is a known candidate for human IgA nephropathy. It appears that the three-group ddY mouse model can be a useful tool for identifying susceptibility genes and also for examining their roles in the pathogenesis of IgA nephropathy.

Mechanisms of Progression in IgA Nephropathy

Factors previously reported to be associated with disease progression include male sex, age, prolonged duration, nephrotic range proteinuria, hypertension and glomerular sclerosis in patients with IgA nephropathy. Other developmental and/or exacerbating factors for patients with IgA nephropathy are: (1) complement activation; (2) blood coagulation activity and/or its inhibition in plasma; (3) activity of cytokines/growth factors; (4) activity of reactive oxygen species (ROS); (5) activation of adhesion molecules; (6) apoptosis; (7) podocyte injury (loss) and (8) interstitial mast cell infiltration (fig. 2). Among them, podocyte injury and interstitial mast cell infiltration from our data are reviewed in this chapter. Loss of Glomerular Epithelial Cells (Podocytes) It is widely assumed that glomerular mesangial cell proliferation and mesangial expansion represent major pathological mechanisms underlying

Pathogenesis of IgA Nephropathy

3

IgA deposition in mesangial areas/cells (activation)

Cell infiltration Genetic factors

Cytokine, chemokine and growth factor ROS (reactive oxygen species) Complement (local production) Platelet aggregation/blood coagulation Adhesion molecule

Repair ?

Mesangial expansion Podocyte damage Chemokine Cytokine Transferrin Complement FcRn (IgG)

?

Cell proliferation Result Glomerulo-tubular cross-talk

Cause

Apoptosis

Proteinuria Interstitial damage Tubulo-interstitial cross-talk

Tubular damage

Macrophage, lymphocyte, mast cell, fibroblast

ESRF

Fig. 2. Progression of IgA nephropathy.

progression to glomerular sclerosis. Marked glomerular mesangial expansion is accompanied by a further increase in total glomerular volume. Broadening of the podocyte foot processes is associated with a reduction in the number of podocytes per glomerulus and an increase in the surface area covered by the remaining podocytes. Podocyte loss appears to contribute to progression of IgA nephropathy. Hypotheses concerning the cause of podocyte loss are: (a) glomerular hypertrophy and hypertension may cause podocyte injury, and (b) mesangial expansion beyond some critical point can presumably cause closure of capillary loops and obliteration of the podocytes [8]. Morphological studies on experimental models of progressive glomerular disease have identified the detachment of podocytes from the glomerular basement membrane (GBM) as a critical step in the development and progression of glomerulosclerosis. Several molecular mechanisms for the detachment have been proposed, including reorganization of the actin cytoskeleton in podocytes, apoptosis of podocytes and oxidation of the GBM [9]. To predict progression in patients with IgA nephropathy, we analyzed glomerular lesions except for sclerosis, adhesion and/or crescents in 34 patients with this disease by morphometric analysis. Levels of urinary protein excretion, creatinine clearance (Ccr), serum creatinine (sCr) and mean blood pressure at the time of renal biopsy were used as clinical parameters. The slope of 1/sCr was also used as a prognostic parameter. Renal

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specimens were obtained by echo-guided biopsy. In PAS-stained light microscopic renal sections, three mid sections of open glomeruli were selected and photographed. Stereologic estimation was performed as follows: absolute values of glomerular volume (V(G)), glomerular surface area (S(G)), podocyte and nonpodocyte cell number per glomerulus [N(G(pod)) and N(G(Non-pod))], glomerular surface area covered by one podocyte [S(G)/N(G(pod))] and glomerular volume occupied by one nonpodocyte cell [V(G)/N(G(Non-pod))]. There was a significant correlation between the levels of urinary protein excretion and the change of podocyte injury parameters [N(G(pod)) and S(G)/N(G(pod))] or N(G(Non-pod)). N(G(pod)) was negatively correlated but S(G)/N(G(pod)) and N(G(Non-pod)) were positively correlated with urinary protein excretion. S(G)/N(G(pod)) and N(G(Non-pod)) were correlated with mean blood pressure. N(G(pod)), S(G)/N(G(pod)), N(G(Non-pod)), urinary protein excretion and mean blood pressure were significantly correlated with the slope of 1/sCr. High specificity was observed for N(G(pod)), S(G)/N(G(pod)) and mean blood pressure. High sensitivity was also observed for N(G(Non-pod)) and urinary protein excretion. It appears that podocyte injury might provide additional prognostic information in patients with IgA nephropathy [10]. Further examinations are warranted to calculate the number of podocytes by electron microscopy to detect the outcome in patients with IgA nephropathy. Interstitial Mast Cell Infiltration Mast cells (MC) are derived from hematopoietic progenitors and migrate into inflammatory lesions. Human MC can be classified into two types according to their protease composition: those containing only tryptase (MC(T)) and those containing both tryptase and chymase (MC(TC)) [11]. MC(T) may play a role in immunological responses, whereas MC(TC) seem to play roles in angiogenesis and tissue remodeling. The role of MC in renal inflammatory and fibrotic processes has recently attracted considerable attention. Although the mechanism of the protection provided by MC is poorly understood, hormonal mediators released from MC are thought to protect against interstitial fibrosis. Heparin, e.g., is one of the molecules released by the secretory granules of MC and is well known for its anticoagulant activity and inhibition of the production of TGF-␤. MC have been observed in the renal interstitium of patients with primary glomerular diseases. Their levels increase with progression of tubulointerstitial fibrosis in patients with IgA nephropathy. Kurusu et al. [12] reported that the number of MC in non-fibrotic tubulointerstitial fields can be a predictor of the renal prognosis of patients with IgA nephropathy. In vitro studies have revealed that MC also produce inflammatory mediators other than histamine, such as

Pathogenesis of IgA Nephropathy

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fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF). Accordingly, MC are assumed to contribute to the development of renal interstitial fibrosis in humans. Angiotensin II (Ang II) is closely involved in the pathogenesis of renal fibrosis and is generated by chymases as well as by angiotensin converting enzyme (ACE). It has been suggested that ACE works mainly in intravascular areas, while chymases work mainly in extravascular areas. Human MC have one ␣-chymase, which generates Ang II by cleaving the terminal His and Leu residues from Ang I, whereas rodents express various kinds of ␤-chymase [13]. Rat ␤-chymase destroys Ang II by cleaving it between Tyr4 and Ile5, but the mouse ␤-chymase MC protease 4 (mMCP-4) generates Ang II in the same way as human ␣-chymase [14]. Sakamoto-Ihara et al. investigated whether human MC contribute to renal fibrosis through local activation of the renin-angiotensin system by assessing their numbers in renal biopsy specimens from patients with IgA nephropathy or minimal change nephrotic syndrome (MCNS). In patients with IgA nephropathy and MCNS, the numbers of tryptase-positive MC (MC(T)) and MC positive for both tryptase and chymase (MC(TC)) were examined histopathologically. sCr, mean blood pressure and the severity of glomerular and tubulointerstitial lesions were also determined. MC(TC) numbers differed between IgA nephropathy patients and MCNS patients. IgA nephropathy patients had more MC(TC) than MC(T). MC were found around but not in the conglomerate of Ang II-positive infiltrating cells. In IgA nephropathy patients with the most severe pathology, the number of Ang II-positive cells was correlated with that of MC(TC) and MC(T). It appears that chymase-dependent Ang II synthesis due to human MC may be involved in the inflammatory and fibrotic processes of IgA nephropathy (submitted).

References 1 2

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Imai H, Nakamoto Y, Asakura K, et al: Spontaneous glomerular IgA deposition in ddY mice: An animal model of IgA nephritis. Kidney Int 1985;27:756–761. Tomino Y, Nakamura T, Ebihara I, et al: Altered steady-state of mRNA coding for extracellular matrices in renal tissues of ddY mice, an animal model for IgA nephropathy. J Clin Lab Anal 1991;5:106–113. Yoshiki T, Mellors RC, Strand M, et al: The viral envelope glycoprotein of murine leukemia virus and the pathogenesis of immune complex glomerulonephritis of New Zealand mice. J Exp Med 1974;140:1011–1027. Izui S, MaConahey PJ, Theofilopoulos AN, et al: Association of circulating retroviral gp70-anti-gp70 immune complexes with murine systemic lupus erythematosus. J Exp Med 1979;149:1099–1116. Takeuchi E, Doi T, Shimada T, et al: Retroviral gp70 antigen in spontaneous mesangial glomerulonephritis of ddY mice. Kidney Int 1989;35:638–646. Shimizu M, Tomino Y, Abe M, et al: Retroviral envelope glycoprotein(gp 70) is not a prerequisite for pathogenesis of primary immunoglobulin A nephropathy in ddY mice. Nephron 1992;62: 328–331.

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Suzuki H, Suzuki Y, Yamanaka T, et al: Genome-wide scan in a novel IgA nephropathy model identifies a susceptibility locus on murine chromosome 10, in a region syntenic to human IGAN1 on chromosome 6q22–23. J Am Soc Nephrol 2005;16:1289–1299. Pagtaluman ME, Miller PL, Jumping-Eagle S, et al: Podocyte loss and progressive glomerular injury in type II diabetes. J Clin Invest 1997;99:342–348. Asanuma K, Shirato I, Ishido K, et al: Selective modulation of the secretion of proteinases and their inhibitors by growth factors in cultured differentiated podocytes. Kidney Int 2002;62: 822–831. Hishiki T, Shirato I, Takahashi Y, et al: Podocyte injury predicts prognosis in patients with IgA nephropathy using a small amount of renal biopsy tissue. Kidney Blood Press Res 2001:24: 99–104. Irani AA, Schechter NM, Craig SS, et al: Two types of human mast cells that have distinct neutral protease compositions. Proc Natl Acad Sci USA 1986;83:4464–4468. Kurusu A, Suzuki Y, Horikoshi S, et al: Relationship between mast cells in the tubulointerstitium and prognosis of patients with IgA nephropathy. Nephron 2001;89:391–397. Chandrasekharan UM, Sanker S, Glynias MJ, et al: Angiotensin II-forming activity in a reconstructed ancestral chymase. Science 1996;271:502–505. Caughey GH, Raymond WW, Wolters PJ, et al: Angiotensin II generation by mast cell alpha- and beta-chymases. Biochim Biophys Acta 2000;1480:245–257.

Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku 113–8421 Tokyo (Japan) Tel. ⫹81 3 5802 1065, Fax ⫹81 3 3813 1183, E-Mail [email protected]

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Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 8–12

Treatment for IgA Nephropathy Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine, Juntendo University, Tokyo, Japan

Abstract The Committee on IgA nephropathy in Japan has published new clinical guidelines (2nd edition) for the diagnosis and treatment of patients with this disease. The nonspecific therapeutic approach involves a reduction of dietary intake of protein in patients with IgA nephropathy who have developed renal failure. At present, the most important therapeutic goal in patients with IgA nephropathy is the control of hypertension. It has been assumed that removal of tonsillar tissues might reduce the production of polymeric IgA and decrease the frequency of renal parenchymal damage resulting from episodes of macroscopic hematuria and proteinuria. Although there have been no randomized controlled trials (RCT) of tonsillectomy, these are necessary to determine the efficacy of tonsillectomy in patients with IgA nephropathy. Copyright © 2007 S. Karger AG, Basel

Current Strategy of Treatment in Patients with IgA Nephropathy in Japan

Nonspecific therapeutic approach involves reduction of dietary intake of protein in patients with IgA nephropathy who have developed renal failure. Long-term dietary restriction is generally considered to reduce the levels of urinary protein and ameliorate glomerular injuries in patients with IgA nephropathy. Previous approaches to drug therapy of IgA nephropathy in Japan have included anti-platelet drugs, anticoagulants, prednisolone (PSL), immunosuppressants, fish oil, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARB) and/or tonsillectomy. At present, the most important therapeutic goal in patients with IgA nephropathy is the control of hypertension. Blood pressure of less than 130/80 mm Hg is the therapeutic target in patients with IgA

nephropathy. Patients with more or less normal renal function, with or without proteinuria or hypertension, have been preferably treated with ACE inhibitors. Several investigators reported that ACE inhibitors reduce the levels of urinary protein excretion and preserve renal function on patients with IgA nephropathy. Furthermore, ACE inhibitors are recommended on the basis of their beneficial effects on the production of cytokines and extracellular matrix (ECM) components, even when hypertension is not present. ACE inhibitors are generally considered to have cardiac and renal protective actions, and they may improve glomerular hypertension due to dilatation of efferent arterioles in the kidneys and suppress glomerular sclerosis. Pulse therapy with high-dose corticosteroids has not been accepted in patients with IgA nephropathy, except in cases presenting as rapidly progressive glomerulonephritis characterized histologically by necrotizing and/or crescent formation, because the majority of patients with IgA nephropathy have an indolent course. Tonsillectomy has been applied in patients with IgA nephropathy for two reasons [1]. First, tonsillar lymphocytes from patients with IgA nephropathy have been found to produce more polymeric IgA than healthy controls. Second, tonsillitis is a frequent precipitating event leading to macroscopic hematuria and, frequently, glomerular crescent formation, acute tubular injury, and/or a reduction in glomerular filtration rate (GFR). As a result of these observations, it has been assumed that removal of tonsillar tissues might reduce the production of polymeric IgA and decrease the frequency of renal parenchymal damage resulting from episodes of macroscopic hematuria [1]. The macroscopic hematuria seen in IgA nephropathy is commonly precipitated by mucosal stimulation (e.g. pharyngitis) suggesting the possibility of aberrant mucosal immunity in the pathogenesis of IgA nephropathy. The tonsils are also a significant source of under-glycosylated IgA1, implicated in the pathogenesis of IgA deposition. Tonsillectomy also decreases the levels of serum IgA. However, there have been no randomized controlled trials (RCT). No recommendations can be made regarding tonsillectomy for disease progression in patients with IgA nephropathy on the basis of currently available retrospective studies and case reports in Kidney Disease Outcomes Quality Initiatives, UK Renal Association, European Best Practice Guidelines, International Guidelines and CARI (Caring for Australasians with Renal Impairment) Guidelines [2]. The Canadian Society of Nephrology guidelines state that tonsillectomy could reduce proteinuria and hematuria in IgA nephropathy patients with recurrent tonsillitis. Tonsillectomy should be performed in patients with appropriate ENT (ear, nose, and throat) indications. Controlled trials are needed before tonsillectomy should be considered for any other group. Kano et al. in my division investigated toll-like receptor (TLR) expression in tonsils from IgA nephropathy and determined their cell types. It is suggested

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that tonsillectomy with steroid pulse therapy may provide rapid and good therapeutic outcomes in IgA nephropathy patients who show high expression of TLR9 in tonsillar plasmacytoid dendric cells. It appears that TLR9 activation in the mucosa may be involved in the pathogenesis of this disease (submitted).

Guidelines for the Treatment of IgA Nephropathy in Japan, 2nd Version (Joint Committee of the Japanese Ministry of Health, Labor and Welfare)

The committee on IgA nephropathy of the Special Study Group on Progressive Glomerular Diseases of the Japanese Ministry of Health, Labor and Welfare published new clinical guidelines (2nd version) for the diagnosis and treatment of patients with IgA nephropathy in Japan [3]. Patients with IgA nephropathy are divided into the following four groups at the time of renal biopsy: (a) good prognosis group: almost no necessity of dialysis; (b) relatively good prognosis group: probability of dialysis requirement is relatively low; (c) relatively poor prognosis group: dialysis is likely to be required within 5–20 years, and (d) poor prognosis group: high probability of dialysis requirement within 5 years. Selection of treatment should be based on renal biopsy and clinical findings (renal function) in each patient [3]. Procedures Good Prognosis Group Regimen. No particular regimen. There are no special restrictions related to status except that extremely vigorous exercise should be avoided. Visits to an outpatient clinic once or twice a year for urinalysis and blood pressure determination are recommended. Diet Therapy. No particular therapy except for the avoidance of excessive salt intake. Drug Therapy. Drug therapy is not required in principle, but antiplatelet drugs are administered as required. Relatively Good Prognosis Group Regimen. No particular regimen is required, but the same recommendations apply as for the good prognosis group mentioned above. Visits to an outpatient clinic should be made at least three or four times a year. Diet Therapy. There is no particular diet therapy, but the same recommendations apply as for the good prognosis group mentioned above. Drug Therapy. Drug therapy is not required in principle, but antiplatelet drugs or adrenocorticosteroids are administered as required.

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Relatively Poor Prognosis Group Regimen. Patients are instructed to avoid overworking. Regular office work or school activities are allowed. Working (especially night work) and sports are restricted based on urinalysis and renal function data. Care is necessary during childbearing and parturition. Visits to an outpatient clinic should be made once a month in principle, and blood chemistry and urinary protein should be tested, in addition to urinalysis and determination of blood pressure. Diet Therapy. Diet therapy includes a low-salt diet of 7–8 g/day, a low protein diet of 0.8–0.9 g/kg ideal body weight (IBW)/day and a calorie intake of 35 kcal/kg IBW/day. Water intake is not restricted unless edema is present. Diet therapy in children is adjusted according to age. Drug Therapy. The drug therapy regimen is summarized below: (a) Antiplatelet agents: Prolonged administration of an antiplatelet agent is common, but each drug must be checked for indications covered by health insurance. (b) Antihypertensive agents: Angiotensin converting enzyme (ACE) inhibitors, angiotensin II receptor blockers (ARB) and/or diuretics should be used in patients without renal failure. For those with insufficient hypotensive effects or with renal failure, calcium antagonists (CCB) or ␣-blockers should be administered. If the decrease in blood pressure is still insufficient, ␣-methyldopa may be used concomitantly. (c) Adrenocorticosteroids: Adrenocorticosteroids should be administered to patients who show slight increases in mesangial matrix and interstitial fibrosis associated with acute inflammatory changes in renal biopsy specimens. In addition to these histological changes, if the patients show more than a moderate degree of proteinuria (⬎0.5 g/day) and their creatinine clearance (Ccr) exceeds 70 ml/min, adrenocorticosteroids should be used. (d) Anticoagulants: When crescent formation, glomerulosclerosis and adhesion to Bowman’s capsule are evident in renal biopsy, warfarin should be used, although heparin may be used for inpatients. (e) Immunosuppressants: Immunosuppressants are not usually used. Poor Prognosis Group Regimen. The regimen is based on that for chronic renal failure (CRF). Pregnancy and parturition should be avoided. Visits to an outpatient clinic must be made at least once a month, and laboratory examinations are basically the same as those for CRF. Diet Therapy. Diet therapy includes a low-salt diet of 7 g/day, a low-protein diet of 0.6 g/kg IBW/day and a calorie intake of 35 kcal/kg IBW/day. Water

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intake is not restricted unless edema is present. Diet therapy in children is adjusted according to age. Drug Therapy. Drug therapy is similar to that for the relatively poor prognosis group. CRF is treated according to the disease state.

References 1 2 3

Silva G, Hogg RJ: IgA nephropathy; in Tisher CC, Brenner BM (eds): Renal Pathology. Philadelphia. Lippincott, 1989, pp 434–491. Thomas M: Specific management of IgA nephropathy: role of tonsillectomy. The CARI Guidelines. Nephrology 2006;11(suppl 1):S146–S148. Tomino Y, Sakai H, Special Study Group (IgA nephropathy) on Progressive Glomerular Disease: Clinical guidelines for immunoglobulin A (IgA) nephropathy in Japan, ed 2. Clin Exp Nephrol 2003;7:93–97.

Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku 113–8421, Tokyo (Japan) Tel. ⫹81 3 5802 1065, Fax ⫹81 3 3813 1183, E-Mail [email protected]

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Clinical Reviews of IgA Nephropathy Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 13–18

International IgA Nephropathy Network Clinico-Pathological Classification of IgA Nephropathy John Feehallya, Jonathan Barratta, Rosanna Coppob, Terry Cookc, Ian Robertsd, on behalf of the International IgA Nephropathy Network a

The John Walls Renal Unit, Leicester General Hospital, Leicester, UK; bNephrology and Dialysis Unit, ‘Regina Margherita’ Children’s Hospital, Turin, Italy; cDepartment of Histopathology, Imperial College London, Hammersmith Hospital, London, and d Oxford Radcliffe Hospitals and University of Oxford, Oxford, UK

Abstract There is no international consensus for the pathological or clinical classification of IgA nephropathy (IgAN). This has limited international comparisons between outcome studies which use different histological classifications, and made it more difficult to identify appropriate patients to enter into clinical intervention trials, as well as making it harder to choose appropriate therapy in individual patients. The International IgA Nephropathy Network [www.igan-world.org], working with members of the Renal Pathology Society, has established an international working group which is developing a consensus classification. Agreement has been reached on definitions of pathological features, and the consistency of scoring these features has been tested by pathologists around the world. Pathological features are now being scored in biopsies from large cohorts of patients from many parts of the world in whom sequential clinical information is available. From the integrated analysis of these clinical and pathological features, a clinico-pathological classification will be proposed which will be further refined and tested in additional cohorts of patients. The goal is to establish a reproducible and clinically effective classification which will gain worldwide acceptance for use in clinical practice and research. Copyright © 2007 S. Karger AG, Basel

Introduction

There is no international consensus for the pathological or clinical classification of IgA nephropathy (IgAN). Nephrologists use clinical information to identify the risk of developing progressive chronic kidney disease (CKD) in

individual patients with IgAN. Pathologists have developed a number of classifications over the last 25 years, some semiquantitative [1–3], some single-grade [4, 5]; each has strengths and limitations in predicting prognosis, and none has gained pre-eminence. This lack of consensus has a number of disadvantages. It has contributed to the slow progress in developing prognostic systems with the sensitivity and specificity to predict outcome for individual patients. It limits opportunities to make international comparisons between different outcome studies which use different histological classifications. It has also limited opportunities to refine the stratification of risk for the design of clinical intervention trials. In 2004, a proposal to develop a consensus classification was developed by the International IgA Nephropathy Network – an informal network of nephrologists and scientists with representation from the majority of nephrology research groups around the world active in the field of IgAN [www.igan-world.org] – working with members of the Renal Pathology Society interested in IgAN. A questionnaire among renal pathologists showed support for the development of a consensus classification of IgAN provided it could be demonstrated to have real clinical benefit. Therefore, representatives of IIgANN and RPS together developed a programme of work leading to a consensus classification. From the beginning this was designed as an international consensus aiming to involve nephrologists and pathologists from as many parts of the world as possible, including all areas where IgAN is known to be of high prevalence.

Goals and Strategy

Our goal is to develop a new clinico-pathological classification for IgAN. The purpose of this new consensus classification would be to identify the risk of progression of renal disease in IgAN, thus enabling clinicians and researchers to improve individual patient prognostication, identify potential for response to immunosuppression or other specific treatments, and refine recruitment to clinical trials. It is necessary to approach this work without preconceptions, in order to test the predictive power of a wide range of pathological and clinical features. The consensus work has also required unity of purpose and a collaborative approach. Organisational challenges have included the need to develop tools allowing consistent data collection, and the need to meet the varying requirements of institutional review boards and ethics committees so that anonymised pathological material and clinical data can be circulated within and beyond its country of origin. In more detail the goals are: (1) to agree a consensus classification of histopathology of IgA nephropathy; (2) to test reproducibility of this classification

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Agree pathological definitions

Data collection

Agree pathological scoring process

Data analysis to identify elements with prognostic predictive power

Agree clinical dataset Select patient cohorts Agree data collection process and data verification

Refine and agree a clinico-pathological classification

Publish Achieve international usage

Test on further patient cohorts

Fig. 1. Overall strategy for development of the IgA nephropathy consensus classification.

among pathologists; (3) to agree a clinical dataset useful for outcome studies in IgA nephropathy; (4) to evaluate the value of pathology parameters combined with clinical parameters in outcome studies in large cohorts of patients with IgA nephropathy, including cohorts varying in age, and in geographical and racial origin; (5) to develop from this work a validated clinico-pathological classification that discriminates patients with IgA nephropathy into groups identifiable from their presenting clinical and laboratory data; and which predicts different outcome out to at least 5 years as estimated by renal survival or by rate of deterioration in renal function, and (6) to promote the use of the new classification once published as the convention to be used in future clinico-pathological outcome reports of IgA nephropathy, as well as in routine clinical practice. The overall strategy for the project is shown in figure 1.

Summary of Achievements up to October 2006

A group of nephrologists and pathologists have committed themselves to achieving these goals, and have met on several occasions: A first meeting of interested nephrologists and pathologists was held in St Louis USA in October 2004; a draft plan was published for web-based consultation in July 2005; a consensus meeting was held in Oxford, UK in September 2005, and pathologists involved in the project met in San Antonio, USA in February 2006. These meetings were attended by 15 nephrologists and 18 pathologists, from 10 countries on 4 continents.

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Progress so far includes the following areas listed below. Pathological Definitions and Scoring The consensus group agreed on the pathological features on light microscopy which need to be evaluated, and definitions for each of those features. The inter-observer reproducibility in identifying those features was then tested by circulating biopsies of 40 patients with IgAN among a worldwide group of 16 pathologists. Inconsistencies were debated at a further meeting, leading to agreement on a scoring system to be applied in subsequent analyses. Clinical Dataset The consensus group agreed on a clinical dataset sufficient to make useful correlations with pathological parameters. This included demographic data, and clinical and laboratory features at the time of diagnostic renal biopsy and annually thereafter. It was recognised that an ideal dataset was not always available from clinical records (e.g., smoking history is inconsistently recorded), but a minimum dataset was defined, including measurements of blood pressure, proteinuria and excretory renal function. Selection of Patient Cohorts for Testing 300 patients with available clinical datasets, available biopsies for review and a minimum of 5 years follow-up from time of biopsy are being selected for the next analysis which will lead to an initial proposal for a classification. The 300 cases comprise 250 adults and 50 children, and are being identified from centres across the world whose staff are involved in the consensus group and from others who have indicated their support. Centres are asked to contribute between 10 and 50 cases. It is necessary to ensure that selected cases include some in whom there is significant deterioration in GFR over 5 years to maximise the opportunity to identify discriminatory clinical or pathological features. Patients who have received a range of treatments are included. So far 15 centres (3 paediatric centres and 12 adult centres) from 9 countries in 4 continents have contributed cases (table 1). Biopsies from these cases are being circulated among pathologists for scoring, after ensuring that the various requirements of institutional review boards and ethics committees have been met, to allow circulation of pathological material and clinical data within and beyond the country of origin. Data Analysis, Definition and Testing of a Proposed Classification The anonymised clinical and laboratory data are being collected centrally with the biopsy scores. From the analysis of these data will emerge a proposed

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Table 1. Centres providing cases for the first phase of IgAN consensus classification 2006/07 Asia PR China

Japan Europe France Italy

United Kingdom North America Canada United States

South America Chile

Beijing Hong Kong Nanjing Tokyo St Etienne Bari Milano Roma Torino Glasgow Toronto Birmingham Mayo Clinic South West Study Group Santiago

classification of IgAN which will then be tested and re-tested in further cohorts of patients until its consistency and clinical utility have been determined as far as is possible with retrospective analysis. If necessary, additional centres will be involved in the study, and it is hoped that the truly international dimension and careful design of this project will encourage other centres to be involved. It will be important to avoid premature publication of the proposed new classification, which will be tested and validated thoroughly before dissemination of the proposal.

Conclusion

The eventual success of this project will depend on the validity and clinical utility of the classification, and also the confidence with which it is received within the worldwide IgAN community. By our inclusive approach and systematic planning, we hope that we have given it the best chance of success.

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References 1

2 3 4 5

Alamartine E, Sabatier JC, Berthoux FC, et al: Comparison of pathological lesions on repeated renal biopsies in 73 patients with primary IgA glomerulonephritis: value of quantitative scoring and approach to final prognosis. Clin Nephrol 1990;34:45–51. Radford MG, Donadio JV, Bergstralh EJ, Grande JP: Predicting renal outcome in IgA nephropathy. J Am Soc Nephrol 1997;8:199–207. Katafuchi R, Kiyposhi Y, Oh Y, et al: Glomerular score as a prognosticator in IgA nephropathy: its usefulness and limitation. Clin Nephrol 1998;49:1–8. Lee SM, Rao VM, Franklin WA, et al: IgA nephropathy: morphologic predictors of progressive renal disease. Hum Pathol 1982;13:314–332. Haas M: Histologic subclassification of IgA nephropathy: a clinicopathologic study of 244 cases. Am J Kidney Dis 1997;29:829–842.

Prof. John Feehally The John Walls Renal Unit, Leicester General Hospital Gwendolen Road Leicester LE5 4PW (UK) Tel. ⫹44 1162 58 4132, Fax ⫹44 1162 58 4764, E-Mail [email protected]

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Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 19–26

IgA Nephropathy: A Clinical Overview Bruce A. Juliana, Robert J. Wyattb, Karel Matousovicc, Zina Moldoveanua, Jiri Mesteckya, Jan Novaka a University of Alabama at Birmingham, Birmingham, Ala., bUniversity of Tennessee Health Sciences Center, Memphis, Tenn., USA; cCharles University School of Medicine and Faculty Hospital, Prague and Pilsen, Czech Republic

Abstract Our understanding of the clinical aspects of IgA nephropathy (IgAN) has advanced since the 10th International Symposium on IgA Nephropathy in 2004. In this review we discuss new developments in areas of familial IgAN, the emerging field of biomarkers, and prognostic features. Familial disease continues to account for a significant number of newly diagnosed patients with IgAN. These patients have clinical manifestations and long-term outcomes similar to those of patients with sporadic disease. Characterization of the IgAN1 gene linked to IgAN in some Italian and American multiplex families has remained elusive. Other multiplex IgAN pedigrees have shown no linkage to any locus. With advances in technology to better measure and characterize polypeptides in small concentrations, the area of biomarkers has generated substantial interest since 2004. New potential disease-specific biomarkers of IgAN include the IgA1 neoepitope at the threonine228 and/or serine230 IgA1 hinge-region residues, serum levels of galactose-deficient IgA, and urinary IgA-IgG immune complexes. Other investigators have used proteomic approaches to find panels of urinary polypeptides (many of which have not been sequenced) that discriminate patients with IgAN from normal healthy controls as well as patients with various other proteinuric renal diseases. These or other related findings may provide the necessary tools to better classify phenotypes in multiplex pedigrees and to improve monitoring of disease progression or response to therapy. Copyright © 2007 S. Karger AG, Basel

Since the 10th International Symposium on IgA Nephropathy in March 2004 in St. Etienne, France, investigators continue to unravel the clinical complexities of IgAN. Here, we review recent advances in the areas of genetics and familial IgAN, the emerging field of proteomic biomarkers, and prognostic markers. Papers elsewhere in this volume address treatment of patients with IgAN.

The proportion of native-kidney biopsies with IgAN in the USA has recently been shown to be higher than that published 10–20 years ago. In an Arkansas referral center, IgAN was diagnosed in 6.9% of the 4,504 biopsy specimens processed from March 2001 to February 2005 from patients older than 20 years [1]. In young adults aged 20–39 years, IgAN was the most common glomerulonephritis, accounting for 14.2% of the 1,082 biopsy specimens.

Genetics and Familial IgAN

As many as 10–15% of newly diagnosed patients with IgAN have a family history of renal disease. Families with multiple members with IgAN have been discovered worldwide, but the precise location and function of a gene linked to IgAN remain elusive. The IgAN1 gene at chromosome 6q22–23 linked to IgAN in Italian and American pedigrees has not been characterized. Other multiplex families have failed to show linkage to any locus. Individuals in some multiplex families have non-IgAN renal disease, including immune-complex glomerulonephritis [2] or thin glomerular basement membrane glomerulopathy [3], whereas renal disease in other families is restricted to IgAN and HenochSchoenlein purpura nephritis [4]. A recent study from Italy found that the longterm outcome is similar in patients with familial and sporadic IgAN [5]. Pursuit of a gene that accounts for familial IgAN has been hampered by the lack of a non-invasive diagnostic marker of disease. Thus, individuals with subclinical renal disease may be misclassified as unaffected, confounding the analysis of the segregation of renal disease with genetic markers.

Biomarkers for IgAN

Advances in the last few years in the methodology for precise analysis of the composition of polypeptides have renewed interest in the field of biomarkers. Biomarkers may be any of several biological characteristics that can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes or pharmacologic response to therapy [6]. Candidates for biomarkers include metabolites, mRNA profiles, lipids, proteins, and polypeptide fragments. The best candidates would be measured with accurate and easy-to-perform assays that can be performed quickly and serially. Biomarkers should be in an easily sampled medium; urine would likely provide the best source for patients with renal disease. For patients with IgAN, ideal biomarkers would obviate the need for a requisite invasive kidney biopsy for

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diagnosis, elucidate pathogenetic mechanisms, forecast clinical course, and monitor response to treatment. A fundamental biochemical abnormality with immunological consequences in patients with IgAN is the increased amount of circulating IgA1 with galactose-deficient hinge-region O-linked glycans [7]. The serum level of this aberrantly glycosylated IgA1 can be measured by its selective binding to Helix aspersa (HAA) lectin that is specific for N-acetylgalactosamine. In a group of 153 Caucasian patients with IgAN, we found that 76% had a serum HAAbinding IgA level above the 90th percentile value for the 150 Caucasian controls (Moldoveanu Z et al., Kidney Int, in press). High levels were detected in patients with familial and sporadic IgAN. About 30% of 133 firstdegree relatives had high levels, possibly indicating a genetically determined predisposition. Another potential serum biomarker for IgAN derives from the galactosedeficient IgA1 hinge-region glycans. Using mass spectrometry and Western blots, we have narrowed the span in the hinge-region that apparently serves as the antigenic epitope in the nephritogenic circulating immune complexes. After digestion of the IgA1 heavy chain from an IgAN patient by site-specific IgA proteases, a Western blot developed with HAA showed that the threonine228 and/or the serine230 were the amino acid residues with attached galactosedeficient glycans [8; also Novak J et al., this volume]. Urine also contains biomarkers for IgAN. Much of the growing interest in this field has centered on proteomics. Proteomics is the assessment of proteins and peptides within a particular compartment, a proteome, using a wide range of analytical methods. The technology has evolved to include two-dimensional gel electrophoresis and other electrophoretic methods, protein arrays, chromatography, and mass spectrometry (fig. 1). Urine is a better fluid than serum for proteomic analysis because it is less complex. In serum, concentrations of various proteins span 10 orders of magnitude, the predominance of a few proteins obscures less abundant peptides, and some potential biomarkers escape detection due to their binding to albumin. Furthermore, the urinary proteome is more stable. Polypeptides do not degrade in urinary samples stored up to six hours at room temperature or for several months at –20⬚C. Proteomics has uncovered urinary biomarkers for several renal diseases, including IgAN. Using a cross-capture ELISA and factoring for creatinine concentration, we found that patients with IgAN excreted more IgA-IgG-containing immune complexes than did disease controls with non-IgA nephropathies with comparable or greater proteinuria [9]. Using capture ELISA and immune complex-specific ELISA, the amounts of urinary IgA and IgA-containing immune complexes were significantly higher in patients with IgAN than in patients with primary non-IgAN glomerulonephritis or healthy controls (Novak J. et al.,

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Sample collection and preparation

Sample separation (electrophoresis, liquid chromatography, chips and microfluidics)

Verification in an independent cohort of patients and controls

Sample analysis (mass spectrometry, image analysis)

Identification of biomarkers for diagnosis of IgAN

Data processing and database searches

Fig. 1. Scheme of proteomic approaches to identification of polypeptide biomarkers for diagnosis of IgAN. Samples (blood, urine, or tissue) from patients with IgAN, healthy controls, and disease controls are collected and processed. Separation by electrophoretic methods (1- or 2-dimensional gel electrophoresis, capillary electrophoresis), liquid chromatography, or chip-based techniques is followed by analytical techniques (image analysis or mass spectrometry) to characterize and quantify differentially present polypeptides. Additional characterization of these polypeptides may include peptide mass fingerprinting and/or sequencing by tandem mass spectrometry and characterization of post-translational modifications. Data analysis by bioinformatic approaches and database searches will assist in the identification of potential biomarkers. Candidate biomarkers must be validated in an independent cohort of patients.

poster at this conference). Furthermore, these urine samples were analyzed by SDS-PAGE and Western blot with IgA- and IgG-specific antibodies. Sample loading was normalized to urinary creatinine concentration. Protein profiles of IgAN samples showed high concentrations of the IgA heavy chain and its proteolytic fragments. In contrast, samples from controls without nephritis had profiles similar to those of healthy controls showing modest IgA and no proteolytic fragments.

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In a study using the classic method of two-dimensional electrophoresis with subsequent isolation of proteins from the gel for analysis by mass spectrometry, Park et al. [10] found 84 protein spots that were differentially expressed in the pooled urine sample from 13 patients with IgAN compared to that from 12 normal controls. Of these protein spots, 42 were over-expressed in the IgAN patients and 42 were under-expressed. By mass spectrometry sequencing, the over-expressed protein spots were found to be derivatives of 27 different proteins. The over-expressed proteins included intracellular or cytoplasmic proteins, nuclear-associated proteins, membrane-associated proteins, and secreted or plasma proteins. Unfortunately, this technology is time-consuming and small proteins (below 10 kDa) may escape detection. Another method, surface-enhanced laser desorption and ionization (SELDI) mass spectrometry, is fast but has low resolution, and many proteins are lost in the process due to the matrices selecting particular polypeptides. Some investigators have turned to capillary electrophoresis coupled on-line to mass spectrometry because it is faster, has high resolution, and can identify up to 600 polypeptides in a urine sample. Haubitz et al. [11] evaluated 45 patients with IgAN, 13 patients with membranous glomerulopathy, and 57 healthy controls. They described 22 urinary polypeptides that discriminated patients with IgAN from normal controls, 12 of which were over-expressed in the patients. Frequencies of the discriminating polypeptides did not differ by magnitude of proteinuria, gender, age, or serum creatinine concentration. Even patients with IgAN in clinical remission without pathological proteinuria were distinguished from healthy controls. After grouping patients by the number of antihypertensive medications, the polypeptide pattern trended toward normal with more intensive therapy, independent of age or magnitude of proteinuria. Comparison of the marker peptides of patients with IgAN to those of patients with membranous glomerulopathy showed a sensitivity of 77% and specificity of 100%. The authors also compared their biomarkers to previously identified markers for other renal diseases compiled in a database. Patients with IgAN were discriminated from patients with focal segmental glomerulosclerosis, minimal-change disease or diabetic nephropathy with a sensitivity of 100% and a specificity of 100%. Sequencing of three of the polypeptides that discriminated between patients IgAN from healthy controls by mass spectrometry identified three different fragments of serum albumin. The basis by which these fragments were restricted to IgAN remains unknown. Perhaps the polypeptides were generated by unique protease activity in inflamed glomeruli. In view of these preliminary studies, disease-specific biomarkers are more likely to be comprised of a panel of several distinct and well-defined peptides than a single molecule. In any event, with proteomics the search for disease-specific biomarkers is not hindered by antecedent bias arising from hypothesized mechanisms of disease.

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Table 1. Recently reported genetic polymorphisms that have been associated with development or progression of IgA nephropathy Polymorphism

Ethnicity

Development of IgAN Megsin A23167G Interleukin-10 promoter

Chinese (14) Korean (15)

Progression of IgAN Megsin A23167G MUC20, a novel mucin protein CC-chemokine receptor 5 Monocyte chemoattractant protein-1 Fc␥RIIa Fc␥RIIIa

Chinese (14) Chinese (16) French (17) Japanese (18) Japanese (19) Japanese (19)

Loss of renal allograft HLA-B8DR3 haplotype

European (20)

Prognosis and Monitoring Progressive Disease in IgAN

In the absence of a validated biomarker to predict prognosis and monitor response to treatment, investigators continue to use the well-known clinical measures to assess the activity of IgAN. There has been a general consensus that hypertension and increasing magnitude of proteinuria, and the pathological findings of glomerular crescents or sclerosis, tubular atrophy and interstitial fibrosis portend a less favorable long-term outcome. Proteinuria is the focus of great attention because it is amenable to therapy. Whether the benefits of twodrug suppression of the effects of angiotensin (with angiotensin-convertingenzyme inhibitors and angiotensin-receptor-type-1 blockers) for preservation of renal function can be monitored by serially measuring proteinuria has not yet been clarified. In the last few years, other features have been proposed as prognostic markers. An increased serum uric acid level appears to be an independent risk factor for progressive disease [12]. Other investigators have found that patients with IgAN and thin glomerular basement membranes have a better outcome than patients with membranes of normal thickness [13]. In addition, the prognosis of IgAN is undoubtedly impacted by genetically determined variations of factors that likely influence the inflammatory and scarring consequences of an immune-complex-mediated glomerular injury. Since 2004, polymorphisms of

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several such factors have been associated with development of IgAN or progressive renal injury (table 1; [14–20]).

Acknowledgments Supported in part by a grant from the National Institutes of Health, PO1 DK61525, by the General Clinical Research Centers of the University of Alabama at Birmingham M01 RR00032 and the University of Tennessee Health Sciences Center, M01 RR00211 (USA), and by a grant from the Research Project MSM 0021620819 (Czech Republic).

References 1 2 3 4 5 6 7 8

9 10 11 12 13

14

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Nair R, Walker PD: Is IgA nephropathy the commonest primary glomerulopathy among young adults in the USA? Kidney Int 2006;69:1455–1458. Izzi C, Sanna-Cherchi S, Prati E, et al: Familial aggregation of primary glomerulonephritis in an Italian population isolate: Valtrompia study. Kidney Int 2006;69:1033–1040. Frasca GM, Soverini L, Gharavi AG, et al: Thin basement membrane disease in patients with familial IgA nephropathy. J Nephrol 2004;17:778–785. Julian BA, Quiggins PA, Thompson JS, et al: Familial IgA nephropathy: evidence for an inherited mechanism of disease. N Engl J Med 1985;312:202–208. Izzi C, Ravani P, Torres D, et al: IgA nephropathy: the presence of familial disease does not confer an increased risk for progression. Am J Kidney Dis 2006;47:761–769. Hewitt SM, Dear J, Star RA: Discovery of protein biomarkers for renal diseases. J Am Soc Nephrol 2004;15:1677–1689. Tomana M, Matousovic K, Julian BA, et al: Galactose-deficient IgAl in sera of IgA nephropathy patients is present in complexes with IgG. Kidney Int 1997;52:509–516. Novak J, Moldoveanu Z, Renfrow MB, et al: Analysis of aberrant O-glycosylation of IgA1 in patients with IgA nephropathy (IgAN). American Society of Nephrology, 39th Renal Week Annual Meeting, San Diego, CA, November 14–19, 2006. Matousovic K, Novak J, Yanagihara T, et al: IgA1-containing immune complexes in the urine of IgA nephropathy patients. Nephrol Dial Transplant 2006;21:2478–2484. Park M-R, Wang E-H, Jin D-C, et al: Establishment of a 2-D human urinary proteomic map in IgA nephropathy. Proteomics 2006;6:1066–1076. Haubitz M, Wittke S, Weissinger EM, et al: Urine protein patterns can serve as diagnostic tools in patients with IgA nephropathy. Kidney Int 2005;67:2313–2320. Myllymaki J, Honkanen T, Syrjanen J, et al: Uric acid correlates with the severity of histopathological parameters in IgA nephropathy. Nephrol Dial Transplant 2005;20:89–95. Linossier M-T, Palle S, Berthoux F: Different glycosylation profile of serum IgA1 in IgA nephropathy according to the glomerular basement membrane thickness: normal versus thin. Am J Kidney Dis 2003;41:558. Xia YF, Huang S, Li X, et al: A family-based association study of megsin A23167G polymorphism with susceptibility and progression of IgA nephropathy in a Chinese population. Clin Nephrol 2006;65:153–159. Chin HJ, Na KY, Kim SJ, et al: Interleukin-10 promoter polymorphism is associated with the predisposition to the development of IgA nephropathy and focal segmental glomerulosclerosis in Korea. J Korean Med Sci 2005;20:989–993. Li G, Zhang H, Lv J, et al: Tandem repeats polymorphism of MUC20 is an independent factor for the progression of immunoglobulin A nephropathy. Am J Nephrol 2006;26:43–49.

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Berthoux FC, Berthoux P, Mariat C, et al: CC-chemokine receptor five gene polymorphism in primary IgA nephropathy: the 32 bp deletion allele is associated with late progression to end-stage renal failure with dialysis. Kidney Int 2006;69:565–572. Mori H, Kaneko Y, Narita I, et al: Monocyte chemoattractant protein-1 A-2518G gene polymorphism and renal survival of Japanese patients with immunoglobulin A nephropathy. Clin Exp Nephrol 2005;9:297–303. Tanaka Y, Suzuki Y, Tsuge T, et al: Fc␥RIIa-131R allele and Fc␥RIIIa-176V/V genotype are risk factors for progression of IgA nephropathy. Nephrol Dial Transplant 2005;20:2439–2445. Andresdottir MB, Haasnoot G, Persijn GG, et al: HLA-B8DR3: a novel risk factor for graft failure after renal transplantation in patients with underlying IgA nephropathy. Transplantation 2006; 82(suppl 2):646–647.

Bruce A. Julian, MD University of Alabama at Birmingham, Division of Nephrology Department of Medicine, 1530 Third Avenue South, THT 643 Birmingham, AL 35294 (USA) Tel. ⫹1 205 934 9045, Fax ⫹1 205 934 7742, E-Mail [email protected]

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Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 27–36

Angiotensin Antagonists and Fish Oil for Treating IgA Nephropathy R. Coppo, A. Amore, L. Peruzzi, D. Mancuso, R. Camilla Pediatric Nephrology School, Nephrology, Dialysis and Transplantation Department, Regina Margherita University Hospital, Turin, Italy

Abstract In IgA nephropathy (IgAN), ACE inhibitors (ACE-I) and angiotensin receptor blockers (ARB) are beneficial against hypertension, and their anti-proteinuric effect has been clearly demonstrated. However, sub-analyses of IgAN patients enrolled in large studies failed to prove a benefit against progression to renal failure. The European Community Biomed Concerted Action – a placebo-controlled randomized controlled trial begun in 1995 – in children and adults (9–35 years old) with proteinuria ⬎ 1 ⬍ 3.5 g/day/1.73 m2 and normal or moderately reduced renal function proved the significant benefit of ACE-I on progression of kidney disease. The combination of ACE-I and ARB in proteinuric normotensive IgAN patients showed greater antiproteinuric effect and the COOPERATE trial also reported a superior effect of combination therapy in protecting against renal function deterioration. Treating IgAN with fish oil has a good rationale for renal inflammation as well as for prevention of cardiovascular morbidity. However, the published reports gave conflicting conclusions and also very recent data did not show significant benefits. In conclusion, ACE-I and ARB have a definite role in treating IgAN, particularly the hypertensive and proteinuric forms. These patients should be treated to target BP to ⬍130/70 mm Hg and proteinuria ⬍0.5 g/day. Copyright © 2007 S. Karger AG, Basel

It is well known that the effects of angiotensin II go beyond the regulation of renal hemodynamics and glomerular permselectivity [1]. Angiotensin II can modulate the immune system, triggering pro-fibrogenic cytokines (TGF-␤), stimulating fibroblast proliferation, acting as a growth factor and leading to cell hypertrophy and proliferation, inducing cytokines (IL-6, IL-8, RANTES, MCP-1), generating ROS, activating the transcription factor NF-␬B, upregulating TLR-4 on mesangial cells, and downregulating nephrin and podocin on podocytes [1]. All these activities, summarized in figure 1, lead to inflammation,

MCP1 RANTES IL-8

Angiotensin II Proteinuria

LM infiltration and activation

↑ Cytokine production

↑ TGF-β and ECM ↑ Glomerular hyperfiltration

Endothelial and mesangial cell exposure to shear stress/stretch

↑ PAI-1

↑ Aldosterone

ROS ↓ ECM degradation

ECM accumulation Inflammation

Injury to glomerular cells

Glomerular and tubulointerstitial fibrosis

Fig. 1. Activities of angiotensin II.

injury of glomerular cells and matrix accumulation. To sum up, the reninangiotensin system (RAS) plays a pivotal role in the progression of renal diseases, promoting both intraglomerular and systemic hypertension, and acting on angiotensin receptors of mesangial and tubular cells triggering progression of interstitial fibrosis. On the other hand, IgA deposits may activate mesangial cells, leading to production of biological effects which contribute to amplification of the angiotensin II-induced damage, suggesting a unique role for RAS in IgA nephropathy (IgAN). Among the intracellular signaling activated by both angiotensin II and deposited IgA, NF-␬B has been supposed to play a major role in IgAN, as it was detected in renal biopsies from these patients, particularly in those with active lesions [2]. NF-␬B was demonstrated to be activated and translocated into the nucleus of mesangial cells upon incubation with aggregated IgA or IgA immune complexes. We previously demonstrated that aberrantly glycosylated IgA, which are thought to play a pathogenetic role in IgAN, when incubated with cultured mesangial cells, trigger the nuclear translocation of the two NF-␬B active subunits p65 and p50 [3] and the coincubation of angiotensin-converting-enzyme inhibitors (ACE-I) or angiotensinreceptor-1 blockers (ARB) significantly blunts the activation of this transcription factor in a dose-dependent manner.

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Hence there is a theoretical disease-specific benefit in IgAN for angiotensin II antagonism, since it is aimed to normalize altered mesangial cell responses enhanced by aberrantly glycosylated IgA. We previously demonstrated that in IgAN, particularly in proteinuric cases, there is a local RAS hyperreactivity [4], and precocious activation of the RAS was also reported. There is a general consensus that high urinary protein excretion is one of the most relevant risk factors for progression of IgAN [5]. Proteinuria mostly precedes the development of other clinical risk factors such as hypertension and reduced renal function, which tend to be more related to irreversible sclerotic changes. Persistent nephrotic-range proteinuria in IgAN – as well as in other nephropathies – has been found to be associated with very poor outcome [6], but also moderate proteinuria has been recognized as a risk factor for progression of IgAN [7]. Proteinuria may activate tubular cells and trigger a local activation of the RAS system and the NF-␬B nuclear translocation with consequent release of a large series of mediators inducing lymphomonocyte interstitial infiltration, and release of mediators which lead to interstitial fibrosis [reviewed in 1]. Finally, hypertension is one of the major risk factors for progression of IgAN [5], as recognized by several investigators, who recently stressed that even border-line high values of BP may be harmful, particularly when proteinuria is associated. For all the above reviewed mechanisms of renal damage related to angiotensin activation and concomitant IgA mesangial deposition, angiotensin II antagonists have a specific strong theoretical indication for IgAN, particularly the hypertensive and proteinuric forms. Table 1 reports a synopsis of the most relevant clinical studies which used angiotensin antagonism (mostly ACE-I) in hypertensive IgAN and the different blood pressure (BP) targets. Most of the retrospective study reported ACE-I superior effects in comparison to other antihypertensive drugs in limiting the glomerular filtration rate (GFR) loss and the amount of proteinuria [8]. Most recent prospective studies detected a stabilization of GFR only in intensive treatment groups, when BP was reduced to ⬍130/70 mm Hg often with a multidrug combination, while patients who had BP ⬎135/75 mm Hg failed to be protected against functional decline [9]. The antiproteinuric effect was reported in early short-term investigations and confirmed in long-term studies (table 2); however, the effect was not reproduced in each patient and did not lead to complete remission of proteinuria, as only 40% of the cases showed a reduction of urinary protein excretion by more than 50%. It was therefore logical to wonder whether this partial effect was enough for a relevant reno-protection. Ruggenenti [10] made a sub-analysis of

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Table 1. Angiotensin antagonism in patients with hypertensive IgAN Author (year)

Target BP

Regimen-FU

Efficacy of ACE-I

Cattran (1994) retrospective

⬍140/90

ACE-I vs. other treatments 30 months FU

Significant reduction of GFR loss and proteinuria

Rekola (1991) retrospective

⬍140/90

ACE-I or ␤-blockers 3 years FU

Significant reduction of GFR loss

ACE-I or CCB 1 year FU

No effect on GFR loss, some reduction in proteinuria

ACE-I plus CCB 3 years FU

Stabilization of GFR only in intensive treatment group (BP ⬍ 130/70), while BP ⬎ 135/75 had GFR decline

Bannister (1995) prospective

Kanno (2000) Non randomized

Different BP targets

ACE-I ⫽ Angiotensin converting enzyme inhibitors; CCB ⫽ calcium channel blockers; BP ⫽ blood pressure; FU ⫽ follow-up.

Table 2. Angiotensin antagonism in patients with proteinuric IgAN Author (year)

uP

Regimen-FU

Efficacy of ACE-I

Maschio (1994)

1–2.5 g

Fosinopril vs. Placebo 12 months FU

Mild significant reduction in proteinuria

Perico (1998)

0.5–4 g

ACE-I or ARB 1 month FU

Significant reduction of proteinuria in both arms

Russo (1994)

1–3 g

ACE-I 1 or x2 ARB 1 or x2 or ACE-I plus ARB

Significant reduction in proteinuria, even more in combination group

Song (2003)

⬎1 g/day

ACE-I plus ARB 33 weeks FU

Significant mild reduction in proteinuria no BP effect

Park (2003)

2–2.3 g

ARB or CCB 12 weeks

ARB reduced proteinuria

ACE-I ⫽ Angiotensin converting enzyme inhibitors; ARB ⫽ angiotensin I receptor blockers; uP ⫽ urinary protein (g/day); FU ⫽ follow-up.

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the response to ACE-I in 352 proteinuric nephropathies enrolled in the REIN prospective, double-blind, placebo-controlled trial. In a subgroup of 75 IgAN patients with 3 ⫾ 2 g/day proteinuria (always ⬎1 g/day), ACE-I had some protective effect, as it saved a loss of 2.3 ml/min/year in comparison to the untreated patients. The effect was not statistically significant, possibly due to the small sample size. In 2001, Dillon [11] performed a meta-analysis of 237 IgAN patients enrolled in 3 short-term crossover trials (3 months), 1 randomized and controlled trial (1 year) and 3 retrospective controlled trials (2 years), and reported that the effect on proteinuria was clearly seen, while, due to defects in the original design, no definitive conclusion could be drawn about the nephro-protective effect. His conclusions were that ‘we do not know which treatment is the most effective’. In 2003, the first randomized controlled trial (RCT) of ACE-I in IgAN was published by Praga et al. [12], but it was not placebo-controlled, and enrolled a limited number of patients (44 subjects) from only one center with a wide range of proteinuria (from 0.5 to 5 g/day) and various degrees of renal function impairment, hence with likely variable prognosis. After a mean follow-up of 75 months, the proportion of patients developing the primary endpoint (50% increase of baseline plasma creatinine) was significantly lower in the treated group than in the control group (12 vs. 57%, respectively). In 1995 we designed a double-blind placebo RCT, supported by the European Community Concerted Action of Biomedicine and Health (Biomed) [13]. At that time there was a definite expectation for a trial on ACE-I in IgAN, since the effect of ACE-I on progression of chronic nephropathies had just been proved, but not yet for IgAN. This was the first multicenter, double-blind, placebo-controlled RCT, investigating, in a selected cohort of IgAN, the effects of ACE-I on renal function decline and proteinuria. The trial strictly selected young patients (3–35 years old) of a very constant level of moderate proteinuria (between 1 and 3.5 g/day/1.73 m2 over the 3 months before enrolment) and normal or moderately reduced renal function (creatinine clearance (CrCl) ⬎ 50 ml/min/1.73 m2). Fifty-seven patients, average age 19.9 years (range 9–35 years), randomized to receive Benazepril 0.2 mg/kg/day (ACE-I) or placebo (PL), completed the trial (median follow-up 42 months). The primary outcome was progression of kidney disease, defined as ⬎30% decrease of baseline CrCl and/or worsening of proteinuria until ⱖ3.5 g/day/1.73 m2. Secondary outcome was proteinuria partial (⬍0.5 g/day/1.73 m2) or total remission (⬍160 mg/day/1.73 m2) for ⬎6 months. The survival to the events was evaluated by univariate (KaplanMeier, log-rank test) and Cox multivariate analysis. Of the 57 subjects who had a follow-up, one single patient (4.3%) in the ACE-I group and 5 (14.7%) in the PL group showed a worsening of CrCl ⬎30%.

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No patient on ACE-I developed nephrotic syndrome, versus 7 (20.6%) on PL. The primary outcome of renal disease progression resulted significantly different between the two groups (log-rank P ⫽ 0.035). Mean levels of proteinuria were significantly reduced, as expected, and a stable partial remission of proteinuria was observed in 13/23 (56.5%) ACE-I patients versus 3/34 (8.8%) PL patients (log-rank P ⫽ 0.033), with total remission in 17.4% of ACE-I treated patients and in none of PL (log-rank P ⫽ 0.029). The multivariate Cox analysis showed that treatment with ACE-I was the independent predictor of prognosis, while no influence on the progression of renal damage was found for gender, age, baseline CrCl, systolic or diastolic blood pressure, mean arterial pressure, or proteinuria [13]. In conclusion, angiotensin antagonism is successful in limiting progression of renal damage in young IgAN patients with proteinuria between 1 and 3.5 g/day. A new trial is ongoing in collaboration with Pozzi et al. [14], to test whether the angiotensin inhibition by both ACE-I and ARB may decrease the risk of progression in patients with IgAN so far considered benign (proteinuria ⬍ 0.5 g/day). Such inhibition will be at first achieved with a unique pharmacological class (ACEI or ARB), then shifting to the association of the two classes as soon as the inhibition with one drug becomes ineffective. Is it possible to increase the angiotensin antagonist effect? We could consider using higher doses, or switching from ACE-I to ARB, or combining ACE-I and ARB. High doses of ACE-I or ARB (up to four-fold increase) have been proved to be of benefit by experimental evidence in a model of remnant kidneys [15] limiting the development of renal sclerosis. The superior effect of higher doses versus conventional ones was, however, not confirmed in other models. In some clinical investigations, doubling ACE-I or ARB doses in 12 adults with IgAN did not improve the anti-proteinuric effect, while the side effects were increased [16]. Inhibition of the RAS can be achieved by means of ACE-I or ARB. Both drugs have positive effects and drawbacks. ACE-I drugs depress aldosterone synthesis, which has independent deleterious effects, and limit the degradation of bradykinin, with some additional favourable effects on reduction of proteinuria and of glomerular hypertension. On the other hand, ACE-I also reduces the effects related to AT2-R stimulation, which are thought to lead to vasodilation and inhibition of fibrosis. This treatment leaves uncontrolled non-ACE produced angiotensin II by chymase and CAGE. ARB have the advantage of being placebo-like, without any relevant side effect, but their main supposed advantage, the lack of inhibition and even stimulation of AT2-R, seems indeed more harmful than benign, after the last reports indicating that AT2-R may trigger NF-␬B. No reduction of aldosterone is induced by ARB, while angiotensin II and angiotensin IV are left active with consequent increase in PAI-1 and

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proteolysis. The combination of these two drugs has the advantage of overcoming the limitations of either individual drug, and an effective blocking of TGF␤ has been observed. The combination of ACE-I and ARB in proteinuric normotensive IgAN patients has obtained much greater effect than each individual drug. The effect of the combination therapy was not further increased by doubling the doses [15]. Information on the effects of combination treatment of ARB and ACE-I in IgAN can be derived by the COOPERATE trial [17], which involved 263 patients of whom 131 (50%) were IgAN. Patients were randomized to maximal antiproteinuric effect ACE-I Trandolapril 3 mg/day or Losartan 100 mg/day, or a combination of both. BP was targeted at 130/80 mm Hg. The end points were doubling of serum creatinine or ESRF. Nakao [17] reported a superior effect of combination therapy over individual drug use in reducing proteinuria as well as in protecting against renal function deterioration. The 3-year renal survival in the combination group was mainly due to reduction of proteinuria and irrespective of patients’ baseline proteinuria levels. The effect was not increased by doses of ARB increased up to threefold. Angiotensin antagonism therapy has still debated points. The relative contribution of BP reduction and other additional in vivo effects has been questioned [18]. It is not clear whether ACE-I remains effective over time or whether there is an escape mechanism [19]. An additional effect has been envisaged for anti-aldosterone drugs. Aldosterone increases after long-term ACE-I, showing an ‘aldosterone escape’ [20]; in is still unclear whether this is relevant to longterm prognosis. Cardiovascular complications are major causes of morbidity and mortality in patients with chronic kidney diseases (CKD). IgAN patients with CKD are more likely to die of cardiovascular events than reach the need of dialysis. We must prevent not only end stage renal failure but, perhaps even more, the risk factors for cardiovascular events: hypertension, proteinuria, smoking, and dyslipemia. Considering these points, treating IgAN with fish oil has a good rationale, since eicosapentanoic (EPA) and docosahexaenoic (DHA) acids shift the cyclooxygenase effects from the synthesis of pro-inflammatory mediators, like thromboxane TxA2 and prostaglandin PGI2, towards the less harmful products TxA3 and PGI3; similarly, the lipoxygenase activity is shifted from the synthesis of TB4 to the less phlogogenic leucotriene LTB5. Fish-oil and omega-3 polyunsaturated fatty acids (PUFA) have been shown to be useful in prevention of cardiovascular events and reduction of proteinuria in several experimental glomerular diseases. In anti-Thy1–1 experimental glomerulonephritis, omega-3 PUFA had suppressive effects on mesangial cell proliferation, effect on reduction of proteinuria, and inhibition of renal inflammation. Preliminary pilot studies gave conflicting results: stabilization of GFR for some

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patients, but no benefit for others. In a controlled trial of fish oil in IgAN [21], 106 IgAN patients with proteinuria ⬎1 g/day, hypertension (60%), and impaired GFR were treated with EPA 1.8 g/day and DHA 1.2 g/day for 2 years and followed up for 5 years. A significant benefit was observed on renal function decline, even though proteinuria levels remained unchanged. The outcome of studies treating IgAN with fish oil evaluated in a meta-analysis by Dillon [22] failed to prove an overall benefit of this therapy. A controlled trial of fish oil and prednisone in IgAN (Hogg R et al., personal communication), on 100 IgAN patients, aged under than 40 years, with a urinary protein/creatinine ratio ⬎0.5 and GFR ⬎50 ml/min, randomized the patients to 4 g/day omega-3 PUFA or alternate day prednisone or placebo for 2 years. No significant difference was found between treatment groups and placebo, as the end-points were reached in 2 patients on prednisone, 8 on fish oil and 4 on placebo. Ron Hogg has recently reported that the effect of Omacor (omega-3 PUFA) in patients with IgAN is dependent upon the serum levels of EPA and DHA, and the antiproteinuric effect is significantly correlated to the dose (mg per kg body weight). However, fish oil in IgAN remains of controversial benefit; it is expensive and carries some side effects; however, it can be of benefit for patients with IgAN since it is of benefit against the cardiovascular deterioration. Hence, it is mostly left to individual choice. In conclusion, ACE-I and ARB have a definite role in treating IgAN, particularly the hypertensive and proteinuric forms. The best strategy for proteinuric and hypertensive IgAN patients includes the following therapies [23]: control BP aggressively by means of ACE-I and/or ARB; give the maximum tolerated doses or target BP to proteinuria ⬍0.5 g/day; control blood lipids with statins; reduce salt intake; reduce excessive body weight, and increase physical activity. BP should be targeted according to concomitant proteinuria levels: in cases with proteinuria ⬍1 g/day, BP should not exceed 135/85 mm Hg (MAP 99 mm Hg); when proteinuria is ⬎1 g/day, BP should not exceed ⬎125/75 mm Hg (MAP 92 mm Hg); target BP is ⬍130/70 mm Hg (MAP 92 mm Hg) when proteinuria is ⬎1 g/day. The target for proteinuria is ⬍0.5 g/day and it should be attained by combination therapy ACE-I and ARB, wait for the effect 12 months. Consider other therapeutic options (steroids) in case of failure.

References 1

Wolf G, Ritz E: Combination therapy with ACE inhibitors and angiotensin II receptor blockers to halt progression of chronic renal disease: pathophysiology and indications. Kidney Int 2005;67: 799–812.

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3 4

5 6 7

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9

10

11 12 13

14

15

16

17

18

19 20 21

Ashizawa M, Miyazaki M, Abe K, Furusu A, Isomoto H, Harada T, Ozono Y, Sakai H, Koji T, Kohno S: Detection of nuclear factor-kappa B in IgA nephropathy using Southwestern histochemistry. Am J Kidney Dis 2003;42:76–86. Coppo R, Amore A: Aberrant glycosylation in IgA nephropathy (IgAN). Kidney Int 2004;65: 1544–1547. Coppo R, Amore A, Gianoglio B, Cacace G, Picciotto G, Roccatello D, Peruzzi L, Piccoli G, De Filippi PG: Angiotensin II local hyperreactivity in the progression of IgA nephropathy. Am J Kidney Dis 1993;21:593–602. Coppo R, D’Amico G: Factors predicting progression of IgA nephropathies. J Nephrol 2005;18:503–512. Radford MG Jr, Donadio JV Jr, Bergstralh EJ, Grande JP: Predicting renal outcome in IgA nephropathy. J Am Soc Nephrol 1997;8:199–207. Usui J, Yamagata K, Kai H, Outeki T, Yamamoto S, Muro K, Hirayama A, Yoh K, Tomida C, Hirayama K, Suzuki S, Kobayashi M, Nagata M, Koyama A: Heterogeneity of prognosis in adult IgA nephropathy, especially with mild proteinuria or mild histological features. Intern Med 2001;40:697–702. Cattran DC, Greenwood C, Ritchie S: Long-term benefits of angiotensin-converting enzyme inhibitor therapy in patients with severe immunoglobulin A nephropathy: a comparison to patients receiving treatment with other antihypertensive agents and to patients receiving no therapy. Am J Kidney Dis 1994;23:247–254. Kanno Y, Okada H, Saruta T, Suzuki H: Blood pressure reduction associated with preservation of renal function in hypertensive patients with IgA nephropathy: a 3-year follow-up. Clin Nephrol 2000;54:360–365. Ruggenenti P, Gherardi G, Benini R, Remuzzi G: Chronic proteinuric nephropathies: outcomes and response to treatment in a prospective cohort of 352 patients with different patterns of renal injury. Am J Kidney Dis 2000;35:1155–1165. Dillon JJ: Treating IgA nephropathy. J Am Soc Nephrol 2001;12:846–847. Praga M, Gutierrez E, Gonzalez E, Morales E, Hernandez E: Treatment of IgA nephropathy with ACE inhibitors: a randomized and controlled trial. J Am Soc Nephrol 2003;14:1578–1583. Coppo R, Peruzzi L, Amore A, Piccoli A, Cochat P, Stone R, Kirschstein M, Linnè T: First prospective double-blind randomized placebo-controlled multicenter trial of ACE-inhibitors (ACE-I) in moderately proteinuric IgA nephropathy in the young (in press). Pozzi C, Del Vecchio L, Casartelli D, Pozzoni P, Andrulli S, Amore A, Peruzzi L, Coppo R, Locatelli F; Adulto e Bambino Study Group; Immunopatologia Renale Study Group of the Italian Society of Nephrology: ACE inhibitors and angiotensin II receptor blockers in IgA nephropathy with mild proteinuria: the ACEARB study. J Nephrol 2006;19:508–514. Ma LJ, Nakamura S, Aldigier JC, Rossini M, Yang H, Liang X, Nakamura I, Marcantoni C, Fogo AB: Regression of glomerulosclerosis with high-dose angiotensin inhibition is linked to decreased plasminogen activator inhibitor-1. J Am Soc Nephrol 2005;16:966–976. Russo D, Pisani A, Balletta MM, De Nicola L, Savino FA, Andreucci M, Minutolo R: Additive antiproteinuric effect of converting enzyme inhibitor and losartan in normotensive patients with IgA nephropathy. Am J Kidney Dis 1999;33:851–856. Nakao N, Seno H, Kasuga H, Toriyama T, Kawahara H, Fukagawa M: Effects of combination treatment with losartan and trandolapril on office and ambulatory blood pressures in non-diabetic renal disease: a COOPERATE-ABP substudy. Am J Nephrol 2004;24:543–548. Casas JP, Chua W, Loukogeorgakis S, Vallance P, Smeeth L, Hingorani AD, MacAllister RJ: Effect of inhibitors of the renin-angiotensin system and other antihypertensive drugs on renal outcomes: systematic review and meta-analysis. Lancet 2005;366:2026–2033. Suissa S, Hutchinson T, Brophy JM, Kezouh A: ACE-inhibitor use and the long-term risk of renal failure in diabetes. Kidney Int 2006;69:913–919. Aldigier JC, Kanjanbuch T, Ma LJ, Brown NJ, Fogo AB: Regression of existing glomerulosclerosis by inhibition of aldosterone. J Am Soc Nephrol 2005;16:3306–3314. Donadio JV Jr, Bergstralh EJ, Offord KP, Spencer DC, Holley KE: A controlled trial of fish oil in IgA nephropathy. Mayo Nephrology Collaborative Group. N Engl J Med 1994;331: 1194–1199.

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22 23

Dillon JJ: Fish oil therapy for IgA nephropathy: efficacy and interstudy variability. J Am Soc Nephrol 1997;8:1739–1744. Nagy J, Kovacs T, Wittmann I: Renal protection in IgA nephropathy requires strict blood pressure control. Nephrol Dial Transplant 2005;20:1533–1539.

Prof. Rosanna Coppo Nephrology Dialysis Transplantation Regina Margherita University Hospital Piazza Polonia 94 IT–10126 Torino (Italy) Tel. ⫹39 011 3135362, Fax ⫹39 011 6635543 E-Mail [email protected]

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Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 37–43

Treatment of IgA Nephropathy: Corticosteroids, Tonsillectomy, and Mycophenolate Mofetil Tetsuya Kawamura Department of Medicine, Division of Kidney and Hypertension, Jikei University School of Medicine, Tokyo, Japan

Abstract Previous studies exploring the potential of glucocorticoid therapy on proteinuria and renal survival of patients with IgA nephropathy (IgAN) indicate that corticosteroid therapy is recommended if the patients show a moderate degree of proteinuria and their creatinine clearance exceeds 70 ml/min, although these studies, most of which are not prospective or randomized, have not provided conclusive results. Recently, Pozzi et al. demonstrated that treatment with glucocorticoids for 6 months significantly improved renal survival and proteinuria for 10 years of follow-up. A recent meta-analysis by Samuels et al. supports the use of corticosteroids in reducing proteinuria and preventing progression to end-stage renal disease. Increasing attention has been drawn to the role of tonsillectomy in the longterm prognosis of IgAN. The notion that tonsillectomy not only helps to prevent episodic macroscopic hematuria in the short-term but also gives long-term renal protection in IgAN is supported by two large retrospective studies from Japan. A study of 329 patients with IgAN by Hotta et al. found that tonsillectomy plus high-dose methylprednisolone was identified as one of the independent variables in predicting remission of clinical findings and lack of renal progression. Moreover, Xie et al. have reported that, for 20 years of follow-up, renal survival was significantly better in IgAN patients who underwent tonsillectomy than those who did not undergo the procedure. However, the role of tonsillectomy in the long-term prognosis of IgAN remains unclear, since it has not yet been tested in a controlled randomized trial. The role of mycophenolate mofetil (MMF) in IgAN has been examined in four major trials. Two prospective randomized studies report no benefit from MMF. The remaining two studies showed a greater reduction of proteinuria in patients treated with MMF compared to prednisone or placebo. In both studies, however, MMF did not effectively modify the progressive course of the disease. Thus, despite promising results in large randomized controlled trials in lupus nephritis, the evidence for the use of MMF in IgAN is inconclusive. Copyright © 2007 S. Karger AG, Basel

Corticosteroids

Many previous studies have examined the renoprotective effects of corticosteroids in patients with IgA nephropathy (IgAN). An early retrospective study by Kobayashi et al. demonstrated the potential of glucocorticoid therapy on proteinuria and renal survival of IgAN patients, especially of those with initial creatinine clearance (Ccr) values of 70 ml/min or more, but not of those with values below than 70 ml/min [1]. The same authors also reported that steroid therapy for an average period of 18 months in IgAN patients with a Ccr of 70 ml/min or more and proteinuria between 1 and 2 g/day resulted in a better renal survival rate 10 years after therapy compared with an untreated group (80 vs. 34%) [2]. On the other hand, most randomized prospective studies clearly demonstrated that corticosteroids have an antiproteinuric effect, but do not preserve the glomerular filtration rate (GFR). This is probably because of small sample sizes and short duration of follow-up. Among those previous prospective studies, an exception to the mixed results comes from the well-designed, randomized, controlled trial by Pozzi et al. [3, 4]. In this study, 86 IgAN patients with serum creatinine of 1.5 mg/dl or less and proteinuria of 1.0–3.5 g/day were randomized to receive ‘pulse’ methylprednisolone (1 g/day for 3 days at the beginning of months 1, 3, and 5) followed by alternate day oral prednisolone (0.5 mg/kg) or placebo for 6 months. After 12 months, proteinuria in 31 patients (72%) of steroid group had dropped below 1 g/day, whereas only 13 patients (30%) of the placebo group experienced a similar improvement in proteinuria. After 10 years of follow-up, the renal survival was significantly better in the steroid group than in the placebo group (97 vs. 53%) [4] (fig. 1). Thus, this well-designed trial strongly suggests a role for a 6-month trial of corticosteroids in IgAN patients with urinary protein excretion more than 1.0 g/day yet with preserved renal function. A recent metaanalysis by Samuels et al. supports the use of corticosteroids in reducing proteinuria and preventing progression to end-stage renal disease [5]. A prospective multicenter trial of corticosteroids in Japanese patients with IgAN was performed in 1996–2001 by the Special Study Group on Progressive Glomerular Disease of the Japanese Ministry of Health, Labor and Welfare [6]. Sixty-nine IgAN patients with proteinuria of 0.5–3.4 g/day and Ccr of 70 ml/min or more were divided into two groups: an anti-platelet drug (Dilazep) group and a corticosteroid (PSL) group. The PSL group received prednisolone for 2 years as well as the anti-platelet drug dilazep hydrochloride. There were no differences between the two groups in terms of blood pressure, proteinuria, Ccr, total protein or albumin at baseline. Proteinuria in the PSL group was significantly attenuated at one year and three years compared to baseline, whereas it was not significantly changed in the Dilazep group. Of note, Ccr significantly decreased in the Dilazep group at four years compared to baseline, while it was

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1.0 Log rank p = 0.0003 0.8 0.6 0.4 0.2

Treatment

Events

Yes No

1/43 13/43

0 0

2

4 6 Follow-up (years)

Treatment Yes No

43 43

42 40

39 33

33 23

20 14

8

10 12 7

Fig. 1. Renal survival estimated on the basis of an increase in plasma creatinine concentrations to ⬎100% above baseline values.

well preserved throughout the course in the PSL group, suggesting that corticosteroids can confer antiproteinuric and GFR-preserving effects in IgAN patients with normal renal function. In contrast to the efficacy of corticosteroids in IgAN patients with normal renal function, it is not well elucidated whether corticosteroids might be effective for patients with advanced IgAN and impaired renal function. The effect of methylprednisolone pulse therapy was retrospectively analyzed by Tamura et al. [7] in 8 IgAN patients with an average serum creatinine level of 2.76 mg/dl. They found that the slopes of reciprocal serum creatinine plotted against time during the 12 months following the steroid pulse therapy improved significantly from ⫺0.0233 to ⫺0.0004 dl/mg/month. Likewise, another retrospective study by Moriyama et al. [8], analyzing 60 IgAN patients with Ccr less than 70 ml/min at the time of renal biopsy, revealed that proteinuria at 1 year after treatment had significantly decreased in the steroid group, but not in the non-steroid group. However, the levels of serum creatinine at 1 year after treatment were higher than those before treatment in both of the two groups. The results from these studies indicate that the GFR-preserving effect of corticosteroids might not be remarkable in IgAN patients with impaired renal function.

Tonsillectomy

There has been ample evidence suggesting a pathogenic role of the tonsils in IgAN. Clinically, macroscopic hematuria often occurs in patients with IgAN

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after acute pharyngitis, and in those patients, acute glomerular lesions such as cellular crescents or capillary necrosis are frequently observed. Previous experimental studies demonstrated that J-chain mRNA-positive IgA-bearing cells were increased in tonsils of IgAN patients, suggesting increased dimeric IgA production. Moreover, tonsilar lymphocytes from patients with IgAN showed a significantly higher proliferative activity and production of IgA1 antibodies than controls after stimulation by H. parainfluenza antigen. Additionally, IgA1 molecules produced by tonsillar lymphocytes from patients with IgAN have been demonstrated to be under-O-glycosylated as well as those in serum or deposited in glomeruli of IgAN patients. On the basis of these findings, increasing attention has been drawn to the role of tonsillectomy in the long-term prognosis of IgAN. The notion that tonsillectomy not only helps to prevent episodic macroscopic hematuria in the short-term, but also gives long-term renal protection in IgAN, is supported by two large retrospective studies from Japan. A retrospective study of 329 patients with IgAN by Hotta et al. [9] found that tonsillectomy plus high-dose methylprednisolone was identified as an independent factor in predicting disapperance of urinary abnormalities, i.e. ‘clinical remission’ and lack of renal progression. Moreover, Xie et al. [10] compared long-term renal survival in 48 patients with IgAN who underwent tonsillectomy and 70 patients who did not undergo the procedure. After 240 months of follow-up, renal survival was significantly better in patients with tonsillectomy than those without tonsillectomy (90 vs. 64%), which had a significant impact on renal outcome by multivariate analysis. However, the role of tonsillectomy in the long-term prognosis of IgAN remains unclear, because it has not yet been tested in a randomized controlled or prospective trial. Recent reviews of the Japanese studies about the effects of tonsillectomy in IgAN insist that tonsillectomy cannot be recommended for widespread use, or there is insufficient data to recommend tonsillectomy for IgAN patients [11, 12]. In this regard, a multicenter randomized controlled trial (RCT) of steroid pulse therapy with tonsillectomy vs. steroid pulse therapy alone has been carefully designed and is now in progress in Japan. The aim of this trial is to clarify whether the combined therapy by tonsillectomy and steroid pulses is more effective than steroid pulses alone in terms of the induction of IgAN patients into clinical remission, i.e. disappearance of both proteinuria and hematuria. Inclusion/exclusion criteria of patients and primary outcomes are summarized in table 1. Eligible subjects were randomly assigned to receive steroid pulses with tonsillectomy (Group A) or steroid pulses alone (Group B). In Group A patients, tonsillectomy was immediately performed. One to 3 weeks after tonsillectomy,

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Table 1. Inclusion/exclusion criteria and primary outcomes in a multicenter randomized controlled trial of steroid pulses with tonsillectomy vs. steroid pulses alone in Japanese patients with IgAN Inclusion criteria Patients with biopsy-proven IgAN and age 15–69 years, urinary protein excretion of 1.0–3.5 g/day and serum creatinine levels of 1.5 mg/dl or less Patients with chronic tonsillitis or recurrent tonsillitis Systolic blood pressure of 140 mm Hg or less and diastolic blood pressure of 90 mm Hg or less with or without antihypertensive drugs Exclusion criteria Patients with nephrotic syndrome or serum creatinine levels of 1.6 mg/dl or more Patients who have been treated with steroids Patients in good prognosis group* Glomerulosclerosis, crescent formation, and adhesion to Bowman’s capsule are absent (from Clinical guidelines for IgA nephropathy) Patients in whom tonsillectomy should not be done by the decision of otorhinolaryngologists because of the difficulty of tonsillectomy or no indication Primary outcomes Percentage of patients with clinical remission, i.e. disappearance of both proteinuria and hematuria Change in 24-hour urinary protein excretion and the degree of microscopic hematuria from baseline *Slight mesangial cell proliferation and increased matrix.

they received 0.5 g of methylprednisolone intravenously for three consecutive days at the beginning of the steroid course and again 2 and 4 months later. They were also given oral prednisone at a dose of 0.5 mg/kg every other day for 6 months. Group B patients received only 0.5 g of methylprednisolone intravenously for three consecutive days at the beginning of the steroid course. The protocol of administration with corticosteroids was identical to Group A.

Mycophenolate Mofetil

The role of mycophenolate mofetil (MMF) in IgAN has been examined in four major trials. Two prospective randomized studies have reported no benefit from MMF in patients with heavy proteinuria and impaired renal function. The effectiveness of MMF in a dose of 2.0 g/day was analyzed in 36 IgAN patients with decreased renal function at diagnosis and/or proteinuria of more than 1.0 g/day. There was no difference between groups in the percentage of patients

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with a decrease of 25% or more in inulin clearance or with a serum creatinine increase of 50% or more over 3 years [13]. Moreover, 32 patients with IgAN with a mean creatinine of 2.4 mg/dl and proteinuria greater than 2.5 g/day on average were randomized to either 1 year of MMF or placebo. Total follow-up was 2 years. No statistically significant differences were observed for any outcome, including primary outcome, a 50% increase in serum creatinine [14]. The remaining two studies have shown a greater reduction of proteinuria in patients treated with MMF compared to prednisone or placebo. Forty patients with proteinuria more than 1.0 g/day were randomized to receive MMF for 24 weeks (group 1) or continue conventional therapy (group 2), and followed for 72 weeks. This study showed a greater reduction of proteinuria in patients in group 1 than in group 2, but no difference in serum creatinine [15]. The last study examined a subset of IgAN patients with proteinuria greater than 2.0 g/day that were randomized to receive either MMF or prednisone. Both proteinuria and cholesterol decreased to a greater extent and serum albumin rose to a higher level in the mycophenolate group [16]. Although these two studies indicated a potential antiproteiuric effect of MMF in IgAN patients with heavy proteinuria, neither demonstrated any GFRpreserving effect of MMF. Thus, despite promising results in large RCTs in lupus nephritis, the evidence for the use of MMF in IgAN is inconclusive.

Summary

Although corticosteroids, tonsillectomy and mycofenolate mofetil are powerful tools for the treatment of progressive IgAN, therapeutic efficacy and responsiveness to each therapy might be quite variable between individuals. Many RCTs with high quality will be needed to clarify the indication and limitation of these promising therapeutic strategies.

References 1 2 3 4 5

Kobayashi Y, Fujii K, Hiki Y, et al: Steroid therapy in IgA nephropathy: retrospective study in heavy proteinuric cases. Nephron 1988;48:12–17. Kobayashi Y, Hiki Y, Kokubo T, et al: Steroid therapy during the early stage of progressive IgA nephropathy. A 10-year follow-up study. Nephron 1996;72:237–242. Pozzi C, Bolasco P, Fogazzi G, et al: Corticosteroids in IgA nephropathy: a randomised controlled trial. Lancet 1999;353:883–887. Pozzi C, Andrulli S, Del Vecchio L, et al: Corticosteroid effectiveness in IgA nephropathy: longterm results of a randomized, controlled trial. J Am Soc Nephrol 2004;15:157–163. Samuels JA, Strippoli GF, Craig JC, et al: Immunosuppressive treatments for immunoglobulin A nephropathy: a meta-analysis of randomized controlled trials. Nephrology (Carlton) 2004;9: 177–185.

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7 8 9 10 11 12 13 14

15 16

Tomino Y, Suzuki H, Horikoshi S, et al: Multicenter trial of adrenocorticosteroids in Japanese patients with IgA nephropathy: results of the special study group (IgA nephropathy) on progressive glomerular disease, Ministry of Health, Labor and Welfare of Japan. Curr Top Steroid Res 2004;4:93–98. Tamura S, Ueki K, Ideura H, et al: Corticosteroid therapy in patients with IgA nephropathy and impaired renal function. Clin Nephrol 2001;55:192–195. Moriyama T, Honda K, Nitta K, et al: The effectiveness of steroid therapy for patients with advanced IgA nephropathy and impaired renal function. Clin Exp Nephrol 2004;8:237–242. Hotta O, Miyazaki M, Furuta T, et al: Tonsillectomy and steroid pulse therapy significantly impact on clinical remission in patients with IgA nephropathy. Am J Kidney Dis 2001;38:736–743. Xie Y, Nishi S, Ueno M, et al: The efficacy of tonsillectomy on long-term renal survival in patients with IgA nephropathy. Kidney Int 2003;63:1861–1867. Locatelli F, Vecchio LD, Pozzi C: IgA glomerulonephritis:beyond angiotensin-converting enzyme inhibitors. Nature Clin Pract Nephrol 2006;2:24–31. Appel GB, Waldman M: The IgA nephropathy treatment dilemma. Kidney Int 2006;69: 1939–1944. Maes BD, Oyen R, Claes K, et al: Mycophenolate mofetil in IgA nephropathy: results of a 3-year prospective placebo-controlled randomized study. Kidney Int 2004;65:1842–1849. Frisch G, Lin J, Rosenstock J, et al: Mycophenolate mofetil (MMF) versus placebo in patients with moderately advanced IgA nephropathy: a double-blind randomized controlled trial. Nephrol Dial Transplant 2005;20:2139–2145. Chen X, Chen P, Cai G, et al: A randomized control trial of mycophenolate mofeil treatment in severe IgA nephropathy. Zhonghua Yi Xue Za Zhi 2002;82:796–801. Tang S, Leung JC, Chan LY, et al: Mycophenolate mofetil alleviates persistent proteinuria in IgA nephropathy. Kidney Int 2005;68:802–812.

Tetsuya Kawamura Department of Medicine, Division of Kidney and Hypertension Jikei University School of Medicine Tokyo, Japan

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Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 44–49

Interpretation of Renal Biopsies in IgA Nephropathy H. Terence Cook Department of Histopathology, Imperial College, Hammersmith Hospital, London, UK

Abstract The renal biopsy is essential for the diagnosis of IgA nephropathy. It should also be possible to derive important information from the biopsy about prognosis and likely response to treatment. Biopsy features that are associated with progression to end-stage renal disease are glomerulosclerosis and tubulointerstitial scarring, marked crescent formation and marked arteriolar hyalinosis. However, with our present classification systems the renal biopsy adds little over and above clinical features in predicting outcome. It may be possible to improve the predictive value of the renal biopsy by adopting the following recommendations in developing new classifications: (1) looking at the ability of the biopsy to predict changes in renal function in the short term after biopsy rather than prediction of progression to end stage renal disease; (2) examining subgroups of patients where the biopsy is likely to be most informative; (3) distinguishing the effects of reversible and irreversible changes particularly with regard to response to treatment; (4) ensuring uniformity of definitions between pathologists, and (5) paying attention to small lesions and considering including additional biopsy features. The most important role for the renal biopsy in the future is likely to be in predicting response to therapy rather than predicting progression to end-stage renal disease. Copyright © 2007 S. Karger AG, Basel

Introduction

IgA nephropathy is defined by the presence of dominant or co-dominant mesangial deposits of IgA. Lupus glomerulonephritis with extensive IgA is excluded. It may be primary, part of the syndrome of Henoch-Schonlein purpura, or secondary to a number of other extra-renal conditions, particularly diseases of the liver or gastro-intestinal tract. It is the most prevalent form of glomerulonephritis worldwide. Frequency of biopsy diagnosis depends both on

policies for screening of asymptomatic individuals and criteria for renal biopsy, particularly in patients with isolated haematuria. By light microscopy the glomeruli may show any of the morphologic manifestations of immune complex glomerulonephritis, from normal through mesangial proliferation, endocapillary proliferation and segmental necrosis to severe crescentic glomerulonephritis. Segmental proliferation and segmental scars are common. Glomerular scarring is associated with tubular atrophy and interstitial fibrosis. By immunohistochemistry IgA is the sole immunoglobulin in 26% of biopsies. 25% have IgA, IgG and IgM. C3 is present in 95%. C1q is present in only 12% of biopsies and, if prominent, should raise the possibility of SLE. It should be noted that mesangial IgA was detected in 16% of kidneys for renal transplantation in Japan [1].

What Is the Purpose of the Renal Biopsy in IgA Nephropathy?

The renal biopsy in IgA nephropathy has a number of potential roles: (1) Diagnosis. The diagnosis of IgA nephropathy can only be made by renal biopsy; (2) exclusion of co-existent renal pathology; (3) prediction of the clinical course for the individual patient; (4) to provide guidance in the selection of appropriate therapy; (5) to allow selection of appropriate groups of patients for inclusion in clinical trials, and (6) to provide insights into pathophysiology that may assist with development of newer and more focused treatment. There are a number of correlations between the appearances seen in the biopsy and the clinical features at the time of biopsy. In particular, as with many renal diseases, serum creatinine is correlated with interstitial fibrosis and globally sclerosed glomeruli. In addition, haematuria and proteinuria are strongly correlated with crescents [2], and proteinuria is correlated with the amount of mesangial proliferation. Features in the biopsy may also be used to predict outcome [3]. Strong predictors of progression to end-stage renal disease are severe glomerular and tubulointerstitial scarring. Weaker predictors are crescents, degree of mesangial proliferation, capillary wall deposits of IgA and marked arteriolar hyalinosis. These weaker predictors are not independent predictors in multivariate analysis. Therefore, a number of histological grading systems have been used with some success to give an indication of likelihood of progression to ESRD [4, 5]. One large study has examined the rate of decline of renal function and shown that this is associated with proteinuria, blood pressure and histological grading [6]. However, only blood pressure and proteinuria during follow up are independent predictors. This means that, in general, the renal biopsy does not add extra

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information to allow the prediction of outcome above that provided by the clinical data alone. This is in contrast to diseases like SLE where the biopsy provides very useful additional information on prognosis and treatment. We can therefore ask why this is.

Why Doesn’t the Biopsy do Better in IgA Nephropathy?

There are a number of reasons that may explain why the renal biopsy does not provide more useful information in IgA nephropathy: (1) Use of ESRD as the main outcome measure. In most series the ability of an initial biopsy to predict progression to end-stage renal failure has been examined. However, in a disease with a long time course and with possible exacerbations and remissions over time it is probably expecting too much for the initial biopsy to do this. It may be better to study the utility of the biopsy in predicting the rate of change of renal function in a shorter time period immediately after the biopsy is taken. (2) Selection of cases. In most series all cases of IgA nephropathy are considered together. However, it is possible that the biopsy adds extra information to the clinical features in only certain groups of patients, and this effect is lost when all patients are considered together. For example, in patients with no, or only mild, abnormalities of renal function at presentation the amount of glomerular sclerosis is an independent predictor of renal outcome [7]. (3) Failure to distinguish reversible and irreversible lesions (table 1). In many of the classification systems there has been little attempt to distinguish those lesions that can be reversed from those that cause irreversible nephron loss. This may not be of importance when considering the likelihood of progression to ESRD where a cellular crescent may have implications that are no different from a fibrous crescent. However such distinctions are likely to be critical in predicting response to therapy. Several studies have examined the effect of immunosuppressive treatment on renal morphology in repeat renal biopsies in IgA nephropathy [8–11]. Most show that treatment is associated with a reduction in mesangial proliferation, mesangial matrix accumulation and cellular crescents. In one there was also a reduction in segmental glomerulosclerosis and tubulointerstitial volume. Studies in diabetes also show the potential for reversibility of matrix increase both in the glomerulus and in the interstitium. Thus, in a group of diabetics given pancreas transplants, examination of renal biopsies at 10 years showed a reduction in mesangial matrix [12] and also in interstitial volume [13]. With regard to segmental glomerulosclerosis,

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Table 1. Reversible and irreversible lesions in IgA Nephropathy Reversible Immune complex deposition Mesangial proliferation Mesangial matrix expansion Endocapillary proliferation Active tubulointerstitial nephritis Irreversible Global glomerulosclerosis Fibrous crescents Tubular atrophy Possibly reversible or functionally repairable Arteriolar hyalinosis Arterial intimal thickening Interstitial fibrosis Segmental glomerulosclerosis

there is evidence that glomeruli with ⬍50% sclerosis of the tuft are capable of growing new capillary loops with both lengthening and branching of glomerular capillaries [14]. A major role for the renal biopsy is in predicting response to treatment, but relatively little attention has been paid to how well the biopsy is able to do this. (4) Differences over definitions of lesions. It is not always clear whether different pathologists apply the same definitions to lesions. For example, while attempting to develop a new classification of IgA nephropathy, it became apparent to our group that there was no clear definition for what constitutes a ‘mesangial area’ and therefore no clear criteria for quantitating mesangial hypercellularity. Similarly, some pathologists would include as a cellular crescent any proliferation of cells in Bowman’s space while others would require that it involves more that 25% of the circumference. It is also worth noting that even crescents of the same size may have different implications depending on their position in the glomerulus. Thus in a study of experimental glomerulonephritis only those crescents that extended onto the tubulo-glomerular junction were associated with tubular atrophy [15]. (5) Reliance on traditional histology. It is possible that there is information that could be provided from the biopsy other than that which is detected by standard histology. Possibilities include the quantitation of glomerular macrophage infiltration, quantitation of podocytes or the staining of biopsies for cytokines either by immunohistochemistry or in situ hybridization.

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A recent publication has shown that activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease [16].

Conclusions

The renal biopsy is essential for the diagnosis of IgA nephropathy. Ideally, the biopsy should also provide useful information on prognosis and on selection of therapy, but with our current analysis of histology the biopsy performs poorly. I have considered a number of reasons why this might be so. Therefore, future classifications of the biopsy in IgA should take these problems into account, and this leads me to suggest a number of recommendations that should be considered in developing new classifications: (1) look at the ability of the biopsy to predict changes in renal function in the short term after biopsy rather than progression to end stage renal disease; (2) examine subgroups of patients where the biopsy is likely to be most informative; (3) distinguish the effects of reversible and irreversible changes particularly with regard to response to treatment; (4) ensure uniformity of definitions between pathologists; (5) pay attention to small lesions that may be important, and (6) consider including additional biopsy features. I would hope that attention to these guidelines will allow us to improve the ability of the renal biopsy to predict outcome and response to treatment in IgA nephropathy. In future, a major role of the biopsy is likely to be in the prediction of response to therapy, and this may require very different classification systems from those designed to predict progression to end stage renal disease.

References 1 2 3 4 5 6 7 8

Suzuki K, Honda K, Tanabe K, et al: Incidence of latent mesangial IgA deposition in renal allograft donors in Japan. Kidney Int 2003;63:2286–2294. Nicholls KM, Fairley KF, Dowling JP, et al: The clinical course of mesangial IgA associated nephropathy in adults. Q J Med 1984;210:227–250. D’Amico G: Natural history of idiopathic IgA nephropathy and factors predictive of disease outcome. Semin Nephrol 2004;24:179–196. Haas M: Histologic subclassification of IgA nephropathy: a clinicopathologic study of 244 cases. Am J Kidney Dis 1997;29:829–842. Lee SMK: Prognostic indicators of progressive renal disease in IgA nephropathy: emergence of a new histologic grading system. Am J Kidney Dis 1997;29:953–958. Bartosik LP, Lajoie G, Sugar L, et al: Predicting progression in IgA nephropathy. Am J Kidney Dis 2001;38:728–735. To KF, Choi PCL, Szeto CC, et al: Outcome of IgA nephropathy in adults graded by chronic histological lesions. Am J Kidney Dis 2000;35:392–400. Hotta O, Furuta T, Chiba S, et al: Regression of IgA nephropathy: a repeat biopsy study. Am J Kid Dis 2002;39:493–502.

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Shoji T, Nakanishi I, Suzuki A, et al: Early treatment with corticosteroids ameliorates proteinuria, proliferative lesions, and mesangial phenotypic modulation in adult diffuse Proliferative IgA nephropathy. Am J Kid Dis 2000;35:194–201. Yoshikawa N, Ito H, Sakai T, et al: A controlled trial of combined therapy for newly diagnosed severe childhood IgA nephropathy. J Am Soc Nephrol 1999;10:101–109. Kuriki M, Asahi K, Asano K, et al: Steroid therapy reduces mesangial matrix accumulation in advanced IgA nephropathy. Nephrol Dial Transplant 2003;18:1311–1315. Fioretto P, Steffes MW, Sutherland DER, et al: Reversal of lesions of diabetic nephropathy after pancreas transplantation. New Engl J Med 1998;339:69–75. Fioretto P, Sutherland DER, Najafian B, et al: Remodeling of renal interstitial and tubular lesions in pancreas transplant recipients. Kidney Int 2006;69:907–912. Fogo AB: Can glomerulosclerosis be reversed? Nat Clin Pract Nephrol 2006;2:290–291. Le Hir M, Besse-Eschmann V: A novel mechanism of nephron loss in a murine model of crescentic glomerulonephritis. Kidney Int 2003;63:591–599. Roos A, Pia Rastaldi A, Calvaresi N, et al: Glomerular activation of the lectin pathway of complement in IgA nephropathy is associated with more severe renal disease. J Am Soc Nephrol 2006;17: 1724–1734.

H. Terence Cook Department of Histopathology, Imperial College Hammersmith Hospital, Du Cane Road London W12 0NN (UK) Tel. ⫹44 208383 2009, Fax ⫹44 208383 4717, E-Mail [email protected]

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Basic Reviews of IgA Nephropathy Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 50–55

Altered Expression of Lymphocyte Homing Chemokines in the Pathogenesis of IgA Nephropathy Marc Burena, Michifumi Yamashitaa, Yusuke Suzukib, Yasuhiko Tominob, Steven N. Emancipatora a

Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA; Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan

b

Abstract Defective adaptive humoral immune responses to mucosal immunogens, but intact systemic responses, are increasingly recognized in patients with IgA nephropathy (IgAN). Reduced expression of IgA⫹, J chain⫹ cells in the gut lamina propria, with collateral increases in these cells in the marrow, is also documented. Thus, there seems to be a derangement in a ‘mucosa-marrow axis’ in IgAN patients. Recent evidence indicates that chemokines regulate the localization of B cells and their progeny into respiratory and intestinal lamina propria, and into other lymphoid organs as well. Particularly, secretory epithelial cells express the chemokine CCL28, whereas small bowel cells uniquely express CCL25. Extramucosal sites preferentially express CXCL12, CXCL13 and/or CXCL16. Reciprocally, plasmablasts committed to IgA synthesis ubiquitously express the receptor (CCR10) for CCL28, and a subset also express the receptor (CCR9) for CCL25; neither of these is present on cells committed to IgG or IgM synthesis. Herein, the potential contributions of virally induced innate responses to defective mucosal immunity and overproduction of oligomeric IgA in the marrow and tonsils will be reviewed, particularly with respect to the influence that viral infection exerts upon the expression of selected chemokine and receptor pairs. The ramifications for pathogenesis of IgAN will be considered. Copyright © 2007 S. Karger AG, Basel

Commentary

No clear mechanistic paradigm exists to elucidate the pathogenesis of IgA nephropathy (IgAN), despite the increasingly sharper focus of international and

multidisciplinary effort applied over nearly forty years [1, 2]. Indeed, more questions than answers have emerged. For example, circulating levels of IgA are increased in patients with IgAN, and IgA is over-produced by short-term culture of patients’ cells. Yet, there is a defect in the humoral IgA immune response to mucosal immunization of IgAN patients, and in secretion of IgA from the mucosa [3–5]. From this particular paradox, a novel variation of an old principle arises. Initially, the IgA deposited in the glomeruli was believed to be synthesized by plasma cells within the lamina propria of secretory mucosa, but evidence for the involvement of extramucosal sites (such as tonsils and bone marrow) has grown [6–10]. The controversy continues amid widening appreciation of the therapeutic value of tonsillectomy in at least some patients [11–13]. We hypothesize that in IgAN patients, IgA-producing cells otherwise destined for the secretory mucosal lamina propria are mis-routed to non-secretory sites, such as the tonsils, bone marrow and spleen. Specifically, aberrant expression of chemokines and/or chemokine receptors diverts plasmablasts from the gut and airways to these extramucosal sites. Trafficking of lymphocytes and plasmablasts, like that of leukocytes in general, is governed by pairings of heterotrophic adhesion molecules and by expression of chemokines and their cognate receptors. For example, the preferential expression of ␤7 integrins by mucosal lymphocytes supports their localization to tissues wherein endothelial cells express the mucosal addressin cellular adhesion molecule, MAdCAM [reviewed in 2, 14]. Restriction of MAdCAM to mucosal sites promotes accumulation of ␤7 integrin-positive lymphocytes at these sites, just as other ligand pairs direct homing to peripheral lymph nodes and skin. The ␤1 and ␤2 integrins can also promote localization to various tissues, albeit with less tissue specificity than the ␤7 integrins. In addition to these well recognized molecules that drive site-specific cell-to-cell contact, less known soluble paracrine proteins recruit lymphocytes and plasma cells to their destinations within particular tissues. These soluble chemokines offer considerable, and in some cases the predominant, specificity for endorgan localization. In healthy adult mice and humans, mRNA encoding thymus expressed chemokine (TECK, CCL25) is also highly expressed in the duodenum, jejunum and ileum, but in no other mucosal or extramucosal tissue [14]. The protein is detected by immunohistochemistry within crypt, but not villous, epithelium, exclusively in the small bowel. Reciprocally, lymphocytes isolated from the small intestine, and to a lesser extent those from other mucosal sites, uniquely express the receptor (CCR9) for CCL25, and migrate towards CCL25 [14]. By contrast, lymphocytes isolated from many other sources, including lung, colon and tonsil, lack CCR9 expression. In addition to T cell populations (whether

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CD4⫹ or CD8⫹) from the intraepithelial or lamina propria lymphocyte compartments of the gut, IgA secreting cells migrate specifically in response to CCL25 and highly express CCR9, regardless of the lymphoid organ from which these cells are isolated [15]. On the other hand, IgG or IgM secreting cells do not migrate to CCL25, and do not express CCR9. A second CC family chemokine, mucosae-associated epithelial chemokine (MEC, CCL28) has homology to CCL25 [16, 17]. The mRNA and protein are expressed by a variety of epithelial cells, including those from the esophagus, large and small bowel, stomach, trachea and bronchi, mammary gland and salivary gland. Epithelial cells from the skin, urinary and reproductive tracts (including kidney) and the placenta express little or no CCL28. Of note, the marrow, tonsils and spleen also express CCL28, presumably in macrophages and/or stromal cells. Two receptors promote intracellular signal transduction and consequent chemotaxis in response to ligation by CCL28: CCR10 and CCR3. Whereas CCR10 also binds the cutaneous chemokine (CTACK, CCL27), CCL28 is much more efficacious at chemotaxis than its equipotent congener CCL27. In contrast, RANTES (CCL5) is much more potent than CCL28, yet the two ligands are equally efficacious at stimulating the migration of CCR3⫹ cells, which include several disparate populations. Although CCR3 is widely distributed, the CCR10 receptor lies principally on lymphocytes and plasma cells from all segments of the gut (including Peyer’s patches), the lung and mesenteric lymph nodes; CCR10 is also present on cells from the spleen. Interestingly, T cells polarized to the type 2 response exhibit much more abundant CCR10 than do Th1 cells. More germane to our interests here, IgA plasmablasts are selectively attracted to CCL28 [18]. Essentially all IgA plasmablasts express CCR10, whereas few IgM⫹ and virtually no IgG⫹ plasmablasts do [18, 19]; migration to CCL28 parallels the expression of CCR10. Notably, tonsillar and marrow IgA⫹ plasmablasts also express CCR10 (among other chemokine receptors), and CCL28 is constitutively produced in the marrow, tonsils and spleen, albeit at levels considerably lower than in secretory mucosal sites [20]. A subset of IgA⫹, CCR10⫹ plasmablasts also express CCR9 [19]; plasmablasts localized to the small bowel are, under normal circumstances, drawn entirely from this CCR9⫹, CCR10⫹ population. On the other hand, IgA⫹ plasmablasts isolated from the marrow and other extramucosal sites exhibit abundant co-expression of CCR3, CXCR4 and CXCR6 along with CCR10, making them capable of responding to CXCL12 and CXCL16, both well expressed in these extramucosal sites [20]. In principle, the relative density of the various chemokine receptors on various plasmablasts, in concert with tissue-dependent expression of their respective ligands, govern the partitioning of the cells among their potential destinations. Among IgA⫹ plasmablasts, characterized by ubiquitous and constitutive

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CCR10, co-expression of CCR9 favors accumulation in the small gut that is heavily laden with CCL25, whereas co-expression of CXCR4 apparently promotes localization to the tonsil and marrow, sites rich in CXCL12. Expression of CCR10 alone, perhaps at higher density, might drive localization to the ‘neutral’ territory of other mucosal sites that express little or no CCL25 or CXCL12, such as the colon, tracheobronchial tree and salivary glands. In healthy individuals, the density of cells committed to IgA⫹ secretion is highest in the lamina propria of the gut and airways, and accessory glands of these viscera, such as the salivary and lacrimal glands. Conversely, in extramucosal sites such as the marrow, spleen and tonsils, IgG⫹ and/or IgM⫹ antibody secreting cells predominate. Although little is currently known about control of CCR9 and CCR10 expression, it is likely that these receptors, particularly CCR10, share features of transcriptional regulation with secretion of IgA and/or synthesis of J chain. Perhaps there is repression of CXCR4 (and/or CXCR6) in conjuntion with synthesis of J chain; this would explain why most extramucosal IgA⫹ plasmablasts and plasmacytes do not produce J chain. In IgAN patients, one can envision reduced density of CCR10, inhibited production of CCR9 and/or increased proclivity towards the synthesis of CXCR4 in IgA⫹, J chain⫹ plasmablasts. Alternately, there might be heterotopic expression of CCL25 and/or markedly increased CCL28 in tonsil and/or bone marrow in IgAN patients, or diminished or altered CCL25 or CCL28 by mucosal epithelium. Any of these changes could by itself shift the migration of J chain⫹, IgA⫹ plasmablasts towards the tonsils, marrow and spleen and away from the mucosa, and especially away from the small bowel. These alterations could promote an inversion in the ratio of pIgA⫹ (expressing J chain) to IgG⫹ plasmablasts in various sites, diminish dramatically the number of antigenspecific plasma cells in mucosal lamina propria and impair excretion of secretory IgA. The predicted consequences, including defective mucosal immunity and heightened levels of pIgA in the bloodstream are well recognized concomitants in IgAN patients [1–10]. Moreover, if any of these effects were combined, synergy in the effect would be likely. The defects could represent constitutive (genetic) alterations in the regulation and/or structure of these chemokines and/or receptors themselves, or they might derive from persistence of normally transient changes in response to extrinsic stimuli that are sustained or durable in IgAN patients relative to healthy individuals. Changes in the synthesis of chemokines and chemokine receptors are well recognized concomitants to a variety of innate responses to components of infectious agents. Although those now known are related principally to phagocytes and memory lymphocytes in the context of inflammation, the homologies among the members of the chemokine families and the receptors for these chemokines suggest that these more recent members might be subject to the same overall regulatory signals.

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In the majority of patients, exacerbation of IgAN occurs in close temporal association with a syndrome that appears to be a viral infection of a mucosal site, such as the respiratory tract or gut. Innate responses to viral infection exert powerful effects on cellular function that transcend anti-viral mechanisms, including the maturation and phenotypic modulation of dendritic cells, the balance of cytokine production by antigen-specific T cells, and differentiation of B cells to plasmablasts. We reasoned that innate responses to viral infection might also influence the expression of the chemokines and/or chemokine receptors responsible for homing of IgA⫹ plasmablasts to the various tissue compartments, and might thereby contribute to the pathogenesis of IgAN. Particularly, we focus on double-stranded (ds) RNA, a by-product of the replication of most pathogenic viridiae. If the action of dsRNA upon various plasmablasts, epithelial cells and/or lymphoid stromal cells has consequences relevant to natural viral infection in vivo, this hypothesis has appreciable ramifications for the pathogenesis of IgAN. New therapeutic initiatives might arise from modulation of such innate anti-viral responses, if the kinetics and mechanisms of control of expression and function of these chemokines and their receptors can be elucidated.

References 1

2

3 4 5 6

7

8 9

10

11

Emancipator SN: IgA nephropathy and Henoch-Schonlein syndrome; in Jennette JC, Olson JL, Schwartz MM, Silva FG (eds): Heptinstall’s Pathology of the Kidney, ed 5. Philadelphia, Pa., Lippincott-Raven, 1998, pp 479–540. Emancipator SN, Mestecky J, Lamm ME: IgA nephropathy and related diseases; in Mestecky J, Bienenstock J, Lamm ME, Mayer L, McGhee JR, Strober W (eds): Mucosal Immunology, ed 3. San Diego, Calif., Elsevier Academic Press, 2005, pp 1579–1600. Waldo FB: Role of IgA in IgA nephropathy. J Pediatr 1990;116:S78–S85. de Fijter JW, Eijgenraam JW, Braam CA, et al: Deficient IgA1 immune response to nasal cholera toxin subunit B in primary IgA nephropathy. Kidney Int 1996;50:952–961. Yasumori R: Measurement of secretory IgA in salivary juice and the localization of secretory IgA in duodenal mucosa in patients with IgA nephropathy. Nippon Jinzo Gakkai Shi 1990;32:171–181. Bene MC, Faure G, Hurault de Ligny B, et al: Immunoglobulin A nephropathy. Quantitative immunohistomorphometry of the tonsillar plasma cells evidences an inversion of the immunoglobulin A versus immunoglobulin G secreting cell balance. J Clin Invest 1983;71:1342–1347. Egido J, Blasco R, Lozano L, et al: Immunological abnormalities in the tonsils of patients with IgA nephropathy: inversion in the ratio of IgA: IgG bearing lymphocytes and increased polymeric IgA synthesis. Clin Exp Immunol 1984;57:101–106. Harper SJ, Pringle JH, Wicks AC, et al: Expression of J chain mRNA in duodenal IgA plasma cells in IgA nephropathy. Kidney Int 1994;45:836–844. Harper SJ, Allen AC, Layward L, et al: Increased immunoglobulin A and immunoglobulin A1 cells in bone marrow trephine biopsy specimens in immunoglobulin A nephropathy. Am J Kidney Dis 1994;24:888–892. Harper SJ, Allen AC, Bene MC, et al: Increased dimeric IgA-producing B cells in tonsils in IgA nephropathy determined by in situ hybridization for J chain mRNA. Clin Exp Immunol 1995;101: 442–448. Bene MC, Hurault de Ligny B, Kessler M, et al: Tonsils in IgA nephropathy. Contrib Nephrol 1993;104:153–161.

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12 13 14

15 16 17 18 19 20

Komatsu H, Fujimoto S, Hara S, et al: Multivariate analysis of prognostic factors and effect of treatment in patients with IgA nephropathy. Ren Fail 2005;27:45–52. Kawasaki Y, Takano K, Suyama K, et al: Efficacy of tonsillectomy pulse therapy versus multipledrug therapy for IgA nephropathy. Pediatr Nephrol 2006;21:1701–1706. Kunkel EJ, Campbell JJ, Haraldsen G, et al: Lymphocyte CC chemokine receptor 9 and epithelial thymus-expressed chemokine (TECK) expression distinguish the small intestinal immune compartment: epithelial expression of tissue-specific chemokines as an organizing principle in regional immunity. J Exp Med 2000;192:761–768. Bowman EP, Kuklin NA, Youngman KR, et al: The intestinal chemokine thymus-expressed chemokine (CCL25) attracts IgA antibody-secreting cells. J Exp Med 2002;195:269–275. Wang W, Soto H, Oldham ER, et al: Identification of a novel chemokine (CCL28), which binds CCR10 (GPR2). J Biol Chem 2000;275:22313–22323. Pan J, Kunkel EJ, Gosslar U, et al: A novel chemokine ligand for CCR10 and CCR3 expressed by epithelial cells in mucosal tissues. J Immunol 2000;165:2943–2949. Lazarus NH, Kunkel EJ, Johnston B, et al: A common mucosal chemokine (mucosae-associated epithelial chemokine/CCL28) selectively attracts IgA plasmablasts. J Immunol 2003;170:3799–3805. Kunkel EJ, Kim CH, Lazarus NH, et al: CCR10 expression is a common feature of circulating and mucosal epithelial tissue IgA Ab-secreting cells. J Clin Invest 2003;111:1001–1010. Nakayama T, Hieshima K, Izawa D, et al: Cutting edge: profile of chemokine receptor expression on human plasma cells accounts for their efficient recruitment to target tissues. J Immunol 2003;170:1136–1140.

Steven N. Emancipator, MD Case Western Reserve University Wolstein Research Building Room 5136 Mail Stop 7288 10900 Euclid Avenue Cleveland, OH 44106 (USA)

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IgA Nephropathy: Current Views of Immune Complex Formation Jiri Mesteckya, Hitoshi Suzukia,b, Takeshi Yanagiharaa,c, Zina Moldoveanua, Milan Tomanaa, Karel Matousovica,d, Bruce A. Juliana, Jan Novaka a

University of Alabama at Birmingham, Birmingham, Ala., USA; bJuntendo University, and cNippon Medical School, Tokyo, Japan; dCharles University, School of Medicine and Faculty Hospital, Prague and Pilsen, Czech Republic

Abstract Characteristic features of IgA nephropathy (IgAN) include IgA1-containing immune complexes (IC) in the circulation, urine, and renal mesangium. IC contain IgA1 deficient in hinge region-associated galactose (Gal) and antibodies specific for antigenic determinants present on the hinge region. The biological effects of IC are primarily related to their molecular size and composition: when added to a culture of human mesangial cells, large IC exhibit a proliferative effect while small complexes are inhibitory. These activities have been observed using IC obtained from sera of IgAN patients or generated in vitro. Specifically, various preparations of human IgA1 with modified glycan moieties formed IC in vitro when incubated with sera from IgAN patients or healthy individuals, cord blood serum, or tissue culture supernatants of EBV-immortalized peripheral blood B cells secreting IgG. Interestingly, IgG antibodies specific for the IgA1 Gal-deficient hinge region are commonly found in sera of hominoid as well as non-hominoid primates and many other vertebrate species, and suggest the evolutionary uniqueness of the human IgA1 hinge region. Because of the molecular defect in IgA1 glycosylation and its subsequent recognition by naturallyoccurring antibodies, experimental approaches that diminish or prevent formation of large immunostimulatory IC should be further explored. Copyright © 2007 S. Karger AG, Basel

Introduction

The presence of IgA-containing immune complexes (IC) in the mesangium of all patients and in the circulation and urine of most patients are characteristic features of IgA nephropathy (IgAN). It has been assumed for several decades that such IC consist of IgA1 as an antibody specific for unknown environmental

antigens. Nevertheless, a search for such antigens typical for IC in IgAN patients proved fruitless: although many exogenous antigens of microbial origin or certain food antigens had been suspected as participants in IC formation, none of the potential candidates was confirmed with an acceptable degree of uniformity and reproducibility [1]. Antigens of endogenous origin, such as basement membrane proteins, have also been considered as components of IC [2, 3]. However, IgA1 autoantibodies, for example to basement membrane collagens, are present in other diseases and are not IgAN-specific [2]. In a similar vein, IgA1-fibronectin-collagen interaction thought to be specific for the detection of IC in the circulation of IgAN patients also proved to be nonspecific for IgAN. Finally, the role of IgA1 rheumatoid factor, defined as an autoantibody to aggregated human IgG, has remained elusive [4]. Based on the biochemical studies (glycan compositions) of IgA1 proteins isolated from sera of IgAN patients and healthy controls, and on detailed analyses of the properties of IC isolated from sera of the patients (including IC composition, dissociability, re-formation and its inhibition), we proposed that IgAN is a disease of an autoimmune character. According to this definition, IgA1 acts as an antigen that is recognized by naturally-occurring antibodies of the IgG, IgA1 or IgM isotypes specific for the hinge region of IgA1 [5]. Here, we present accumulated evidence for this contention, with supportive experimental data, dissenting opinions, and approaches for potential interference of IC formation.

IgA1 as an Antigen

The most striking structural feature that differentiates human IgA1 from immunoglobulins of all other isotypes is its unique hinge region. Comparative evolutionary studies of IgA hinge regions of many species (for reviews, see [6, 7]) clearly indicate a relatively recent insertion of a gene segment encoding for an additional thirteen amino acids into the phylogeneticaly older IgA2 gene [7]. The origin of this insertion remains obscure, although by its amino acid composition and sequence and the presence of O-linked glycans, the human IgA1 hinge region remotely resembles mucin. However, other properties, such as susceptibility to proteolytic enzymes of bacterial origin [8], are distinct and absolutely unique. It should be stressed that only IgA1 of humans, and of some primates such as chimpanzee and gorilla, contain hinge regions, with minor differences in their primary structures. Furthermore, primary structures of human IgA1 and IgA2 differ, besides the hinge region, in only 20 of approximately 360 amino acid residues of CH 1–3 domains and C-terminal tails [6]. The structural or functional advantage (or disadvantage) conferred by the hinge region insertion into the hominoid primate hinge region remains obscure.

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Our studies of the antigenic determinants on human IgA1 that are recognized by naturally occurring antibodies revealed the dominant role of O-linked glycan moieties [5]. Thus, the enzymatic removal of O-linked glycans by endoglycosidases results in the loss of reactivity with antibodies, and the reformation of acid-dissociated IC can be partially inhibited by Gal-deficient IgA1 or other glycoproteins bearing O-linked glycans with terminal N-acetylgalactosamine (GalNAc). However, the exact localization and relative involvement of individual O-linked glycan chains have not been precisely determined. Deficiency of Gal resulting in the exposure of GalNAc (either as the terminal glycan or perhaps in its sialylated form) is essential for reactivity with corresponding antibodies. Furthermore, it is clear that exposed GalNAc is not expressed in the form of Tn antigen, which consists of three vicinary GalNAc residues [9]; monoclonal anti-Tn antigen antibodies do not react with human IgA1 irrespective of the nature of the enzymatically modified glycan form (H. Classen, Copenhagen, personal communication; Mestecky et al., unpublished results). Specifically, monoclonal antibodies to the Tn and sialyl-Tn antigens (gift from Dr. H. Classen and purchased from Dako) reacted with desialylated ovine submaxillary mucin (containing terminal GalNAc) or human O Rh⫺ erythrocytes treated with relevant glycosidases, but not with identically treated human IgA1 or IgA2 myeloma proteins. Furthermore, in all of our studies, human myeloma IgA1 proteins have been used. Despite their monoclonal character, such proteins still display a marked heterogeneity in the number, sites of attachment, and composition of O-linked glycans [10, 11]. Limited studies concerning the localization of antigenic determinants using Fab and Fc fragments generated by various bacterial proteases suggest that Ser/Thr residues 228 and 230 are recognized by anti-GalNAc antibodies. However, it should be kept in mind that myeloma proteins instead of natural IgA1 obtained from IC of IgAN patients were used. Recently, we obtained additional evidence, using IgA1 isolated from the circulation of IgAN patients, which suggested that glycans at Ser/Thr residues 228 and 230 are Gal-deficient and thus likely neo-epitopes (Novak et al., this volume). Another important point concerns the number of Gal-deficient O-linked glycan chains. We detected Gal-deficient IgA1 in complexes with IgG in the high-molecular-mass fractions of sera from IgAN patients by its reactivity with Helix aspersa (HAA) lectin, which binds terminal GalNAc [5, 12]. Therefore, not all terminal GalNAc residues in the IgA1 hinge region are occupied by the corresponding antibodies. It is reasonable to speculate that in addition to the terminal GalNAc, conformation and/or sequence of the polypeptide chain may differentially affect exposure of glycans to anti-glycan antibodies and reactivity with HAA. Obviously, additional experiments addressing these points must be performed.

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The possibility that the absence of the Gal residue is the result of extracellular modification of IgA1, for example by endogenous or exogenous glycosidases, was excluded by analyses of extracellular and intracellular forms of IgA1 produced by lymphocytes obtained from peripheral blood of IgAN patients and healthy controls after transformation by Epstein-Barr virus (EBV) (Suzuki et al., this volume pp 129–133). Reactions with lectins and antibodies demonstrated that IgA1 from supernatants and IgA1-producing cell lines is Gal-deficient. Furthermore, EBV-transformed lymphocytes from IgAN patients, but not healthy controls, display enhanced production of Gal-deficient IgA1. The size-exclusion chromatographic analysis, the presence of a J chain, and the lack of reactivity of monomeric IgA with HAA suggested that Galdeficiency is restricted to high-molecular-mass IC containing polymeric IgA1 [5]. This view was further supported by the finding that EBV-transformed IgA1-producing cells from IgAN patients secreted a higher proportion of polymeric IgA1 compared to the cells from healthy controls (Suzuki et al., this volume pp 129–133).

GalNAc-Specific Antibodies

Although Gal-deficiency of IgA1 molecules in serum IC and mesangial deposits in IgAN patients has been reported by several investigators (for reviews, see [13, 14]), our previous contention that Gal-deficient IgA1 is an antigen recognized by GalNAc-specific naturally occurring antibodies has received limited attention. The unquestionable nature of IgA1-IgG (or IgA1 and IgM) interactions as true antigen-antibody reaction is of particular importance. Alternatively, IgA, due to its well-known tendency to form complexes with a broad spectrum of proteins and glycoproteins (e.g., proteolytic enzymes, amylase, lactate dehydrogenase, serum albumin, fibronectin, lactoferrin, and many others), may interact with other immunoglobulins by formation of easily-dissociable (1 M NaCl) complexes [15]. Results from our laboratory favor the possibility that IgA1-immunoglobulin interactions are based on the complementary specificities of the antigen (Gal-deficient IgA1) and naturally-occurring antiGalNAc antibody. IC isolated from sera of IgAN patients are dissociable only at acidic pH [5, 12] and not in 1 M NaCl. Anti-GalNAc antibodies of the IgG isotype are present in sera of all IgAN patients and healthy individuals, and in cord blood. IC formed in vitro display a stimulatory effect on mesangial cells [16] in a dosedependent fashion. Clones of EBV-transformed peripheral blood lymphocytes from IgAN patients and normal controls secrete IgG antibodies specific for Gal-deficient IgA1, as detected by ELISA and ELISPOT (using plates coated

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with native, Gal-deficient IgA1 [5]; and Moldoveanu et al., unpublished results) and Western blotting (denatured, isolated ␣ chain on SDS gels; Suzuki et al., this volume). Furthermore, cells secreting antibodies specific for Gal-deficient IgA1 can be easily detected and enumerated in peripheral blood from IgAN patients and, at lower frequency, from normal individuals by ELISPOT or immunofluorescence. Finally, antibodies specific for Gal-deficient human IgA1, but not IgA2, are present in IgG from sera of many phylogenetically diverse vertebrate species, including pigs, rabbits, cows, donkeys, goats, sheep, mice, and rats (Moldoveanu et al. – unpublished results). Thus, a critical compilation of existing experimental data provides a solid base for the nature and specificity of IC.

Generation of IC

Because Gal-deficient IgA1 is detectable only in high-molecular-mass fractions of serum, whereas anti-GalNAc antibodies are easily detectable in their free form, it is obvious that IC present in the patients’ sera were formed in the antibody-excess zone. We stress that our studies focused on IC which contain GalNAc-specific antibodies of the IgG isotype and therefore do not fully consider IC containing other isotypes, which probably differ in both physicochemical and biological properties. Nevertheless, biological effects of IC in vivo are likely to depend on the proportion of IC that exhibit either stimulatory or inhibitory effects on cultured human mesangial cells [16]. This, in turn, might reflect the composition (including immunoglobulin isotype representation), charge, size, tissue and body fluid distribution, and reactivities with relevant cellular receptors. The latter point is of particular importance: both, terminal Gal and GalNAc on glycoprotein molecules of limited size are recognized and internalized by the hepatic asialoglycoprotein receptor (ASGP-R) [17]. Although we proposed that anti-GalNAc antibodies may inhibit the removal of Gal-deficient IgA1 in the liver, there are still free terminal GalNAc residues available, as evidenced by their HAA reactivity [5, 12]. Therefore, it is likely that the size of IC and/or inaccessibility of GalNAc to ASGP-R due to the potential hindrance conferred by bound IgG prevent effective IC clearance and increases their elimination through the kidneys [18]. Indeed, IgA-containing IC are present in the urine of IgAN patients in significantly higher amounts than in the urine of patients with other renal diseases with comparable proteinuria, or of healthy controls [18]. With disease progression, proteinuria increases and is often non-selective, so that high-molecular-weight proteins (such as immunoglobulins) cross the glomerular capillary wall and appear in the urine. These observations indicate that kidneys of IgAN patients are

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Table 1. Experiments supporting the nature of antigen-antibody interactions between Gal-deficient IgA1 (antigen) and corresponding GalNAc-specific antibodies Experiment

References

Sera of IgAN patients contain IgG and IgM antibodies to IgA1 but not IgA2 myeloma proteins. Antibodies react with the Fab fragments, which carry the hinge region (detected by ELISA).

[5, 12, 19, 20]

Anti-IgA1 antibodies are directed against the hinge-region glycans Enzymatic removal of O-linked glycans reduces the reactivity. Proteolytic fragments generated by IgA1 proteases react with IgG antibodies according to the presence of hinge-region glycans (determined by ELISA and western blots).

[5, 12]

Anti-hinge-region glycan antibodies are specific for terminal GalNAc (or sialyl-GalNAc). Enzymatic removal of SA and Gal exposes terminal GalNAc and increases reactivity with antibodies (ELISA and Western blot). Re-formation of acid-dissociated IC can be inhibited by terminal GalNAc-bearing glycoproteins (ELISA). Antibody-producing cells from peripheral blood secrete antibodies of the IgG isotype specific for Gal-deficient IgA1.

[5, 12]

[Unpublished]

exposed to a high load of IgA-containing IC which are excreted in the urine by filtration across the damaged glomerular barrier. Indeed, their urinary levels positively correlated with proteinuria (p ⬍ 0.001). These IC have a molecular mass between 650–850 kDa. Experimental models of IC diseases and clinical and laboratory evaluation of human IC diseases (e.g., serum sickness) have convincingly demonstrated that the proportion of antigen to antibody plays a decisive role in the formation of pathogenic IC and disease progression. If IgAN belongs to the category of IC diseases, the manipulation of antigen to antibody ratios, and consequently the generation of non-nephritogenic complexes, may provide a rationale for causespecific therapy. For example, introduction of monoclonal Fv fragment antibodies with high affinity for terminal GalNAc may result in the formation of nonpathogenic small-molecular IC without concomitant stimulation of mesangial cells. Alternatively, small peptides carrying a single GalNAc residue at an appropriate position may bind, without pathological consequences, naturallyoccurring GalNAc-specific antibodies and thus block their interaction with endogenous Gal-deficient IgA1 molecules. In both scenarios, the formation of large and pathogenic IC may be selectively and specifically prevented with no pathologic sequelae (table 1).

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Acknowledgments This work was supported by NIH grant P01 DK061525 (USA), and Research project MSMT – #0021620819 (Czech Republic).

References 1

2 3 4

5

6

7 8

9 10 11

12 13 14 15

16 17 18

Mestecky J, Waldo FB, Britt WJ, et al: Exogenous antigens deposited in the glomeruli of patients with IgA nephropathy; in Sakai H, Sakai O, Nomoto Y (eds): Pathogenesis of IgA Nephropathy. Tokyo, Harcourt Brace Jovanovich, 1990, pp 247–257. van den Wall Bake AWL, Kirk KA, Gay RE, et al: Binding of serum immunoglobulins to collagens in IgA nephropathy and HIV infection. Kidney Int 1992;42:374–382. Ballardie FW, Brenchley PEC, Williams S, et al: Autoimmunity in IgA nephropathy. Lancet 1988;ii:588–592. Czerkinsky C, Koopman WJ, Jackson S, et al: Circulating immune complexes and immunoglobulin A rheumatoid factor in patients with mesangial immunoglobulin A nephropathies. J Clin Invest 1986;77:1931–1938. Tomana M, Novak J, Julian BA, et al: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies. J Clin Invest 1999;104: 73–81. Mestecky J, Moro I, Kerr MA, et al: Mucosal immunoglobulins; in Mestecky J, Bienenstock J, Lamm ME, et al (eds): Mucosal Immunology, ed 3. Amsterdam, Elsevier/Academic Press, 2005, pp 153–181. Sumiyama K, Saitou N, Ueda S: Adaptive evolution of IgA hinge region in primates. Mol Biol Evol 2002;19:1093–1099. Kilian M, Mestecky J, Russell MW: Defense mechanisms involving Fc-dependent functions of immunoglobulin A and their subversion by bacterial immunoglobulin A proteases. Microbiol Rev 1988;52:296–303. Nakada H, Numata Y, Inoue M, et al: Elucidation of an essential structure recognized by an antiGalNAc␣-Ser(Thr) monoclonal antibody (MLS 128). J Biol Chem 1991;266:12402–12405. Novak J, Tomana M, Kilian M, et al: Heterogeneity of O-glycosylation in the hinge region of human IgA. Mol Immunol 2001;37:1047–1056. Renfrow MB, Cooper HJ, Tomana M, et al: Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation Fourier transform-ion cyclotron resonance mass spectrometry. J Biol Chem 2005;280:19136–19145. Tomana M, Matousovic K, Julian BA, et al: Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG. Kidney Int 1997;52:509–516. Barratt J, Feehally J, Smith AC: Pathogenesis of IgA nephropathy. Semin Nephrol 2004;24: 197–217. Novak J, Julian BA, Tomana M, et al: Progress in molecular and genetic studies of IgA nephropathy. J Clin Immunol 2001;21:310–327. Iwase H, Yokozeki Y, Hiki Y, et al: Human serum immunoglobulin G3 subclass bound preferentially to asialo-, agalactoimmunoglobulin A1/Sepharose. Biochem Biophys Res Commun 1999;264: 424–429. Novak J, Tomana M, Matousovic K, et al: IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells. Kidney Int 2005;67:504–513. Stockert RJ: The asialoglycoprotein receptor: relationships between structure, function and expression. Physiol Rev 1995;75:591–609. Matousovic K, Novak J, Yanagihara T, et al: IgA-containing immune complexes in the urine of IgA nephropathy patients. Nephrol Dial Transplant 2006;21:2478–2484.

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19 20

Jackson S, Montgomery RI, Mestecky J, et al: Antibodies directed at Fab of IgA in the sera of normal individuals and IgA nephropathy patients. Adv Exp Med Biol 1987;216B:1537–1544. Jackson S, Montgomery RI, Mestecky J, et al: Normal human sera contain antibodies directed at Fab of IgA. J Immunol 1987;138:2244–2248.

Jiri Mestecky, MD, PhD University of Alabama at Birmingham, Departments of Microbiology and Medicine BBRB 757 – Box 1, 845 19th Street South Birmingham, AL 35294–2170 (USA) Tel. ⫹1 205 934 2225, Fax ⫹1 205 934 3894, E-Mail [email protected]

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Pathogenic Role of IgA Receptors in IgA Nephropathy Renato C. Monteiroa,b a

INSERM U699 and bMedical School of Denis Diderot University, Bichat Campus, Paris, France

Abstract The pathogenesis of IgA nephropathy (IgAN) involves the deposition of aberrant glycosylated IgA1 and/or of IgA1-immune complexes (IC) in the glomerular mesangium. The mechanisms involved in the generation of IgA1-IC and how they are deposited in the mesangium are just emerging. We propose a model whereby two types of IgA receptors participate in sequential steps to promote the development of IgAN, with soluble Fc␣RI (CD89) being initially involved in the formation and/or amplification of the size of circulating IgAIC and, subsequently, transferrin receptor (CD71), in mediating mesangial deposition of nephritogenic IgA1-IC. Activation of transmembrane Fc␣RI associated with FcR␥ adaptor following interaction with IgA-IC is involved in aggravation of IgAN through induction of leukocyte infiltration. Taken together, understanding of the role of IgA receptors in physiology and physiopathology will open new avenues for therapeutic strategies in IgAN. Copyright © 2007 S. Karger AG, Basel

The pathogenesis of IgAN involves the presence of aberrant glycosylated IgA1 under the form of immune complexes in the glomerular mesangium [1]. Since the last decade it has become clear that mechanisms implicated in the pathogenesis of this disease directly implicates IgA receptors [2]. Participation of IgA receptors can be divided in three essential steps of IgAN pathogenesis: (1) the generation of nephritogenic circulating IgA1 complexes; (2) interaction of IgA1 complexes with mesangial IgA receptors, and (3) induction of inflammatory mediators resulting in leukocyte infiltration and progression towards end-stage renal disease (ESRD) (see fig. 1). The first step, essential in the initiation phase of the disease, is characterized by generation of nephritogenic circulating IgA1-IC. Indeed evidence for such harmful complexes has been highlighted by clinical features of the frequent

Aberrant pIgA1 and/or IgA immune complexes

Transmembrane Fc␣RI␥2 activation

Cytokines/chemokines ␥-less Fc␣RI shedding

Mesangial transferrin receptor

Circulating IgA-sol Fc␣RI complexes

Mesangial IgA deposits

Leukocyte infiltration Inflammatory reaction

Progression of IgAN

Fig. 1. Hypothetical role of IgA receptors in IgA nephropathy.

recurrence of mesangial IgA deposits in patients after transplantation of a normal kidney [3]. IgA-IC are characterized mainly by IgA of the IgA1 subclass with abnormalities of glycosylation in their hinge region. However, the composition of such IgA1-IC remains poorly identified. It has been demonstrated that formation of IgA1 complexes may involve at least two distinct events: (1) the self-aggregation of abnormal glycosylated IgA1 and/or induction of immune response to neo-epitopes on the abnormal IgA1, and (2) IgA1 binding to soluble IgA Fc receptor I (Fc␣RI), resulting in formation and/or amplification of the size of these complexes. The first event is characterized by the fact that hypogalactosylation generates antigenic determinants that can be recognized by naturally occurring IgG and IgA1 antibodies, thereby leading to the formation of circulating immune complexes [4]. However, IgG is not always found to be co-deposited in the mesangium of IgAN patients and IgA anti-IgA antibodies are also not always found in these patients. While the first event is directly linked to an abnormal structure of IgA1 possibly arising during their generation [4], the second is dependent on the interaction with the Fc␣RI (CD89) that is expressed on cells of the myeloid lineage [2]. This receptor represents a heterogeneously glycosylated transmembrane protein that binds both IgA subclasses with low affinity. In patients with IgAN, a reduced expression of Fc␣RI was observed on the surface of their circulating monocytes in spite of normal Fc␣RI transcription levels [5]. The presence of plasma IgA seems essential for the reduction in Fc␣RI expression on monocytes. This was shown by incubating IgAN patients’ cells without or with homologous plasma and by incubating purified polymeric IgA with monocytes from normal individuals [5]. The mechanism proposed to explain this phenomenon involves the shedding of the extracellular domain of the Fc␣RI [2]. Support for this hypothesis was provided by the demonstration of

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soluble Fc␣RI in the serum of IgAN patients, but not in serum from normal individuals. This protein was identified as a glycosylated Fc␣RI of 50–70 kDa with a 24 kDa protein core [6]. Production of this soluble Fc␣RI could be induced by incubating polymeric IgA with transfected cells expressing Fc␣RI. These results indicate that interaction of pathogenic IgA with Fc␣RI promotes cleavage of the extracellular domain of Fc␣RI releasing IgA/Fc␣RI complexes into the circulation. Such cleavage may involve yet uncharacterized proteases, thereby explaining the decreased Fc␣RI membrane expression. The detection of circulating complexes containing IgA and soluble Fc␣RI in IgAN patients serum raises the possibility that these complexes may be involved in the development of this disease. To demonstrate this we bred human-Fc␣RI transgenic (Tg) mice that serve as a novel animal model for spontaneous IgAN [6]. Human Fc␣RI interacts with mouse polymeric IgA, albeit with very low affinity, to form complexes that are deposited in the mesangium of the Fc␣RI Tg mice. Human-Fc␣RI Tg mice develop mesangial IgA deposition, hematuria, mild proteinuria and macrophage infiltration around the renal glomeruli [6]. The disease can be transferred to wild-type recipents by infusion of serum IgA/soluble Fc␣RI complexes from these Tg mice. To examine the contribution of patient IgA, a model of SCIDFc␣RI Tg mice was created. These mice do not develop IgAN spontaneously, but they develop the manifestations of IgAN when they receive IgA from IgAN patients. Interestingly, IgA from healthy subjects did not result in IgAN in the SCID-Fc␣RI Tg mice, thereby inferring that abnormally glycosylated IgA coupled with Fc␣RI participates in the pathogenesis of IgAN. It is noteworthy that other types of soluble Fc␣RI exist but are not involved in the pathogenesis of IgAN [7, 8]. The role of IgA receptors in clearance of IgA1-IC remains poorly studied. It has been proposed that immune complexes may impede the recognition of IgA by the asialoglycoprotein receptor (ASGPR) [4]. The ASGPR serves as an IgA receptor on hepatocytes by its ability to bind terminal galactose or N-acetylgalactosamine on O-linked and some N-linked glycans. However, ASGPR seems to play an important role in the clearance of IgA2 rather than of IgA1 [9]. Furthermore, no dysfunction of ASGPR has been demonstrated in IgAN. The second step is constituted by the implication of mesangial IgA receptors in the trapping of circulating IgA1-IC. Mesangial IgA1 deposits have been shown to be primarily of the IgA1 isotype and to be composed mainly of polymeric IgA [2, 10]. As expected from the above, these deposits contain abnormally glycosylated IgA1 [1]. Although several candidate proteins have been identified as putative mesangial IgA receptors, none of them were found in the mesangium. We have recently shown that the transferrin receptor, TfR or CD71, can serve as an IgA1 receptor [11, 12]. The TfR binds only polymeric IgA1, but

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not monomeric IgA1 [13]. In contrast to Fc␣RI, this IgA receptor is not expressed on mature blood leukocytes and is only weakly expressed on quiescent mesangial cells [12]. Interestingly, in patients with IgAN, TfR expression is strongly induced in the mesangium and correlates with the severity of the disease [12]. Because overexpression of this IgA1 receptor (TfR/CD71) is found in mesangium of patients with IgAN [11, 12], it may participate in the selective deposition of IgA1 complexes. Similar observations have now been made in renal biopsies of patients with Henoch-Schoenlein purpura GN with mesangial IgA deposition [12]. Interestingly, overexpression of TfR was also seen in patients with lupus, but only in those with IgA deposition. Our recent studies [14] indicate that abnormally glycosylated IgA1 and IgA1 complexes may favor the interaction with TfR as observed on cultured normal human mesangial cells. Furthermore, IgA1 polymers can induce TfR expression, cytokine release and cell proliferation, which could in part be responsible for the observed injury and recurrence of deposits after transplantation [14]. Another receptor for IgA and IgM, Fc␣/␮R, has been also described [10]. Transcripts for this receptor may also be expressed by human mesangial cells. This receptor has been found to be upregulated by IL-1, thereby implying another basis for IgA deposition. However, no Fc␣/␮R protein has been found on cultured human mesangial cells [15]. We cannot exclude that Fc␣/␮R can be expressed in some cases of IgAN with IgA and IgM deposits, especially those involving a very aggressive inflammatory process. The third step reveals that IgA receptors participate in the progression of this disease towards end-stage renal disease (ESRD). Indeed, one of the remarkable features of IgAN is the heterogeneity of the disease regarding its evolution towards ESRD. Between 20 to 40% of IgAN patients progress towards ESRD after 20–25 years of disease activity. The mechanisms involved in disease progression remains mostly unknown. It is interesting that the severity of IgAN is often associated with leukocyte infiltration in the kidneys. Indeed, the presence of monocytes, macrophages and T cells correlates with the progression of IgAN [16, 17]. Interestingly, although transmembrane Fc␣RI expression is decreased on blood phagocytes of patients, increased IgA bound to these cells are linked to the appearance of glomerulosclerosis and mesangial proliferation [5]. Defective Fc␣RI endocytosis rates and increased IgA recycling towards the cell surface have been demonstrated on blood phagocytes from patients with IgAN [18]. As a consequence, cells from these patients express high levels of bound IgA. Our recent data indicate that activation of transmembrane Fc␣RI associated with FcR␥ adaptor following interaction with IgA-IC aggravates IgAN in Fc␣RI-transgenic animals through induction of the cytokine/chemokine cascade and leukocyte infiltration [19]. Another factor that may also contribute to evolution towards ESRD is the overexpression of TfR by mesangial cells and its

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capacity to mediate mesangial IgA1 complex deposition, which seems to correlate with disease severity [12]. This interaction could thus lead to an inflammatory response by promoting the release of pro-inflammatory cytokines, such as IL-1, IL-6 and TNF-␣, with consequent proliferation and progression towards fibrosis and renal impairment [2]. This hypothetical cycle of events could thus explain the progression and chronicity of the disease. In conclusion, we propose that pathogenic mechanisms in IgAN are initiated by alterations in IgA1 structure followed by complex formation involving IgA1, soluble Fc␣RI and other components. An in situ secondary event then takes place with the induction of a mesangial IgA receptor, the TfR, on mesangial cells. This represents a crucial step for the formation of IgA deposits and eventually contributes to the generation of the third event concerning progression of IgAN towards ESRD. The elucidation of the mesangial TfR-IgA1 interaction as a mechanism for selective mesangial IgA1 deposition suggests new avenues for drug design and treatment of IgAN.

References 1 2 3 4 5

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Barratt J, Feehally J: IgA nephropathy. J Am Soc Nephrol 2005;16:2088–2097. Monteiro RC, Moura IC, Launay P, et al: Pathogenic significance of IgA receptor interactions in IgA nephropathy. Trends Mol Med 2002;8:464–468. Berger J, Yaneva H, Nabarra B, et al: Recurrence of mesangial deposition of IgA after renal transplantation. Kidney Int 1975;7:232–241. Novak J, Julian BA, Tomana M, et al: Progress in molecular and genetic studies of IgA nephropathy. J Clin Immunol 2001;21:310–327. Grossetete B, Launay P, Lehuen A, et al: Down-regulation of Fc␣ receptors on blood cells of IgA nephropathy patients: evidence for a negative regulatory role of serum IgA. Kidney Int 1998;53: 1321–1335. Launay P, Grossetete B, Arcos-Fajardo M, et al: Fc␣ receptor (CD89) mediates the development of immunoglobulin A (IgA) nephropathy (Berger’s disease). Evidence for pathogenic soluble receptor-IgA complexes in patients and CD89 transgenic mice. J Exp Med 2000;191:1999–2009. Van Der Boog PJ, De Fijter JW, Van Kooten C, et al: Complexes of IgA with Fc␣RI/CD89 are not specific for primary IgA nephropathy. Kidney Int 2003;63:514–521. van der Boog PJ, van Kooten C, de Fijter JW, et al: Role of macromolecular IgA in IgA nephropathy. Kidney Int 2005;67:813–821. Rifai A, Fadden K, Morrison SL, et al: The N-glycans determine the differential blood clearance and hepatic uptake of human immunoglobulin (Ig)A1 and IgA2 isotypes. J Exp Med 2000;191:2171–2182. Gomez-Guerrero C, Suzuki Y, Egido J: The identification of IgA receptors in human mesangial cells: in the search for ‘Eldorado’. Kidney Int 2002;62:715–717. Moura IC, Centelles MN, Arcos-Fajardo M, et al: Identification of the transferrin receptor as a novel immunoglobulin (Ig)A1 receptor and its enhanced expression on mesangial cells in IgA nephropathy. J Exp Med 2001;194:417–425. Haddad E, Moura IC, Arcos-Fajardo M, et al: Enhanced expression of the CD71 mesangial IgA1 receptor in Berger disease and Henoch-Schonlein nephritis: association between CD71 expression and IgA deposits. J Am Soc Nephrol 2003;14:327–337. Moura IC, Arcos-Fajardo M, Sadaka C, et al: Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy. J Am Soc Nephrol 2004;15: 622–634.

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Moura IC, Arcos-Fajardo M, Gdoura A, et al: Engagement of transferrin receptor by polymeric IgA1: evidence for a positive feedback loop involving increased receptor expression and mesangial cell proliferation in IgA nephropathy. J Am Soc Nephrol 2005;16:2667–2676. McDonald KJ, Cameron AJ, Allen JM, et al: Expression of Fc␣/␮ receptor by human mesangial cells: a candidate receptor for immune complex deposition in IgA nephropathy. Biochem Biophys Res Commun 2002;290:438–442. Arima S, Nakayama M, Naito M, et al: Significance of mononuclear phagocytes in IgA nephropathy. Kidney Int 1991;39:684–692. Falk MC, Ng G, Zhang GY, et al: Infiltration of the kidney by ␣␤ and ␥␦ T cells: effect on progression in IgA nephropathy. Kidney Int 1995;47:177–185. Monteiro RC, Grossetete B, Nguyen AT, et al: Dysfunctions of Fc␣ receptors by blood phagocytic cells in IgA nephropathy. Contrib Nephrol 1995;111:116–122. Kanamaru Y, Arcos-Fajardo M, Moura IC, et al: Fc␣ receptor I activation induces leukocyte recruitment and promotes aggravation of glomerulonephritis through the FcR␥ adaptor. Eur J Immunol (in press).

Renato C. Monteiro INSERM U699 and Medical School of Denis Diderot University Bichat Campus 16, rue Henri Huchard, BP416 FR–75870 Paris Cédex 18 (France) Tel. ⫹33 1 44 85 62 61, Fax ⫹33 1 44 85 62 60, E-Mail [email protected]

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Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 70–79

The Mucosa-Bone-Marrow Axis in IgA Nephropathy Yusuke Suzuki, Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan

Abstract Large numbers of studies have demonstrated that abnormal humoral and cellular immunity may contribute to the pathogenesis of IgA nephropathy. On the other hand, clinical evidence indicates that bone marrow cells and mucosal immunity may also play a key role. Based on these findings, impaired immune regulation in the ‘mucosa-bone-marrow axis’ has been postulated in IgA nephropathy patients. However, the underlying mechanisms still remain unclear. This is mainly due to difficulties in the clinical approach to the complicated immune system. Therefore, appropriate animal models are required. We recently established several useful animal models. Using these models, our group is approaching underlying mechanisms in which bone marrow and mucosal cells interrelate and finally induce this disease. Up to now, results from these models and its clinical feedback have suggested that mucosal IgA responses to antigens may be altered by Th2-biased background or dysregulation of innate immunity in this disease. This abnormal mucosal IgA immune system may result in failure of mucosal antigen elimination and thus increases in memory cells in the bone marrow. Copyright © 2007 S. Karger AG, Basel

Introduction

Since the first description of IgAN by Berger in 1965, there have been two major questions regarding its pathogenesis. The first is ‘what is the major pathogenic mechanism of IgAN?’ Recurrent IgA deposition in the allograft and recurrence of IgAN in recipients are seen after kidney transplantation in approximately 50% of recipients [1]. On the other hand, one report demonstrated that accidental transplantation of a kidney from an IgAN patient into a recipient with primary renal

disease other than IgAN resulted in the disappearance of IgA [2, 3]. These clinical observations indicate that IgAN represents a consequence of host susceptibility, including abnormalities of IgA immune system, rather than being a disease with intrinsic renal abnormality. On the other hand, we know that many IgAN patients show episodic macrohematuria, which coincides with mucosal infection especially in the upper respiratory tract or gastrointestinal tract [4, 5]. In addition, we now know that tonsillectomy has a favorable effect on long-term renal survival in IgAN patients, especially from recent Japanese studies [6, 7]. Previous clinical studies demonstrated that mucosal vaccination of IgAN patients resulted in impaired mucosal IgA response, compared with healthy subjects. The important message of these studies is that dysregulation of the mucosal immune response might play a role in the pathogenesis of IgAN. It is known that the properties of the IgA molecule in IgAN patients are different from those in healthy adults. It is generally accepted that mesangial IgA1 is underglycosylated polymeric IgA1 (pIgA1). Moreover, several studies demonstrated the presence of high pIgA1 levels in serum and tonsillar tissues of IgAN patients [8]. However, the origin of mesangial pIgA1 in IgAN remains unclear at present. There is a general agreement that plasma IgA is produced mainly in the bone marrow, while the mucosal contribution to circulating IgA levels is small. Previous clinical studies provided some clues. For example, systemic antigen challenge results in increased titers of circulating pIgA1 antibodies with normal levels in mucosal secretions [9, 10]. Large numbers of pIgA1-positive plasma cells are found in the IgAN bone marrow [11]. Moreover, bone marrow transplantation in patients with leukemia and IgAN resulted in the cure of not only leukemia but also IgAN [12]. Accordingly, these findings suggest that overproduction of pIgA1 seems to be based in systemic immune sites, such as the bone marrow. There is a growing body of evidence to suggest that mucosal type pIgA1 is produced in the bone marrow of IgAN patients. Therefore, the crosstalk between mucosa and bone marrow should be carefully discussed.

Is There Any Abnormality in the Mucosa-Bone-Marrow Axis of IgAN Patients?

About 20 years ago, Van Den Wall Bake, Daha and Van Es proposed the mucosa-bone marrow axis based on their series of elegant studies [10, 11, 13, 14]. They also hypothesized an impaired IgA immune response in the ‘mucosa-bone-marrow axis’ of IgAN patients. At that time, such proposals

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were based on experimental models of IgAN. However, the presence of such an axis has not yet been confirmed in human IgAN. However, clinical and experimental studies published during the last decade revealed continued traffic of antigen-specific lymphocytes and antigenpresenting cells between the mucosa and bone marrow in humans [15]. During differentiation of naïve B cells to IgA memory B cells or IgA plasmablasts, these cells express specific integrins and chemokine receptors. The patterns of integrins and chemokine receptors in IgA plasma cells are slightly different and depend on the site of their induction. For example, IgA plasma cells primed in salivary glands and tonsils can move to bone marrow and salivary glands through specific chemokine recognition and appropriate adhesion/homingreceptor engagement [15]. Similar mechanisms have been reported for memory T cells. Therefore, the mucosa-bone marrow axis is currently considered to exist in the human system. In this axis, mucosal and systemic IgA immune responses are intricately linked and regulated. If we believe in the presence of the mucosa-bone marrow axis in humans, then one should ask ‘is there any abnormality in the mucosa-bone marrow axis of IgAN patients?’ In this regard, previous immunization and vaccination studies in IgAN could provide some clues for this question. Previous studies reported the results of mucosal immunization in IgAN using a neoantigen [16, 17]. These studies demonstrated the presence of impaired mucosal and systemic IgA responses, but normal IgG and IgM responses, in such patients. These results mean that IgAN patients are hyporesponders to mucosal neoantigens. On the other hand, systemic or mucosal immunization with recall antigens resulted in exaggerated systemic IgA responses with increased and prolonged production of specific IgA [9, 10, 18, 19]. These results mean that IgAN patients are hyperresponders to recall antigens. The above-mentioned clinical observations allowed us to formulate the hypothesis shown in figure 1. In IgAN patients, impaired mucosal IgA response may result in impaired elimination of mucosal antigens. Accumulation of these antigens could increase immunologic memory for IgA1 production in the mucosa and finally in the bone marrow or other lymphoid tissues. These memory cells could induce hyperresponsiveness to recall antigens and increase nephritogenic IgA1 in IgAN. To verify this hypothesis, we are investigating three issues: (1) Is the bone marrow a reservoir of nephritogenic memory cells? (2) Is dysregulation of cellular immunity involved in IgAN? (3) Is dysregulation of innate immunity involved in IgAN? In the rest of this paper, we would like to discuss each of these questions with our new data.

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IgAN patients have impaired IgA immune responses in mucosa?

Impaired elimination/exclusion of mucosal antigens

Increased levels of immunologic memory for IgA1 production in bone marrow or other lymphoid tissues

IgA1 hyperresposiveness to recall antigens

Increased nephritogenic IgA1 production?

Fig. 1. Hypothesis about induction of IgA nephropathy.

Is the Bone Marrow a Reservoir of Nephritogenic Memory Cells?

There are significant differences in the structure of IgA between animals and humans. For example, all non-primate species lack a hinge region analogous to human IgA1, and the control of IgA production is different. However, animal models provide important information about the complex immune system. In our research work, we found that it was practically very difficult to approach the human system. Therefore, we tried to find appropriate models for our purpose. We are lucky to have recently established a new model of ‘grouped ddY’ mice [20]. The ddY mouse is a well-known model of spontaneous IgAN [21]. These mice show mesangial proliferative glomerulonephritis with mesangial IgA and IgG co-deposition. However, the ddY mice have several problems such as high variability in onset of spontaneous IgAN. Our group obtained serial biopsies from more than 300 ddY mice, and found that these mice could be divided into three groups. We found that about 35% of ddY mice had glomerulonephritis with mesangial IgA deposition before 20 weeks of age. We defined these mice as earlyonset mice. About 30% mice were defined as late-onset mice, showing glomerulonephritis with mesangial IgA deposition after 40 weeks of age. The remaining 35% mice were defined as quiescent mice, which did not show any glomerulonephritis or IgA deposition, even at 60 weeks of age. The serum levels of IgA were not different in these three groups. In addition, the degree of glomerular IgA deposition did not correlate with the serum level of IgA in the three groups. We also conducted an association study between early-onset and quiescent mice. Surprisingly, the marker D10MIT86 on chromosome 10 in ddY mice was

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located in the region syntenic to human 6q22–23 with IGAN1, which is the responsible candidate for familial IgAN [20, 22]. In addition, D1MIT16 on chromosome 1 was located close to the locus of the selectin gene, which is a known candidate for human IgAN. These findings suggest that IgAN in both human and ddY mouse is regulated, at least in part, by certain genes and/or mechanisms. Thus, the ddY mouse model is a useful tool for studying human IgAN. The onset of IgAN in ddY mice was associated with glomerular co-deposition of IgA and IgG2a. Interestingly, serum levels of the IgA-IgG2a complex correlated well with the severity of renal injury. The onset of IgAN in ddY mice was also associated with strong Th1 polarization. Although IgG2a is known as an IgG subtype regulated by Th1 cytokines, it is still not clear whether IgG2a is a product of systemic Th1 polarity. We then employed this model to test our hypothesis. Transplantation of bone marrow from early-onset into quiescent mice induced glomerular IgA deposition and proteinuria (H. Suzuki et al., Kidney Int 2007, in press). Such transplantation also induced Th1 polarization in the recipient quiescent mice. In contrast, transplantation of bone marrow from quiescent into early-onset mice reduced glomerular IgA deposition and proteinuria. The results of bone marrow transplantation studies suggest that the bone marrow is a reservoir of memory cells that induce glomerular IgA deposition and subsequent GN, and that bone marrow cells are essential for continuous delivery of pathological IgA. Next, we examined whether bone marrow transplantation induces IgAN through additional priming in the mucosa or secondary lymphoid tissues (A part of this study was presented by M. Aizawa et al. at the 2006 annual meeting of the American Society of Nephrology). To approach this matter, we employed the aly/aly mice in the next series of experiments. This mouse lacks systemic secondary lymphoid tissues, including lymph nodes, Peyer’s patches and isolated lymphoid follicles in the lamina propria, because of NIK mutation. Another striking feature of this mouse is complete deficiency of serum and mucosal IgA. We transplanted bone marrow from early-onset ddY mice into aly/aly mice. We found glomerular IgA deposition in the transplanted aly/aly mice but no proteinuria or Th1 polarization at 24 weeks after the transplantation. Based on these data, we concluded that bone marrow cells induce glomerular IgA deposition independent of homing steps to secondary lymphoid tissues or mucosa. However, glomerular injury after deposition seems to require secondary lymphoid tissues. These findings suggest that the bone marrow contains memory cells, presumably long-lived plasma cells or memory B cells, producing nephritogenic IgA.

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Is Dysregulation of Cellular Immunity Involved in IgAN?

Recently, there has been a trend toward an increase in allergic and autoimmune diseases, especially in industrialized countries. Paradoxically, this increase is occurring in association with improved public health measures, sanitization and quality of life. The hygiene hypothesis was originally proposed as a mechanism to explain the marked increase in allergies in Western countries [23]. Specifically, the hypothesis proposes that early and frequent exposure to bacterial and other antigens, especially at the mucosal level, which is common in developing nations, leads to a normal Th1 response. However, better public hygiene and fewer infections observed in industrialized nations may lead to persistence of the Th2 phenotype, thereby increasing the risk of the development of various allergies. It is known that the prevalence of IgAN correlates with socioeconomic status. Recently, Johnson et al. proposed that the high incidence of IgAN and minimal change disease could be explained by the hygiene hypothesis [24, 25]. They hypothesized that in Western countries, less exposure to infections may lead to a Th2-dominant phenotype and increased risk for IgAN and minimal change disease. There have been many studies about Th1 and Th2 balance in IgAN. Many suggest that IgAN is a Th2-biased disease, but occasional studies have found evidence for some Th1 activation in IgAN. These findings indicate that clinical studies are practically difficult to determine the role of Th2 bias in this disease because of the timing of human sample collection. Therefore, we approached this matter using our mouse model. We transplanted bone marrow from early-onset ddY mice into Th1-prone C57Bl6 mice and Th2-prone BALB mice. Interestingly, Th2 prone BALB/C mice showed more severe glomerular lesions and stronger Th1 polarization after bone marrow transplantation. One could speculate that the different severity is due to differences in mouse strains. Therefore, we examined this issue using a different animal model. GATA3 is known as a transcriptional factor that specifically regulates Th2 immune response (a part of this study was presented by T. Yamanaka et al. at the 2005 Annual Meeting of the American Society of Nephrology). We bred GATA3 transgenic (Tg) mice and crossed them with Tg mice with OVAspecific TCR genes. We used these double-Tg mice with GATA3 and OVA TCR genes. Repeated mucosal or parenteral OVA challenges were performed in these Th2-biased double-Tg mice and in control wild-type OVA-TCR Tg mice. Only mucosal antigen challenge in Th2-biased mice resulted in glomerular IgA deposition and mild glomerular lesions. Indeed, only mucosally immunized GATA3/OVA TCR double-Tg mice had high serum levels of OVA-specific IgA.

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Cytokine assays using spleen cells and Payer’s patch cells suggested that glomerular IgA deposition was linked to disruption of systemic Th1 tolerance in the Th2-biased mucosa. These findings suggest that mucosal antigen challenge in Th2-biased hosts may induce dysregulation of systemic tolerance, followed by glomerular IgA deposition and injury. In addition to Th2 polarity in systemic immune response, Th2 cytokines have a direct effect on glycosylation of IgA in human B cells. ␤1,3 galactosyltransferase is known as a key enzyme for galactosylation of the IgA1 hinge region. Our group recently showed that Th2 cytokine IL-4 increased underglycosylated IgA1 in a human IgA-positive B cell line via downregulation of ␤1,3 galactosyltransferase and its molecular chaperone Cosmc (a part of this study was presented by K. Yamada et al. at the 2006 Annual Meeting of the American Society of Nephrology).

Is Dysregulation of Innate Immunity Involved in IgAN?

The innate immune system is an important player in mucosal immunity. To approach this question, we maintained ddY mice under conventional conditions and super-pathogen-free (SPF) conditions (a part of this study was presented by H. Suzuki et al. at the 2005 Annual Meeting of the American Society of Nephrology). Interestingly, ddY mice under conventional conditions had high serum levels of IgA, but not IgM and IgG. Next, we checked activation of the innate immune systems. Since Toll-like receptors 2, 4 and 9 are reported to be involved in IC-mediated glomerulonephritis, we checked the activation of these receptors in our mice. Interestingly, the mRNA expression levels of TLR9 and its signaling association molecule MyD88 correlated with the incidence of glomerulonephritis. No such relationship was found for TLR2 and TLR4. Activation of both TLR9 and MyD88 was also linked to the severity of glomerulonephritis. The ligand for TLR9 is CpG DNA, which is unmethylated DNA and mainly found in viruses and bacteria. Next, we nasally challenged ddY mice with CpG DNA. Nasal immunization exacerbated murine IgAN with elevation of glomerular IgA and serum IgA levels. This immunization with CpG DNA also enhanced systemic activation of TLR9/MyD88 and induced strong Th1 polarization. The results of nasal antigen challenge tests in ddY mice indicated that mucosal antigen challenge exacerbated glomerular IgA deposition and injury through activation of TLR9 and MyD88. Next, we investigated the role of innate immunity, especially TLR9 activation, in human IgAN. In this study, we quantified TLR expression in tonsillar tissues obtained from 16 patients with IgAN. These patients had completed

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three courses of steroid pulse therapy after tonsillectomy. Overexpression of TLR9 was noted in the tonsils of three of the 16 patients. The expression level of TLR9 correlated significantly with those of IFN-␣ and IFN-␥ in the tonsils. Next, we determined the type of cells that expressed TLR9 in the tonsils. Compatible with the results of mRNA expression, we found many TLR9-positive cells in the tonsils of patients of the high-TLR9 group. CD123 is an IL-3 receptor-␣ subunit and is mainly expressed in dendritic cells and monocytes/macrophages. Large numbers of CD123-positive cells were found in the tonsils of the high TLR9 group. On the other hand, BDCA2 is a C-type lectin transmembrane glycoprotein restricted to human plasmacytoid DC. Interestingly, most of the TLR9positive cells in the tonsils also expressed BDCA2. Therefore, TLR9-positive cells in the tonsils are mainly plasmacytoid dendritic cells. Next, we checked the clinical characteristics of patients of the high TLR9 group. We could not find any correlation between TLR9 expression and time from onset to treatment, severity of hematuria and proteinuria at tonsillectomy or severity of glomerular lesions (a part of this study was presented by T. Kano et al. at the 2006 Annual Meeting of the Japanese Society of Nephrology). However, this group showed rapid and good therapeutic outcome as assessed by the level of proteinuria and hematuria. In addition, these patients had significantly low serum IgA levels. Our clinical studies using tonsils of IgAN patients indicated that TLR9 activation in mucosal pDC may be involved in the pathogenesis of IgAN. Although we have to analyze the underlying mechanisms, this finding suggests that innate immunity and mucosal IgA immune responses are closely linked and play key roles in IgAN. Involvement of TLR activation suggests the possibility of infection and IC formation with a specific antigen. By using LC-ESI mass spectrometry, we also checked whether the specific antigen is involved in IgA-binding protein from serum of IgAN patients (a part of this study was presented by K. Obayashi et al. at the 2006 Annual Meeting of the Japanese Society of Nephrology). However, in our system, we did not find any obvious exogenous antigen in the IgA binding protein. Interestingly, IgA1 binding IgG, C3 and C4bp decreased after the tonsillectomy with steroid pulse therapy. Although future studies with larger numbers of patients is required, this data suggests that IgA1 binding IgG and its IC may play an important role in the pathogenesis of IgAN.

Conclusion

In IgAN patients, mucosal IgA responses to antigens may be altered by Th2-biased background or dysregulation of innate immunity such in dendritic cells or consequence of their abnormal interaction. This abnormal mucosal IgA

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Priming site

Memory site

Mucosa pIgA B cell Long-lived plasma cell? Memory B cell?

Th2-bias T cell

TLR9 DC Bone marrow

Underglycosylated IgA

Effector site LN APC? Kidney

Fig. 2. Mucosa-bone marrow axis in IgA nephropathy. LN ⫽ Lymph node; APC ⫽ antigen presenting cell; DC ⫽ dendritic cell; pIgA ⫽ polymeric IgA.

immune system could result in failure of mucosal antigen elimination and thus increases in memory cells, such as long-lived IgA plasma cells, in the bone marrow. IgAN patients may produce mesangial pIgA, presumably underglycosylated IgA, and independent additional priming in secondary lymphoid tissues (fig. 2). Further studies are required to analyze the mucosa-bone marrow axis and elucidate the pathogenic mechanisms of IgAN.

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Floege J, Burg M, Kliem V: Recurrent IgA nephropathy after kidney transplantation: not a benign condition. Nephrol Dial Transplant 1998;13:1933–1935. Cuevas X, Lloveras J, Mir M, et al: Disappearance of mesangial IgA deposits from the kidneys of two donors after transplantation. Transplant Proc 1987;19:2208–2209. Sanfilippo F, Croker BP, Bollinger RR: Fate of four cadaveric donor renal allografts with mesangial IgA deposits. Transplantation 1982;33:370–376. Nicholls KM, Fairley KF, Dowling JP, et al: The clinical course of mesangial IgA associated nephropathy in adults. Q J Med 1984;53:227–250. Feehally J, Beattie TJ, Brenchley PE, et al: Sequential study of the IgA system in relapsing IgA nephropathy. Kidney Int 1986;30:924–931. Hotta O, Miyazaki M, Furuta T, et al: Tonsillectomy and steroid pulse therapy significantly impact on clinical remission in patients with IgA nephropathy. Am J Kidney Dis 2001;38:736–743.

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Xie Y, Chen X, Nishi S, et al: Relationship between tonsils and IgA nephropathy as well as indications of tonsillectomy. Kidney Int 2004;65:1135–1144. Barratt J, Feehally J, Smith AC: Pathogenesis of IgA nephropathy. Semin Nephrol 2004;24: 197–217. Layward L, Finnemore AM, Allen AC, et al: Systemic and mucosal IgA responses to systemic antigen challenge in IgA nephropathy. Clin Immunol Immunopathol 1993;69:306–313. van den Wall Bake AW, Beyer WE, Evers-Schouten JH, et al: Humoral immune response to influenza vaccination in patients with primary immunoglobulin A nephropathy. An analysis of isotype distribution and size of the influenza-specific antibodies. J Clin Invest 1989;84:1070–1075. van den Wall Bake AW, Daha MR, Evers-Schouten J, et al: Serum IgA and the production of IgA by peripheral blood and bone marrow lymphocytes in patients with primary IgA nephropathy: evidence for the bone marrow as the source of mesangial IgA. Am J Kidney Dis 1988;12:410–414. Iwata Y, Wada T, Uchiyama A, et al: Remission of IgA nephropathy after allogeneic peripheral blood stem cell transplantation followed by immunosuppression for acute lymphocytic leukemia. Intern Med 2006;45:1291–1295. van den Wall Bake AW, Daha MR, van Es LA: Immunopathogenetic aspects of IgA nephropathy. Nephrologie 1989;10:141–145. van Es LA, van den Wall Bake AW, Stad RK, et al: Enigmas in the pathogenesis of IgA nephropathy. Contrib Nephrol 1995;111:169–175; discussion 175–176. Kunkel EJ, Butcher EC: Plasma-cell homing. Nat Rev Immunol 2003;3:822–829. de Fijter JW, Eijgenraam JW, Braam CA, et al: Deficient IgA1 immune response to nasal cholera toxin subunit B in primary IgA nephropathy. Kidney Int 1996;50:952–961. Roodnat JI, de Fijter JW, van Kooten C, et al: Decreased IgA1 response after primary oral immunization with live typhoid vaccine in primary IgA nephropathy. Nephrol Dial Transplant 1999;14: 353–359. Layward L, Allen AC, Hattersley JM, et al: Elevation of IgA in IgA nephropathy is localized in the serum and not saliva and is restricted to the IgA1 subclass. Nephrol Dial Transplant 1993;8:25–28. Leinikki PO, Mustonen J, Pasternack A: Immune response to oral polio vaccine in patients with IgA glomerulonephritis. Clin Exp Immunol 1987;68:33–38. Suzuki H, Suzuki Y, Yamanaka T, et al: Genome-wide scan in a novel IgA nephropathy model identifies a susceptibility locus on murine chromosome 10, in a region syntenic to human IGAN1 on chromosome 6q22–23. J Am Soc Nephrol 2005;16:1289–1299. Muso E, Yoshida H, Takeuchi E, et al: Enhanced production of glomerular extracellular matrix in a new mouse strain of high serum IgA ddY mice. Kidney Int 1996;50:1946–1957. Gharavi AG, Yan Y, Scolari F, et al: IgA nephropathy, the most common cause of glomerulonephritis, is linked to 6q22–23. Nat Genet 2000;26:354–357. Wills-Karp M, Santeliz J, Karp CL: The germless theory of allergic disease: revisiting the hygiene hypothesis. Nat Rev Immunol 2001;1:69–75. Johnson RJ, Hurtado A, Merszei J, et al: Hypothesis: dysregulation of immunologic balance resulting from hygiene and socioeconomic factors may influence the epidemiology and cause of glomerulonephritis worldwide. Am J Kidney Dis 2003;42:575–581. Hurtado A, Johnson RJ: Hygiene hypothesis and prevalence of glomerulonephritis. Kidney Int 2005;97:S62–S67.

Yusuke Suzuki, MD, PhD Division of Nephrology, Department of Internal Medicine Juntendo University School of Medicine, Hongo 2-1-1, Bunkyo-ku 113–8421, Tokyo (Japan) Tel./Fax ⫹81 3 5802 1065, E-Mail [email protected]

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Searching for IgA Nephropathy Candidate Genes: Genetic Studies Combined with High Throughput Innovative Investigations F.P. Schenaa, G. Cerulloa, D.D. Torresa, G. Zazaa, S. Coxa, L. Biscegliab, F. Scolaric, G. Frascàd, G.M. Ghiggerie, A. Amorosof, on behalf of the European IgA Nephropathy Consortium a

Renal, Dialysis and Transplant Unit, Department of Emergency and Organ Transplant, University of Bari, Bari, bMedical Genetic Service, IRCCS – Casa Sollievo della Sofferenza, San Giovanni Rotondo, cRenal Unit, Spedali Civili, Brescia, d Renal Unit, Ospedale Riuniti, Ancona, eLaboratory of Pathophysiology of Uremia, G. Gaslini Children’s Hospital, Genoa, and fGenetic Unit, University of Turin, Turin, Italy

Abstract Idiopathic IgA Nephropathy (IgAN) is the most common biopsy-proven glomerulonephritis worldwide. All races with the exception of Blacks and Indians are involved. Families with two or more relatives affected by IgAN may be observed in 15–20% of pedigrees of IgAN patients. Genome wide linkage study has been considered the most promising approach to identify IgAN susceptibility genes. Therefore, some European investigators constituted the European IgAN Consortium which was initially funded by the European Union. Data from linkage analysis studies, family association studies and case-control association studies are reported. To date, the Consortium has identified two loci (located on chromosomes 4q26–31 and 17q12–22), in addition to the previous study which described the first IgAN locus on chromosome 6q22–23. The functional mapping of genes involved in the disease proceeds from the identification of susceptibility loci identified by linkage analysis (step 1) to the isolation of candidate genes within gene disease-susceptibility loci, after obtaining information by microarray analysis carried out on peripheral leukocytes and renal tissue samples (step 2). Then, the process will proceed from the design of RNA interferenceagents against selected genes (step 3) to the application of systematically tested effect of RNA agents on functional cellular assay (step 4). The above combined high-throughput technologies will give information on the pathogenic mechanisms of IgAN. In addition, these data may indicate potential targets for screening, prevention and early diagnosis of the disease and more appropriate and effective treatment. Copyright © 2007 S. Karger AG, Basel

Idiopathic IgA nephropathy (IgAN) is the most common biopsy-proven glomerulonephritis, which is defined by the predominant deposition of the sub-class IgA1 in the mesangial area of the glomeruli occurring in individuals with recurrent episodes of macroscopic hematuria in concomitance of upper respiratory tract infections or other mucosal infections. However, the disease may be diagnosed in the presence of persistent microscopic hematuria with or without proteinuria. Clinical and laboratory findings should exclude other renal diseases characterized by glomerular IgA deposits such as Schönlein-Henoch purpura, lupus nephritis and chronic hepatitis. Renal biopsy reports from different parts of the world and from renal registries show that IgAN is the most common primary glomerulonephritis among all races from Europe, Asia and Australia, with the exception of the Blacks and Indians [1–9]. A recent paper from North America demonstrated that IgAN is a common primary glomerulonephritis in USA with particular reference to the midwestern and southern states [10]. Therefore, IgAN may be considered the most common cause of end-stage kidney disease in young adult American Caucasians. Families with two or more relatives affected by IgAN, first described in 1973 by de Werra et al. [11], were then reported by other investigators in later years [12–15]. Thus, today it is possible to distinguish familial IgAN from the sporadic form which is more frequent. Nevertheless, familial IgAN may be diagnosed when physicians accurately analyze the history of all relatives of at least 3 generations who receive the routine urinalysis to identify persistent urinary abnormalities [15]. Previous reports demonstrated that outcomes such as rapid progression of renal damage and end-stage kidney disease are more frequent in patients with familial IgAN [16]. A more recent paper, which analyzed a higher number of familial and sporadic forms of IgAN, demonstrated that there is no difference in outcome severity of the disease [17]. The contradiction between the papers could be attributed to the low number of families analyzed in the first paper, even though it documented a higher relative risk of disease in first degree relatives.

European IgAN Consortium Biobank

Genome-wide linkage study involving IgAN families with large pedigrees was considered the most promising approach to identify IgAN susceptibility genes. The first genome-wide scan study was performed in Japanese IgAN families and demonstrated the existence of some chromosomal locus candidates containing genes responsible for the disease. However, those data remained only in an abstract presented at the 1996 ASN Congress [18]. Then, a well organized

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genome-wide scan study, performed in 30 large extended IgAN multiplex families (24 from Italy and 6 from the USA), identified the locus called IGAN1 located on chromosome 6q22–23 in linkage with IgAN [19]. It yielded a significant peak LOD score of 5.6, with 60% of families linked, assuming an autosomal dominant mode of inheritance with incomplete penetrance. This mode of inheritance of familial IgAN is more consistent with the involvement of a single gene with a large effect located in IGAN1. Nevertheless, multifactorial determination, with the interplay of many genes, each conferring a small effect, cannot be excluded. The knowledge gained from the studies of Mendelian diseases has shown that genetic dissection of a complex trait is more powerful when combined linkage-based, association-based and sequence-based approaches are performed. Moreover, considering that no single study design consistently produces more significant results, multivariate analysis carried out by Artmüller et al. [20] showed that the only factors independently associated with increased study success are (a) an increase in the number of individuals studied and (b) study of a sample drawn from only one ethnic group. Gene discovery in complex human disease is complicated by substantial etiological heterogeneity; in addition, the possibility of genes of small effect and the concomitant requirement for large samples make a DNA Bank absolutely necessary. In consideration of the above statements we believed it opportune to organize a IgAN-oriented Biobank [21]. Therefore, some European investigators, involved in the study of IgAN, constituted the European IgAN Consortium which was initially funded by the European Union. The collaborative study group includes expert nephrologists from Italy, Germany and Greece, and geneticists from Italy and Germany. Additional funds were obtained from other institutions. The organization of a multi-center biobank for the collection of biological samples and clinical data from IgAN patients and relatives following a common protocol was considered the start-up for the identification of the disease susceptibility genes. DNA samples of IgAN patients and relatives belonging to 74 multiple extended pedigrees were collected. Moreover, 166 trios (affected sons or daughters and their healthy parents), 1,085 patients with biopsy-proven IgAN and 1,125 healthy subjects were included in the Biobank. An electronic database was created to include data on the enrolled individuals, laboratory findings and other information from the collected biological fluids (blood, urine and saliva). A website (www.igan.net) was constructed to allow scientific information to be shared between partners and to divulge obtained data [22]. The European IgAN Consortium competency increased in time involving genetic epidemiologists, statisticians and bioinformaticians. We hope that all involved specialists, working together, can avoid problems in study design, data management, analysis and interpretation that make gene discovery and replication of findings so difficult.

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Linkage Analysis Studies

Genome-wide linkage strategy identifies regions of the human genome that are likely to contain gene(s) conferring susceptibility to the disease. It is based on extended pedigrees and is effective in localizing gene(s) that are highly penetrant and affect mainly Mendelian diseases. This method alone is often inefficient in discovering genes responsible for complex diseases, such as IgAN, diabetes and hypertension. In combination with linkage analysis, we are also applying familybased candidate gene association studies. The European IgAN Consortium has performed the first genome-wide scan involving 22 Italian multiplex IgAN families [23]. A total of 186 individuals (59 affected and 127 unaffected) were genotyped and included in a two-stage linkage analysis. The regions 4q26–31 and 17q12–22 exhibited the strongest evidence of linkage by non-parametric analysis (best p values of 0.0025 and 0.0045, respectively). These localizations were also supported by multipoint parametric analysis where a peak LOD score of 1.83 (␣ ⫽ 0.50) and of 2.56 (␣ ⫽ 0.65), respectively, were obtained using the affectedonly dominant model, and by allowing for the presence of genetic heterogeneity. These regions are becoming the second (IGAN2) and third (IGAN3) genetic locus candidates to contain causative and/or susceptibility genes for familial IgAN. Other regions did not reach the threshold of a suggestive or significant LOD score; however, the enrolment of additional IgAN families means that these chromosomal regions may be explored in the near future. Our results provide further evidence for genetic heterogeneity among IgAN families. Evidence of linkage to multiple chromosomal regions is consistent with both an oligo/polygenic and a multiple susceptibility gene model for familial IgAN with small/moderate effects in determining the pathological phenotype. The analysis of the known genes located in these two novel loci (positional information procedure), carried out consulting the National Center for Biotechnology Information, identified some potential candidate genes such as the transient receptor potential channel 3 (TRPC3) gene, the interleukin-2 (IL-2) gene, and the IL-21 gene located in 4q26–31, which could be largely involved in the unbalanced Th1/Th2 immune response reported in IgAN patients. In addition, we will also consider the histone deacetylase 5 (HD5) gene and the granulin (GRN) gene located on the 17q12–22 region, which could be involved in the immune-response deregulation. Family-based association studies, evaluating the distribution of these candidate gene polymorphisms, are in progress.

Microarray Studies

Different high-throughput gene analysis techniques can be used for obtaining transcriptome profiling of renal diseases. Microarray analysis represents the

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best and the latest approach to gain information on global gene expression. IgAN is a complex disease which results from the joint influence of genetic and environmental factors. Important categories of environmental exposures such as mucosal infections have been identified, but the genetic architecture of the disease remains obscure. Genome-wide linkage analyses have identified at least three locus candidates containing IgAN susceptibility genes, although no specific gene(s) have been identified. Microarrays are now in use to fingerprint the pathological process. A recently published study postulated that changes in gene expression patterns in circulating leukocytes of IgAN patients may correlate with renal disease activity [24]. The investigators identified 14 upregulated genes. The BTG2, NCUBE1, FLJ2948, SRPK1, LYZ, GIG2 and IL-8 genes correlated mathematically with serum creatinine levels and the PMAIP1, SRPK1, SSI-3, LYZ and PTGS2 genes correlated with higher values of creatinine clearance, thus implying that the latter group of genes may provide a protective effect, while the overexpression of other genes such as B3GNT5, AXUD1 and GIG-2 indicates a worse prognosis. This gene signature reflected kidney function and did not correlate with hematuria or proteinuria. The authors concluded that studies carried out on large populations of IgAN patients will be necessary to confirm that the leukocyte gene expression profile can be used as a marker for diagnosis and for predicting outcome. The European IgAN Consortium has recently organized a protocol for studying gene expression in peripheral blood mononuclear cells (PBMC) and their subclasses from IgAN patients with different clinical and histological patterns. Some genes overexpressed in PBMC are located in the chromosomal regions linked with IgAN. Extensive studies are in progress in a large population of IgAN included in the database of the IgAN Consortium. Expression profiling using serial analysis of gene expression (SAGE) and microarray techniques allows global description of expressed genes present in renal tissue. This is a high throughput genomics technology which enables the simultaneous determination of a large number of genes from tissue samples. Waga et al. identified 13 upregulated genes in IgAN renal biopsy samples. The cluster analysis identified 3 clusters with 7, 12 and 1 involved gene, respectively [25]. The expression levels of these genes were then examined on expanded RNA samples from other renal biopsies, leukocyte samples and cultured primary cells. Data demonstrated the involvement of the genes GABP and STAT3 in cluster I, and gp330 (megalin), MBP45K, MEF2, Oct1 and GABX in cluster II. We recently used laser-capture microdissection applied to renal biopsy samples in combination with extraction of intact RNA and differential gene expression analysis. First, we defined the clinical groups based on differences in the manifestation of characteristic phenotypes; then, we proceeded

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with gene expression analysis by gene chips. The analysis of the data is in progress.

Association Studies

The IgAN Consortium takes care of the collection of biological samples from large homogeneous cohorts of IgAN patients, their parents and their first degree relatives, and family-based association studies are preferred to analyze the role of some candidate genes. A family-based association study, including 53 patients, 45 complete trios, 4 incomplete trios and 36 discordant siblings, evaluated the impact of some Th1/Th2/Th3/TR-type lymphocyte and monocyte/macrophage cytokines on IgAN susceptibility [26]. Cytokine gene polymorphisms with a potential regulatory role on their production were investigated using the family-based association test (FBAT): IFN␥ intron-1 CA-repeat at position 1349–1373; IL-13 –1055C/T; TGF␤ 915G/C; IL-10 5⬘-proximal and distal microsatellites; TNF␣ –308G/A, –238G/A. The FBAT multi-allelic analysis showed an association between IFN␥ polymorphism and susceptibility to IgAN (p ⫽ 0.03). The bi-allelic analysis showed that the 13-CA repeat allele was preferentially transmitted to the affected individuals (p ⫽ 0.006; Bonferroni p value ⫽ 0.04). The direct sequencing of IFN␥ amplicons showed a strict association between the 13-CA repeat allele and the A variant of the ⫹874T/A single nucleotide polymorphism (SNP rs2430561) directly adjacent to the 5⬘ end of the microsatellite. The in vitro production of IFN␥ evaluated in PBMC from 10 genotyped patients demonstrated a correlation between the ⫹874A allele and a lower production of IFN␥ (p ⫽ 0.028). This SNP affects IFN␥ production lying within a binding site for the transcription factor NF-␬B. The occurrence of the ⫹874A variant is responsible for the low production of IFN␥ and predisposes to a preferential Th2-mediated immune response. The predominance of this variant in individuals with IgAN may be responsible for the onset of the disease. This unbalanced Th2 cytokine production in response to upper respiratory tract infections, which may be a significant pathogenic factor in human IgAN, has recently been confirmed by another paper on Th2 predominance produced by our group. This case-control association study shows a significantly higher frequency of the IL-10–1082 G/G genotype in IgAN patients [27]. The high producer IL-10 genotype (–1082 GG) was significantly more prevalent in IgAN patients than controls (OR 2.41; CI 1.45–4.00; p ⫽ 0.0008). Since this genotype is characterized by high production of IL-10, which is a Th2 cytokine, our data support once again the hypothesis that individuals

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genetically determined to an overproduction of Th2 cytokines may be predisposed to IgAN. The prevalence of Th2 cytokines may also explain the abnormality in IgA1 glycosylation occurring in IgAN patients and the concomitant formation of circulating IgA1-IgG immune complexes. Hyperfunction of Th2 cells and cytokine polarity are linked to a more nephritogenic pattern of IgA1 glycosylation in the animal model, and the decreased glycosylation of IgA1 elicited by Th2 cytokines is blunted in vitro by the addition of IFN␥ [28]. The core 1 ␤1,3-galactosyltransferase (C1GALT1) is suspected to be involved in the abnormal glycosylation process of IgA1 in IgAN. The C1GALT1 gene complete sequence analysis was performed in 284 IgAN patients and 234 healthy controls. We found a statistically significant association of the genotype 1365G/G with susceptibility to IgAN (␹2 ⫽ 17.58, p ⬍ 0.0001, odds ratio 2.57 [95% CI: 1.64–4.04]). No association was found with the progression of the disease. Our case-control association study demonstrates that the low expression of C1GALT1 seems to confer susceptibility to IgAN [29].

Conclusions

The application of genome-wide expression analysis requires some infrastructure such as: (1) the collaboration between scientists and clinicians with different skills to develop an effective methodological strategy; (2) the accumulation of sufficient technical expertise to generate high-quality, large-scale, biochemical, genetic and physiological data; (3) the development of effective mechanisms and tools to properly store, disseminate and analyze the data that will be generated from large-scale scientific projects. These appropriate infrastructures have been set up over the last five years by the European IgAN Consortium and the actual high-throughput innovative program is articled in the following projects: (1) management of the European IgAN Biobank collecting large samples of biological specimens from well-characterized IgAN patients, their relatives, and healthy subjects. This material is an invaluable source for genetic and genomic studies in IgAN; (2) the fine mapping of the chromosomal regions in linkage with familial IgAN and further linkage analyses of new multiplex IgAN families to receive additional information; (3) the global gene expression analysis performed in PBMC and renal tissue to define a set of genes, up- or downregulated in IgAN patients, of which some could be located in the linked chromosomal regions and could be responsible for the onset and/or progression of the disease; (4) the association studies organized and partially followed by the applied functional genomics of individualized

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Step 1

Step 2

Step 3

Step 4

Identification of disease susceptibility loci

Selection of candidate genes within the individualized loci

Design of RNAi agents against the selected genes

Functional genomic analysis

si RNA mRNA Chromosome 4, 6, 17

Linkage analysis

Microarray techniques on PBMCs and renal biopsy specimens

Screening of libraries of small interfering RNAs (si RNA)

Functional cell-based assay

Fig. 1. The high-throughput innovative program of the European IgAN Consortium.

genes using all the available and most innovative techniques will give a fingerprint of genes involved in the disease. Therefore, the functional mapping of genes involved in IgAN proceeds from the identification of susceptibility loci by linkage analysis (step 1) to the selection of candidate genes within the disease-susceptibility loci after obtaining accurate information by microarray analysis carried out on PBMC and renal tissue samples (step 2). Then, the process will proceed to the design of RNA interference-agents against selected genes (step 3) and finally to the application of systematically tested effect of RNA agents on functional cellular assay (step 4) (fig. 1). The above combined high-throughput technologies aim to produce knowledge on the mechanisms linking causal determinants of disease and disease progression. In addition, they may indicate potential targets for screening, prevention and early diagnosis of the disease and more appropriate and effective treatment. At the end of this article it is a great pleasure to invite scientists to the international scientific harmonization between IgAN Biobanks, because the pooling of data offers several benefits, of which one is the generation of a minimum of 5,000 cases, and ideally 10,000. This number is required to provide 80% power to detect sized interaction effect [30]. In addition, it will be possible

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to carry out powerful analyses based on homogeneous sub-groups within the disease, e.g. based on age, gender or ethnic origin. Large genetic cohort studies have an important role in furthering our understanding of the complex disease which is IgAN. References 1

2 3 4

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12 13 14 15 16 17 18 19 20 21

Schena FP: Survey of the Italian Registry of Renal Biopsies. Frequency of the renal diseases for 7 consecutive years. The Italian Group of Renal Immunopathology. Nephrol Dial Transplant 1997;12:418–426. Rivera F, Lopez-Gomez JM, Perez-Garcia R, Spanish Register of Glomerulonephritis: Frequency of renal pathology in Spain 1994–1999. Nephrol Dial Transplant 2002;17:1594–1602. Simon P, Ramee MP, Boulahrouz R, et al: Epidemiologic data of primary glomerular diseases in western France. Kidney Int 2004;66:905–908. Research Group on Progressive Chronic Renal Disease: Nationwide and Long-Term Survey of Primary Glomerulonephritis in Japan as Observed in 1,850 Biopsied Cases. Nephron 1999;82: 205–213. Sinniah R, Javier AR, Ku G: The pathology of mesangial IgA nephritis with clinical correlation. Histopathology 1981;5:469–490. Li LS, Liu ZH: Epidemiologic data of renal diseases from a single unit in China: analysis based on 13,519 renal biopsies. Kidney Int 2004;66:920–923. Briganti EM, Dowling J, Finlay M, et al: The incidence of biopsy-proven glomerulonephritis in Australia. Nephrol Dial Transplant 2001;16:1364–1367. Jennette JC, Wall SD, Wilkman AS: Low incidence of IgA nephropathy in blacks. Kidney Int 1985;28:944–950. Seedat YK, Nathoo BC, Parag KB, et al: IgA nephropathy in Blacks and Indians of Natal. Nephron 1988;50:137–141. Nair R, Walker PD: Is IgA nephropathy the commonest primary glomerulopathy among young adults in the USA? Kidney Int 2006;69:1455–1458. de Werra P, Morel-Maroger L, Leroux-Robert C, et al: Glomerulonephritis with diffuse IgA deposits in the mesangium. Study of 96 adult cases. Schweiz Med Wochenschr 1973;103: 761–768. Julian BA, Quiggins PA, Thompson JS, et al: Familial IgA nephropathy. Evidence of an inherited mechanism of disease. N Engl J Med 1985;312:202–208. Levy M, Lesavre P: Genetic factors in IgA nephropathy (Berger’s disease). Adv Nephrol Necker Hosp 1992;21:23–51. Scolari F, Amoroso A, Savoldi S, et al: Familial occurrence of primary glomerulonephritis: evidence for a role of genetic factors. Nephrol Dial Transplant 1992;7:587–596. Schena FP: Immunogenetic aspects of primary IgA nephropathy. Kidney Int 1995;48:1998–2013. Schena FP, Cerullo G, Rossini M, et al: Increased risk of end-stage renal disease in familial IgA nephropathy. J Am Soc Nephrol 2002;13:453–460. Izzi C, Ravani P, Torres D, et al: IgA nephropathy: the presence of familial disease does not confer an increased risk for progression. Am J Kidney Dis 2006;47:761–769. Fukushima T, Nomura S, Kawai S, et al: Whole genome scanning for IgA nephropathy (IgAN) (abstract). J Am Soc Nephrol 1996;7:1333. Gharavi AG, Yan Y, Scolari F, et al: IgA nephropathy, the most common cause of glomerulonephritis, is linked to 6q22–23. Nature Genet 2000;26:354–357. Altmüller J, Palmer LJ, Fischer G, et al: Genomewide scans of complex human diseases: true linkage is hard to find. Am J Hum Genet 2001;69:936–950. Schena FP, Cerullo G, Torres DD, et al, on behalf of the European IgA nephropathy Consortium: The IgA nephropathy Biobank. An important starting point for the genetic dissection of a complex trait. BMC Nephrology 2005;6:14.

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European IgAN Consortium website (http://www.igan.net). Bisceglia L, Cerullo G, Forabosco P, et al, on behalf of the European IgA Nephropathy Consortium: Genetic heterogeneity in Italian families with IgA nephropathy: suggestive linkage for two novel IgA nephropathy loci. Am J Hum Genet 2006;79:1130–1134. Preston GA, Waga I, Alcorta DA, et al: Gene expression profiles of circulating leukocytes correlate with renal disease activity in IgA nephropathy. Kidney Int 2004;65:420–430. Preston GA, Waga I, Alcorta DA: Gene expression profiles of circulating leukocytes correlate with renal disease activity in IgA nephropathy. Kidney Int 2004;65:420–430. Schena FP, Cerullo G, Torres DD, et al, on behalf of the European IgA Nephropathy Consortium: Role of interferon-gamma gene polymorphisms in susceptibility to IgA nephropathy: a familybased association study. Eur J Hum Genet 2006;14:488–496. Capasso M, Boschetto L, Di Noce F, et al, on behalf of the European IgAN Consortium: Influence of the Inteleukin-10 and Tumor necrosis factor-␣ polymorphisms on primary IgA nephropathy susceptibility (submitted). Ebihara I, Hirayama K, Yamamoto S, et al: Th2 predominance at the single-cell level in patients with IgA nephropathy. Nephrol Dial Transplant 2001;16:1783–1789. Pirulli D, Ulivi S, Zadro C, et al, on behalf of the European IgA Nephropathy Consortium: A polymorphism in the gene coding for the Core 1 beta 1,3 galactosyltransferase T1 contributes to the genetic susceptibility to IgA Nephropathy (submitted). Davey Smith G, Ebrahim S, Lewis S, et al: Genetic epidemiology and public health: hope, hype, and future prospects. Lancet 2005;366:1484–1498.

F. Paolo Schena Renal Dialysis and Transplantation Unit, Department of Emergency and Organ Transplants University of Bari, Piazza G. Cesare 11 IT–70124 Bari (Italy) Tel. ⫹39 080 547 8869, Fax ⫹39 080 557 5710, E-Mail [email protected]

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Clinical Advances (Update) Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 90–93

Clinicopathological Influence of Obesity in IgA Nephropathy: Comparative Study of 74 Patients Mari Tanakaa, Tomomi Tsujiia, Toshiyuki Komiyaa, Yukako Iwasakia, Takeshi Sugishitaa, Satomi Yonemotoa, Tatsuo Tsukamotoa, Satoshi Fukuib, Akimasa Takasub, Eri Musoa Departments of aNephrology and bPathology, Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka, Japan

Abstract The pathological role of obesity has rarely been studied in primary glomerular diseases. The purpose of this study is to examine the clinicopathological influence of obesity in IgA nephropathy (IgAN). 74 patients with IgA nephropathy in our institution from October 2000 to January 2004 were retrospectively divided into two groups according to body mass index (BMI): the non-obese group (group N) with BMI ⬍ 25 kg/m2, and the obese group (group O) with BMI ⱖ 25 kg/m2. There were 50 patients in group N and 24 patients in group O. Clinical analysis showed no significant difference between these two groups in blood pressure, serum cholesterol, creatinine clearances or grade of hematuria. However, urinary protein excretion and serum creatinine were significantly greater in group O than in group N. Although semiquantitative analysis of light-microscopical findings showed no significant differences in the severity of mesangial proliferation, matrix expansion, glomerulosclerosis or crescent formation, image analysis showed that total glomerular area and tuft area were significantly larger in group O. In addition, ultrastructural study revealed significantly higher glomerular basement membrane thickness in group O. 62 patients (46 patients, group N; 16 patients, group O) were followed in our institution for one year. Urinary protein was significantly decreased only in patients who received steroid in both groups. Although administration of ACE inhibitor or ARB tended to decrease urinary protein in group O, the change was not statistically significant. Our findings indicate that obesity may accelerate the increase of proteinuria in IgAN through ultrastructural modification of the glomerular basement membrane. Copyright © 2007 S. Karger AG, Basel

Introduction

The pathological role of obesity in the progression of glomerular lesions has been suggested in obesity-related nephropathy, but has rarely been studied in primary glomerular diseases. The purpose of this study is to examine the clinicopathological influence of obesity in IgA nephropathy (IgAN). Patients and Methods 74 patients with renal biopsy-proven IgAN in our institution from October 2000 to January 2004 were retrospectively divided into two groups according to body mass index (BMI): the non-obese group (group N) with BMI ⬍ 25 kg/m2 and the obese group (group O) with BMI ⬍ 25 kg/m2. Patients with diabetes mellitus or autoimmune diseases were excluded. For all patients, we collected the following data at the period of renal biopsy: (1) physiological findings: gender, age, height, body weight, the presence of hypertension; (2) laboratory findings: 24-hour protein excretion, creatinine clearance adjusted for body surface area, hematuria (red blood cell counts per high-power field in the urine sediment), serum creatinine, blood urea nitrogen, serum albumin, serum immunoglobulin A (IgA), total cholesterol; (3) pathological findings: light-microscopical examination, electron-microscopical examination of glomerular basement membrane (GBM) thickness, immunohistochemical examination of ␣-smooth muscle actin (␣-SMA). Hemoglobin A1c (HbA1c), plasma renin activity (PRA) and plasma aldosterone were collected from 45 patients (30 patients, group N; 15 patients, group O). 62 patients (46 patients in group N and 16 patients in group O) were followed-up in our institute for one year. The change in proteinuria was estimated. These data were compared between the two groups using unpaired Student’s t test. Statistical significance was considered at p ⬍ 0.05.

Results

There were 50 patients in group N (17 males and 33 females) and 24 patients in group O (18 males and 6 females). Clinical analysis showed no significant differences between the two groups regarding blood pressure, albumin, IgA, creatinine clearance, HbA1c, plasma renin activity and grade of hematuria. However, 24-hour urinary protein excretion and serum creatinine were significantly greater in group O than in group N (0.43 ⫾ 0.50 vs. 0.74 ⫾ 0.83 g, p ⬍ 0.05; 0.73 ⫾ 0.23 vs. 0.97 ⫾ 0.44 mg/dl, p ⬍ 0.001). Although there were no significant differences in light-microscopical findings such as severity of mesangial proliferation, matrix expansion, glomerulosclerosis or crescent formation, computer-aided image analysis showed that total glomerular area and tuft area were significantly larger in group O (21,209 ⫾ 5,013 vs. 26,770 ⫾ 5,974 ␮m2, p ⬍ 0.0001; 16,146 ⫾ 4,175 vs. 20,523 ⫾ 4,853 ␮m2, p ⬍ 0.0001).

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b

a Fig. 1. Representative glomeruli stained with periodic acid-Schiff (original magnification ⫻200). An obese patient (b) shows marked glomerulomegaly compared with a nonobese patient (a).

Representative micrographs of glomeruli stained with periodic acid-Schiff are shown in figure 1. In addition, ultrastructural study revealed significantly higher glomerular basement membrane thickness in group O than in group N (322 ⫾ 82 vs. 402 ⫾ 100 nm, p ⬍ 0.001). The percentage of ␣-SMA positive area per tuft area was not different between the two groups. 62 patients (46 patients, group N; 16 patients, group O) were followed-up in our institution for one year. We started steroid therapy when cellular and fibrocellular crescents were more than 10% of all glomeruli. In group N, steroid was administered in 13 patients, ACE inhibitor or ARB without steroid in 6 patients, anti-platelet drugs in 11 patients and no drugs in 16 patients. In group O, steroid was administered in 4 patients, ACE inhibitor or ARB without steroid in 9 patients, and no drugs in 2 patients. In both groups, proteinuria was significantly decreased only in patients who received steroid (726.2 ⫾ 144.1 vs. 236.5 ⫾ 78.3 mg/gCre, p ⬍ 0.001; 598.2 ⫾ 169.8 vs. 151.3 ⫾ 27.0 mg/gCre, p ⬍ 0.001). Although 7 of 9 patients who received ACE inhibitor or ARB in group O showed ameliorated proteinuria, the change was not statistically significant (815.6 ⫾ 474.9 vs. 259.5 ⫾ 74.9 mg/gCre, p ⫽ 0.109).

Discussion

In obesity-related nephropathy, there have been few observations on GBM thickness. Henegar [1] reported obese dogs showing thickening of GBM and TBM; however, these findings were not quantitative. In our study, quantitative analysis revealed significant GBM thickening in obese IgAN patients.

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In IgAN, local TGF␤1 is known to be highly expressed in glomeruli due to mesangial IgA deposition [2, 3]. On the other hand, in obesity, expression of glomerular TGF␤1 is also known to be upregulated, possibly through the elevation of leptin [4], AT [1, 2] or insulin [1]. Therefore, it is highly suspected that in obese IgAN patients, local TGF␤1 expression is synergistically upregulated in glomeruli, which might contribute to increase GBM thickness. The GBM thickening we observed in obese IgAN patients may provide membrane dysfunction and increased urinary protein excretion which will accelerate disease progression.

Conclusion

Our findings indicate that obesity may accelerate the increase of proteinuria in IgAN through ultrastructural modification of the glomerular basement membrane.

References 1 2

3 4

Henegar JR: Functional and structural changes in the kidney in the early stage of obesity. J Am Soc Nephrol 2001;12:1211–1217. Lai KN: Polymeric IgA1 from patients with IgA nephropathy upregulates transforming growth factor-beta synthesis and signal transduction in human mesangial cells via the renin-angiotensin system. J Am Soc Nephrol 2003;14:3127–3137. Wang Y: Binding capacity and pathophysiological effects of IgA1 from patients with IgA nephropathy on human glomerular mesangial cells. Clin Exp Immunol 2004;136:168–175. Wolf G: Leptin stimulates proliferation and TGF-␤ expression in renal glomerular endothelial cells: potential role in glomerulosclerosis. Kidney Int 1999;56:860–872.

Mari Tanaka, MD The Tazuke Kofukai Medical Research Institute Department of Nephrology Kitano Hospital 2-4-20 Ohgimachi, Kita-ku Osaka (Japan)

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A Multicenter Prospective Cohort Study of Tonsillectomy and Steroid Therapy in Japanese Patients with IgA Nephropathy: A 5-Year Report Mariko Miyazakia, Osamu Hottaa, Atsushi Komatsudab, Shigeru Nakaic, Tatsuya Shojid, Chikao Yasunagae, Yoshio Tagumaa, Japanese Multicenter Study Group on the Treatment of IgA Nephropathy (JST-IgAN) a Department of Nephrology, Sendai Shakaihoken Hospital, Sendai; bThird Department of Internal Medicine, Akita University School of Medicine, Akita; cDepartment of Nephrology, Osaka General Medical Center, Osaka; dDepartment of In-Home Medicine, Nagoya University School of Medicine, Nagoya; eThe Kidney Center, Saiseikai Yahata Hospital, Kitakyusyu, Japan

Abstract Tonsillectomy combined with corticosteroids has been performed for IgA nephropathy (IgAN) mainly in Japan. We, the Japanese Multicenter Study Group on the Treatment of IgA Nephropathy (JST-IgAN), have conducted a multicenter prospective cohort study including the combination therapy from 1999. A total of 101 patients (43 male, 58 female) were observed for 5 years. Their average age was 34.4 ⫾ 11.8 (15–55). Subjects were classified by daily proteinuria (UP) and serum creatinine (sCr); UP excreted below 0.5 g/day was defined as stage 1, 0.5–1.0 g/day defined as stage 2, more than 1.0 g/day and sCr ⱕ1.2 mg/dl in females or ⱕ1.4 mg/dl in males defined as stage 3, and sCr ⬎1.2 mg/dl in females or ⬎1.4 mg/dl in male defined as stage 4. Both tonsillectomy and high-dose corticosteroid were performed in 75 patients. Of these, 29 patients were stage 1, 26 were stage 2, 16 were stage 3, and 4 were stage 4. The number of subjects with steroid monotherapy were 1 (stage 1), 7 (stage 2), 9 (stage 3) and 1 (stage 4). The primary endpoint in this study was normalized urinalysis as clinical remission. The remission rate treated with combined therapy was 86.2% in stage 1, 73.1% in stage 2, and 43.8% in stage 3. On the other hand, it was 71.4% in stage 2, and 11.1% in stage 3 in steroid monotherapy subjects. Although the number in each treatment group varied because of the non-randomized study, tonsillectomy combined with 3 courses of high-dose corticosteroid therapy was more effective for clinical remission in the higher proteinuria group. We concluded that the goal should be cure and release from disease at an earlier stage of IgAN. Copyright © 2007 S. Karger AG, Basel

Introduction

The therapeutic strategy for IgA nephropathy (IgAN) is still discussed by many investigators. Tonsillectomy combined with corticosteroids has been reported to be effective for the disappearance of urinary abnormality in earlier stages of IgAN [1]; however, there has been no prospective study about the combined therapy before. The randomized controlled trial (RCT), the most reliable method to inspect the effects, is difficult because a surgical operation to asymptomatic tonsils is included in the protocol. Therefore, we conducted a multicenter prospective cohort study, JST-IgAN. Patients and Methods Patients This study was a 60-month, multicenter, non-randomized prospective cohort study designed to verify the efficacy of tonsillectomy combined with steroids. The inclusion criteria on entry were: age between 15 and 55, and diagnosed IgAN by kidney biopsy between April 1999 and March 2001. All patients provided informed consent to entry and to report the results of annual examination to the center. The patients with purpura, diabetes, positive HBs antigen, and positive HCV antibody were excluded from the study. Methods Treatment was chosen according to the physician’s decision at each center. There were 6 treatments: group T3: tonsillectomy and 3 courses of corticosteroid pulse therapy (pulse); group T1: tonsillectomy and 1 course of pulse; group T0: tonsillectomy without pulse; group P3: 3 courses of pulse without tonsillectomy, group P1: 1 course of pulse without tonsillectomy, and group P0: without pulse and without tonsillectomy. Daily doses of methylpredonisolone for pulse were 500 mg for 40–70 kg body weight, 750 mg for 70–80 kg, and 1,000 mg for over 80 kg. Corticosteroids were tapered off at 1 year. In this protocol, use of both ACE inhibitor and ARB was not permitted from 0 to 2 years as initial therapy; however, they were available after 2 years if necessary. Initially, the subjects were divided into 4 clinical groups by proteinuria (UP) and serum creatinine (sCr) at baseline. Subjects with UP of 0.3–0.5 g/day and sCr ⱕ1.2 mg/dl in females or ⱕ1.4 mg/dl in males were defined as stage 1. Subjects with UP of 0.5–1.0 g and the same range of sCr were defined as stage 2. Subjects with UP ⬎1.0 g/day and the same range of sCr were defined as stage 3. Subjects with elevated sCr ⬎1.2 mg/dl in females or ⬎1.4 mg/dl in males were defined as stage 4. In annual examination, category ‘0’ was set up as the primary endpoint of clinical remission. When sCr rose 150% from baseline, we defined ‘Deterioration’ as the composite endpoint.

Results

The number of initially enrolled patients was 185. Their annual reports were collected by the center; however, 42 were lost to follow-up within 2 years,

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Table 1. Effect of tonsillectomy and steroid on complete remission (remission cases/total cases) ⫹ ⫹

⫺ ⫹

⫹ ⫺

⫺ ⫺

Total

Stage 1 Stage 2 Stage 3 Stage 4

25/29 19/26 7/16 1/4

1/1 5/7 1/9 0/1

1/3

1/2 1/2 0/1

28/35 25/35 8/26 1/5

Total

52/75

7/18

1/3

2/5

Tonsillectomy Steroids

22 were observed for 3 years, and 20 were observed for 4 years. In total, 101 patients were analyzed at final outcome at 5 years. There were 43 male and 58 female patients. The average age was 34.4 years. Serum Cr and Ccr were within the normal range. Mean UP was 0.86 g. Median category of urinary red blood cell sediment was 30–49 per hpf. 75 of 101 patients were treated with combined therapy of tonsillectomy and pulse, 18 patients were treated without tonsillectomy but pulse. 3 were treated with tonsillectomy without pulse, and 5 were treated with neither tonsillectomy nor pulse. The outcome of each initial stage is shown in table 1. In stage 1, the complete remission rate was 80%. In stage 2, the rate was 71.4%. In stage 3, it is difficult to obtain complete remission; however, combination therapy is more likely to succeed than steroid monotherapy. On the other hand, in stage 4, which included high-sCr patients, only 1 patient showed an improvement in spite of combined therapy. The remission of hematuria was the remarkable effect of tonsillectomy in any clinical stage. The relative risk of hematuria was 3.68 without tonsillectomy. Furthermore, the evaluation focused on the treatment with pulse. The complete remission rate and relapse rate were 68 and 4% in groups T3 and T1, but 39 and 17% in groups P3 and P1, respectively. The relative risk for urinary abnormality was 2.6 (95% CI: 1.11–6.09) and p ⫽ 0.022 by the ␹2 test.

Discussion

Diagnosis of IgAN is currently confirmed by renal biopsy. However, the histological features change from onset to diagnosis. Fortunately in Japan, it is possible to detect the histological diagnosis in CKD-1/KDOQQI patients via the systematic health check up system and low threshold for renal biopsy. They continued urinary abnormality and showed a slow naturally progressive course

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Aim: complete remission (Tonsillectomy and steroids)

Aim: slowing progression (ACEI, ARB, LPD etc.)

120 Progressive factor

GFR (ml/min)

100 Glomerular hyperfiltration

80

Ischemic nephropathy

60 40 20

Protein loading tubulopathy Glomerular capillary inflammation

0 Onset

Time

Renal death

Fig. 1. Therapeutic strategy for IgAN. Tonsillectomy as upstream intervention and high-dose corticosteroid as downstream immunosupression is effective for the early inflammatory stage. The role of nonimmunological mechanisms is more important, and the aim of treatment is to slow progression to renal failure in the stage of scar formation.

over 20–25 years [2]. Most patients at the early stage excreted ⬍1 g/24 h proteinuria at diagnosis and were considered to require no specific treatment until now [3]. However, prolonged follow-up is required to evaluate the treatment to prevent end-stage renal failure (ESRF). General treatment such as antihypertensive or antihyperlipidemic therapy was beneficial for the risk of ESRF and cardiovascular disease for all stages of chronic renal disease [4]. On the other hand, the therapeutic strategy for upstream mechanisms of IgAN should adapt their clinical and histological stage or complicated disorder (fig. 1). Hotta et al advocated complete remission by combination of tonsillectomy and high-dose corticosteroid therapy [1]. No patients fell into renal impairment as long as complete remission continued. This new paradigm was correlated with a lower index of glomerular lesions and serum creatinine values at baseline. We evaluated the combined therapy using a prospective cohort design. The surgical procedure for pathological focal infection was difficult to randomize. However, this study showed that the combined therapy showed remission equally in 5 years observation. The patients were really diagnosed as IgAN averages 30’s of age. Their ultimate goal of treatment should be not only favorable prognosis but cure without long-term medication. The urinary mass screening system is effective to pick up asymptomatic early stages of glomerulonephritis which has the potential for complete remission.

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In future, it is necessary to reveal which clinical or histological features will most likely derive the most benefit from tonsillectomy and high-dose corticosteroid therapy. It will confirm the best strategy for each stage of IgAN.

Members of JSTIgAN Yamabe H., Osawa H.: Hirosaki University. Wakui H., Komatsuda A.: Akita University. Masakane I., Konta T.: Yamagata University. Ito S., Sato H.: Tohoku University. Abe K., Taguma Y., Hotta O., Miyazaki M.: Sendai Shakaihoken Hospital. Watanabe T., Kato T.: Fukushima Medical College. Suzuki S., Kikuchi Y.: National Defense Medical College. Kobayashi S., Moriya H.: Shonan Kamakura Hospital. Maeda K., Nakai S.: Nagoya University. Tsubakihara Y., Shoji T.: Osaka Medical Center. Nakamoto M., Yasunaga C.: Saiseikai Yahata Hospital. Harada T., Miyazaki M., Furusu A.: Nagasaki University. Imai H.: Aichi Medical University. Akagi H.: Minami-Okayama Medical Center. Matasutani S.: Sendai Red-cross Hospital. Arakawa Y., Hanada N., Sasaki Y.: National Institute of Infectious Diseases.

References 1 2 3 4

Hotta O, Miyazaki M, Furuta T, et al: Tonsillectomy and steroid pulse therapy significantly impact on clinical remission in patients with IgA nephropathy. Am J Kidney Dis 2001;38:736–743. D’Amico G: Natural history of idiopathic IgA nephropathy: role of clinical and histological prognostic factors. Am J Kidney Dis 2000;36:227–237. Barratt J, Feehally J: Treatment of IgA nephropathy. Kidney Int 2006;69:1934–1938. Kundhal K, Lok CE: Clinical epidemiology of cardiovascular disease in chronic kidney disease. Nephron Clin Pract 2005;101:c47–c52.

Mariko Miyazaki, MD Department of Nephrology Sendai Shakaihoken Hospital Tsutsumimachi 3-16-1, Aoba-ku Sendai 981–8501 (Japan)

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Tonsillectomy and Corticosteroid Therapy with Concomitant Methylprednisolone Pulse Therapy for IgA Nephropathy Tatsuya Suwabea, Yoshifumi Ubaraa, Yoko Sogawaa, Yasushi Higaa, Kazufumi Nomuraa, Shohei Nakanishia, Jyunichi Hoshinoa, Naoki Sawaa, Hideyuki Katoria, Fumi Takemotoa, Shigeko Harab, Kenichi Ohashic, Kenmei Takaichia a

Nephrology Center, bHealth Care Unit, and cPathology Center, Toranomon Hospital, Tokyo, Japan

Abstract IgA nephropathy (IgAN) is the most common chronic kidney disease in Japan, but the optimum treatment remains controversial. Our objective was to evaluate the effect of tonsillectomy and corticosteroid therapy combined with methylprednisone pulse therapy in patients at our hospital who had IgAN. Tonsillectomy plus pulse therapy was evaluated in 72 patients (33 men and 39 women) with a diagnosis of IgAN based on renal biopsy who were followed up for more than 1 year. The mean age of the patients was 35.2 ⫾ 10.9 years (range: 20–58 years) and the mean observation period after tonsillectomy was 20.3 ⫾ 9.7 months (range: 12–36 months). After tonsillectomy, steroid pulse therapy was administered (methylprednisolone at 500 mg daily for 3 days) 1–3 times and was followed by oral prednisolone from an initial dose of 30 mg on alternate days that was tapered gradually over one year. At 2 years after tonsillectomy, serum creatinine was unchanged or improved in the majority of patients, but worsened in 5 patients. Hematuria (erythrocytes/HPF) improved from Grade 3.76 (11–30/HPF) to Grade 1.94 (1–5/HPF) on average (we defined the grade of hematuria). None of the patients experienced exacerbation of hematuria. Proteinuria decreased from 1.32 g/day to 0.86 g/day (65% of the pretreatment value), and only 4 patients showed an increase of proteinuria. Mean protein loss decreased to less than 0.5 g/day in patients with creatinine clearance ⱖ90 ml/min and/or patients with initial protein excretion ⱕ1.0 g/day. Copyright © 2007 S. Karger AG, Basel

Background

IgA nephropathy (IgAN) is the most common chronic kidney disease in Japan, but the optimum treatment remains controversial. The combination of

tonsillectomy and corticosteroid therapy with concomitant methylprednisone pulse therapy is commonly employed in Japan, although it is unpopular in other countries [1]. Objective

Our objective was to evaluate the effect of tonsillectomy plus corticosteroid therapy with concomitant methylprednisone pulse therapy in patients at our hospital who had IgAN. Patients Seventy-two patients (33 men and 39 women) with a diagnosis of IgAN based on renal biopsy were enrolled in this study. They were followed up for more than 1 year. The mean age was 35.2 ⫾ 10.9 years (range: 20–58 years) and the mean observation period after tonsillectomy was 20.3 ⫾ 9.7 months (range: 12–36 months).

Methods We followed Hotta’s regimen [2]. After tonsillectomy, steroid pulse therapy was administered (methylprednisolone 500 mg daily for 3 days) 1–3 times and was followed by oral prednisolone at an initial dose of 30 mg on alternate days that was tapered gradually over one year. Creatinine clearance (Ccr) was calculated by the Cockcroft-Gault formula. Results are expressed as mean ⫾ SD. Statistical analysis was performed using Microsoft Excel 2003 and Statview version 4.5. p ⬍ 0.05 was considered to be significant.

Results

The serum creatinine level increased in 5 patients, but was unchanged in the other patients. Proteinuria decreased in most patients and improved dramatically in some patients, with difference after treatment being significant (table 1). At 2 years after tonsillectomy, hematuria (erythrocytes/HPF) improved from Grade 3.76 (11–30/HPF) to Grade 1.94 (1–5/HPF). None of the patients experienced exacerbation of hematuria. The serum IgA level decreased in all patients, with the mean serum IgA level falling from 308.9 ⫾ 91.3 mg/dl to 262.9 ⫾ 88.1 mg/dl over 2 years. Blood pressure was controlled at the same level throughout the observation period. We divided the patients into groups according to their initial Ccr (table 2). Among the patients with an initial Ccr of more than 90 ml/min, serum creatinine worsened in one patient and remained stable in the others. Among the patients with an initial Ccr of 60–90 ml/min, one patient worsened and the other

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Table 1. Changes of laboratory data Before treatment

After 1 year (% of initial value)

After 2 years (% of initial value)

Cr (mg/dl)

1.07 ⫾ 0.55

1.10 ⫾ 0.60 (102.8%)

1.01 ⫾ 0.41 (94.4%)

IgA (mg/dl)

308.9 ⫾ 91.3

223.3 ⫾ 76.5 (72.3%) p ⬍ 0.001

262.9 ⫾ 88.1 (85.1%) p ⬍ 0.001

Proteinuria (g/day)

1.32 ⫾ 1.10

0.83 ⫾ 0.66 (62.9%) p ⬍ 0.005

0.86 ⫾ 0.46 (65.2%) p ⬍ 0.005

Hematuria (Grade)

3.76 ⫾ 1.24

2.12 ⫾ 0.99 (56.4%) p ⬍ 0.001

1.94 ⫾ 1.05 (51.6%) p ⬍ 0.001

Grade of hematuria. Grade1: RBC ⬍ 1/HPF Grade2: 1–5/HPF Grade3: 6–10/HPF Grade4: 11–30/HPF Grade5: ⬎30/HPF

patients either remained the same, or showed an improvement in the serum creatinine level. When the initial Ccr was 40–60 ml/min, two patients worsened and the other patients showed the same serum creatinine level or improved. When the initial Ccr was less than 40 ml/min, one patient worsened and the others maintained the same creatinine level. Among the patients with an initial Ccr of more than 90 ml/min, proteinuria improved in most cases and dramatically in some. When the initial Ccr was 60–90 ml/min, proteinuria improved in most patients, but remained at more than 0.5 g/day after 2 years in some patients. Among the patients with an initial Ccr of less than 40 ml/min, proteinuria did not improve much. We also divided all patients according to their initial protein excretion. Among the patients with an initial protein loss of 0.5–1.0 g/day, proteinuria improved in most cases and worsened in 2 patients. When initial proteinuria was 1–2 g/day, the mean protein excretion did not decrease to less than 0.5 g/day. In the patients with initial protein loss of 2–3 g/day, protein excretion did not decrease much. In the case of initial protein loss of more than 3 g/day, the number of patients followed for more than 2 years was small. When the relation between annual mean protein loss and Ccr was investigated, proteinuria decreased to less than 0.5 g/day in the patients with an initial Ccr of more than 90 ml/min, but it remained at more than 0.5 g/day in the other groups. Regarding the relation between annual mean protein loss and initial proteinuria, the mean protein loss decreased to less than 0.5 g/day in patients with

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Table 2. Changes of annual mean protein loss Before treatment (g/day)

After 1 year (g/day)

After 2 years (g/day)

1.32 ⫾ 1.10

0.83 ⫾ 0.66 (62.9%)

0.86 ⫾ 0.46 (65.2%)

Correlation between annual mean protein loss and Ccr Ccr ⱖ 90 1.02 ⫾ 0.78 0.48 ⫾ 0.48 (n ⫽ 39) (47.1%)

0.41 ⫾ 0.47 (40.2%)

Ccr ⱖ 60, to ⬍90 (n ⫽ 15)

1.45 ⫾ 1.08

0.83 ⫾ 0.78 (57.2%)

0.90 ⫾ 0.67 (62.1%)

Ccr ⱖ 40, to ⬍60 (n ⫽ 14)

1.89 ⫾ 1.55

0.73 ⫾ 0.55 (39.0%)

0.72 ⫾ 0.37 (38.5%)

Ccr ⬍ 40 (n ⫽ 4)

1.59 ⫾ 1.10

0.43 ⫾ 0.11 (27.0%)

0.86 ⫾ 0.84 (54.1%)

All patients (n ⫽ 72)

Correlation between annual mean protein loss and initial proteinuria Proteinuria 0.23 ⫾ 0.11 0.16 ⫾ 0.12 ⬍0.5 g/day (69.6%) (n ⫽ 12)

0.10 ⫾ 0.06 (43.4%)

ⱖ0.5, to ⬍1.0 (n ⫽ 20)

0.63 ⫾ 0.18

0.38 ⫾ 0.21 (60.3%)

0.39 ⫾ 0.45 (61.9%)

ⱖ1.0, to ⬍2.0 (n ⫽ 19)

1.33 ⫾ 0.57

0.84 ⫾ 0.65 (63.2%)

0.84 ⫾ 0.54 (63.2%)

ⱖ2.0, to ⬍3.0 (n ⫽ 13)

2.10 ⫾ 0.57

0.91 ⫾ 0.61 (43.3%)

1.02 ⫾ 0.52 (48.6%)

ⱖ3.0 (n ⫽ 8)

3.34 ⫾ 1.09

1.16 ⫾ 0.82 (34.7%)

0.89 ⫾ 0.77 (26.6%)

initial proteinuria of less than 1 g/day, but the mean loss remained at more than 0.5 g/day in the other groups. As complications, severe bleeding occurred after tonsillectomy in one patient. Acne occurred after 1–2 months of steroid therapy and subsided when steroid treatment was discontinued. None of the patients developed new infections.

Conclusion

Tonsillectomy plus pulse therapy was evaluated in 72 patients with IgAN diagnosed by renal biopsy, who were followed up for more than 1 year. In the

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majority of patients, serum creatinine was unchanged or improved, but the creatinine level increased in 5 patients. Hematuria (erythrocytes/HPF) improved from Grade 3.76 (11–30/HPF) to Grade 1.94 (1–5/HPF). None of the patients experienced exacerbation of hematuria. Proteinuria decreased from 1.32 g/day to 0.86 g/day (65% of the pretreatment value), and only 4 patients showed an increase of proteinuria. Mean protein loss decreased to less than 0.5 g/day in patients with a Ccr ⱖ90 ml/min and/or patients with initial protein loss of ⱕ1.0 g/day.

References 1 2

Barratt J, Feehally J: Treatment of IgA nephropathy. Kidney Int 2006;69:1934–1938. Hotta O, Miyazaki M, Furuta T, et al: Tonsillectomy and steroid pulse therapy significantly impact on clinical remission in patients with IgA nephropathy. Am J Kidney Dis 2001;38:736–743.

Tatsuya Suwabe, MD Nephrology Center Toranomon Hospital Kajigaya 1-3-1 Kajigaya, Takatsu-ku Kawasaki-shi, Kanagawa-ken, 213–0015 (Japan) Tel. ⫹81 44 877 5111, Fax ⫹81 44 877 5333, E-Mail [email protected]

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Impact of Annual Urine Health Check-Up System to Obtain Clinical Remission in Patients with IgA Nephropathy Norio Ieiri, Osamu Hotta, Yoshio Taguma Department of Nephrology, Sendai Shakaihoken Hospital Tsutsumimachi, Aoba-ku, Sendai, Miyagi, Japan

Abstract Purpose: In Japan, the annual urine health check-up system is well developed. Recently we reported a significant impact of tonsillectomy and steroid pulse therapy on clinical remission in our IgA nephropathy (IgAN) patients and indicated that clinical remission might terminate the progressive deterioration in renal function. We surveyed whether early detection of urinary abnormalities by annual urine health check-ups contribute to clinical remission in IgAN patients treated with tonsillectomy and steroid pulse therapy. Methods: We investigated 380 IgAN patients in whom the onset year was identified by annual urine check-ups. Group A consisted of 264 patients in whom treatment intervention was initiated within 3 years after the first appearance of urinary abnormalities, and group B consisted of 116 patients who were diagnosed after more than 3 years duration of urinary abnormalities. We also classified our 380 patients by the degree of glomerular lesions; 233 patients with mild, 83 with moderate, and 64 with severe glomerular lesions. All patients were treated with tonsillectomy and steroid pulse therapy in our renal unit. Results: The clinical remission rate of group A was 87.1%, while that of group B was 54.3%. In the mild glomerular lesion group, group B had a significantly lower remission rate than group A. Even in the severe glomerular lesion group, the remission rate of group A was significantly higher than that of group B. Conclusion: Our results indicate the annual health check-up system to be very useful for achieving clinical remission in IgAN patients, if they are treated with tonsillectomy and steroid pulse therapy. Copyright © 2007 S. Karger AG, Basel

Background

The annual urine health check-up system for the general population in Japan is well developed [1], while the value of this system based on its costeffectiveness is now being questioned in other countries including the USA [2].

Approximately 70% of Japanese IgA nephropathy (IgAN) patients were detected by routine urinary tests [3], but unfortunately IgAN is still one of the most common causes of end-stage renal disease even now. Recently, we reported a significant impact of tonsillectomy and steroid pulse therapy on clinical remission in our IgAN patients, especially those in a relatively early stage of nephropathy, and indicated that clinical remission could terminate the progressive deterioration in renal function [4]. In present study, we surveyed whether early detection of urinary abnormalities by annual urine health checkups contribute to clinical remission in IgAN patients treated with tonsillectomy and steroid pulse therapy. Method We investigated 380 IgAN patients in whom the onset was identified by annual urine check-ups documented in medical records. Group A consisted of 264 patients in whom treatment intervention was initiated within 3 years after the first appearance of urinary abnormalities, and group B consisted of 116 patients who were diagnosed after more than 3 years duration of urinary abnormalities. We also classified 380 patients by the degree of glomerular lesions; 233 patients (191 patients of group A and 42 of group B) with mild, 83 (45 of group A and 38 of group B) with moderate, and 64 (28 of group A and 36 of group B) with severe glomerular lesions. All patients were treated with tonsillectomy and steroid pulse therapy in our renal unit from 1988 to 2000, and their follow-up periods were at least 5 years. All patients gave their informed consent to participate in this study. The glomerular filtration rate (GFR) of each patient was calculated by the Cockcroft-Gault equation. The degree of glomerular lesion in each case was evaluated using the index of the glomerular lesion (IGL) according to the method originally proposed by Suwa and Takahashi [5], with a modification to evaluate sclerotic changes as previously described [4]. Remission of urinary abnormalities or clinical remission was defined as negative proteinuria and hematuria by dipstick and urinary erythrocytes detected less than 1/high-power field (hpf). We evaluated the count of urinary erythrocytes semi-quantitatively as follows: 1) ⬍1/hpf; 2) 1–4/hpf; 3) 5–9/hpf; 4) 10–29/hpf; 5) 30–49/hpf; 6) ⬎50/hpf.

Results

At the time of diagnosis, mean ages were 29.2 ⫾ 13.7 years in group A and 33.7 ⫾ 12.6 years in group B (p ⫽ 0.003). The GFR of group B before treatment initiated showed significant deterioration compared with that of group A (89.94 ⫾ 24.50 ml/min vs. 102.70 ⫾ 27.55 ml/min, p ⬍ 0.001), and IGL was significantly more severe in group B than in group A (1.81 ⫾ 0.56 vs. 1.43 ⫾ 0.43, p ⬍ 0.001). Urinary protein excretion of group A before treatment was statistically less than that of group B (0.74 ⫾ 0.77 g/day vs. 1.06 ⫾ 1.05 g/day, p ⫽ 0.007), and the count of urinary red blood cells of group A was more than group B (4.6 ⫾ 1.3

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Clinical remission rate (%)

100 89.5 80

** *

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88.9 67.9

71.1

64.3

40

*** 20 0

25.0

A B

–1.50

1.51–2.00

2.01–

Group

IGL

Fig. 1. The clinical remission rate at the final observation of group A and B classified by the degree of glomerular lesions. In every degree of glomerular lesion, the clinical remission rate of group A was significantly higher than that of group B. Even in the mild glomerular lesion group (IGL ⱕ 1.50), a later initiation of intervention therapy lowered the clinical remission rate; in the severe glomerular lesion group (IGL ⱖ 2.01), an earlier initiation of treatment led to a remarkably high clinical remission rate. *p ⬍ 0.001; **p ⫽ 0.040; ***p ⫽ 0.001 vs. group A of same IGL.

vs. 4.2 ⫾ 1.6, p ⫽ 0.018). The values of IgG, IgA, and IgM were not statistically significantly different group A and B. The clinical remission rate at the final observation of group A reached 87.1%, while that of group B came to a stand at 54.3%. In every degree of glomerular lesions, the clinical remission rate of group A was significantly higher than that of group B (fig. 1). Even in the mild glomerular lesion group, a later initiation of intervention therapy lowered the clinical remission rate; in the severe glomerular lesion group, an earlier initiation of treatment led to a remarkably high clinical remission rate (fig. 1). In the severe glomerular lesion group, interestingly, there was a significant difference between group A and B regarding the rate of glomerulo-capillaritis as determined by a count of urinary red blood cells (4.9 ⫾ 1.2 vs. 4.0 ⫾ 1.6, p ⫽ 0.026) and by the percentage of the number of glomeruli with necrosis and/or crescent formation of that of the total residual glomeruli (19.35 ⫾ 16.56% vs. 6.72 ⫾ 8.52%, p ⫽ 0.001).

Discussion

Geddes CC et al. [6] reported the geographical variability in renal survival of IgAN patients, and stated that the disease was detected at different phases in

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its course because of the different frequency of opportunities for urine health check ups and the different indications for renal biopsies. Accordingly their report showed that a greater renal survival requires an earlier detection of urinary abnormalities and an earlier diagnosis of the disease. Because a great number of IgAN patients are silent at onset of the disease and are detected by urinary health examination [3], an earlier detection of the disease requires annual urine check-ups. Fortunately the annual urine health check-up system for the general population is well developed in Japan [1], so that we could detect and diagnose IgAN at an earlier stage. Recently, we reported a significant impact of tonsillectomy and steroid pulse therapy on clinical remission in our IgAN patients and indicated that clinical remission could terminate the progressive deterioration in renal function [4]. In the present study, earlier-initiated treatment intervention cases (group A) maintained GFR and showed less glomerular injuries compared with the laterinitiated group (group B), and consequently the clinical remission rate was superior in earlier-initiated cases treated with tonsillectomy and steroid pulse therapy. If intervention therapy were initiated earlier, it could have achieve a greater clinical remission rate in any degree of glomerular lesion, even with severe glomerular lesions, because of the intense but acute phase of glomerulocapillaritis. Therefore it is suggested that the annual urine health check-up system is very valuable for induction of the disease into a state of complete remission, if we use intervention therapy in our IgAN patients aimed at achieving clinical remission such as tonsillectomy and steroid pulse therapy.

Conclusion

Our results indicate the annual urine health check-up system to be very useful for achieving clinical remission in IgAN patients, if they have effective treatment interventions such as tonsillectomy and steroid pulse therapy.

References 1 2 3 4

Health and welfare statistics association: Statistical abstracts on health and welfare in Japan 2004. Health and welfare statistics association, Tokyo, 2004. Boulware LE, Jaar BG, Tarver-Carr ME, et al: Screening for proteinuria in US adults. A costeffectiveness analysis. JAMA 2003;290:3101–3114. Koyama A, Igarashi M, Kobayashi M, et al: Natural history and risk factors for immunoglobulin A nephropathy in Japan. Am J Kidney Dis 1997;29:526–532. Hotta O, Miyazaki M, Furuta T, et al: Tonsillectomy and steroid pulse therapy significantly impact on clinical remission in patients with IgA nephropathy. Am J Kidney Dis 2001;38:736–743.

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Suwa N, Takahashi T: Morphological and Morphometrical Analysis of Circulation in Hypertension and Ischemic Kidney. München-Berlin-Wien, Germany, Urban & Schwarzenberg, 1971, pp 108–116. Geddes CC, Rauta V, Gronhagen-Riska C, et al: A tricontinental view of IgA nephropathy. Nephrol Dial Transplant 2003;18:1541–1548.

Norio Ieiri Department of Nephrology, Sendai Shakaihoken Hospital Tsutsumimachi 3-16-1, Aoba-ku, Sendai Miyagi, 981–8501 (Japan) Tel. ⫹81 22 275 3111, Fax ⫹81 22 275 6033, E-Mail [email protected]

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Sequential Immunosuppressive Therapy in Progressive IgA Nephropathy Franz Maximilian Raschea, Frieder Kellera, Lutz von Müllerb, David Czocka, Philipp M. Lepperc a

Division of Nephrology, Department of Internal Medicine I, bInstitute of Microbiology, Virology and Immunology, University Hospital of Ulm, Ulm, Germany; cDepartment of Intensive Care Medicine, University Hospital of Berne, Berne, Switzerland

Abstract Backgrounds: Cyclophosphamide and high-dose steroids have been used as limited induction therapy in progressive IgA nephropathy (IgAN) to reduce the loss of renal function and proteinuria. We evaluated the effect of cyclophosphamide pulses (CyP) and mycophenolic acid (MPA) as sequential therapy on renal function in patients with progressive IgAN. Methods: Twenty patients with progressive IgAN and advanced renal failure (median GFR 22 ml/min per 1.73 m2) and further disease activity (⌬GFR ⫺0.8 ml/min per month) after cyclophosphamide (CyP; n ⫽ 18) or steroid pulse therapy (n ⫽ 2) were treated with mycophenolate mofetil 1 g per day for a median of 27 months. Results: The monthly loss of renal function was significantly reduced in linear regression analysis from ⫺2.4 ml/min before CyP to ⫺0.12 ml/min with CyP/MPA (p ⫽ 0.0009). Estimated renal survival time was significantly prolonged by a median of 65 months (p ⫽ 0.0014). Proteinuria decreased significantly from 1.7 to 0.4 g/l during MPA treatment (p ⫽ 0.015). In Cox regression analysis, only proteinuria ⬎1.0 g/l was an independent risk factor for doubling of creatinine during CyP/MPA treatment (p ⫽ 0.03). Conclusion: A sequential therapy with CyP/MPA may arrest or slow down the loss of renal function and reduces proteinuria even in patients who passed the so called ‘point of no return’ with progressive IgAN. Copyright © 2007 S. Karger AG, Basel

Introduction

In progressive advanced IgA nephropathy, high-dose steroids or cyclophosphamide, e.g. cyclophosphamide pulses (CyP) [1], have been shown to reduce loss of renal function and proteinuria as a limited, initial immunosuppressive therapy [1–6]. The potential role of azathioprine or mycophenolic acid (MPA) as

a sequential therapy is largely unknown. However, the effect of MPA in IgAN is being discussed controversially [7–10]. We investigated in this prospective study the effect on renal function of sequential therapy with MPA in patients with progressive IgAN.

Patients We treated in a prospective study 20 consecutive patients (Caucasians, mean age 56 ⫾ 13 years, 19 males and 1 female) with biopsy-proven, primary IgAN and further progression, defined as an increase in serum creatinine by more than 10% within three months and/or as proteinuria ⱖ0.7 g/l, after prior cyclophosphamide pulse therapy (CyP; n ⫽ 18) [1] or steroid pulse therapy (n ⫽ 2) [2] with MPA continuously as a sequential maintenance therapy between 1997 and 2004 [10]. Exclusion criteria were age ⬍18 years, serum creatinine ⬎4.5 mg/dl (⬎400 ␮mol/l), kidney size ⬍9 cm in ultrasound, rapidly progressive and extracapillary proliferative forms of IgAN, secondary mesangioproliferative IgAN, acute or chronic infections, carcinoma, leukocyte counts ⬍3.0 /nl, platelet counts ⬍80 /nl, gastrointestinal bleeding, hemolytic anemia, pregnancy, lactation or women with childbearing potential. In renal biopsies, Lee grade IV was present in 90% of the patients [11] and Haas classification grade IV in 72% [12]. The course of renal function was assessed by linear regression analysis of the glomerular filtration rate (GFR MDRD 2) [13]. Statistical significance for all tests was set at a level of p ⬍ 0.05. The study protocol was approved by the institutional review board and informed consent was obtained from all patients. Mycophenolic acid (MPA, 1.0 g/day mycophenolate mofetil, CellCept™, Hoffmann-La Roche, Basel, Switzerland) was administered continuously to the end of the study concomitantly with prednisolone 5 mg/day, and both were discontinued at serum creatinine levels exceeding 7.0 mg/dl. Starting at the diagnosis of IgAN, an ACE inhibitor or an AT1 receptor blocker were administered for 16 (3–51) months prior to MPA treatment.

Results

The overall observation time was 6.3 years (3.3–13.3) and MPA was given for a median of 27 (6–74) months. Disease progression after induction therapy was observed after 4 (0–33) months. Serum creatinine improved in twelve patients. Five patients developed end-stage renal failure after a median of 23 (14–38) months while still on MPA therapy, and three patients were nonresponders to MPA. The loss of renal function was significantly reduced from ⫺2.4 (⫺9.8 to ⫺0.6) ml/min per month with CyP/MPA to ⫺0.12 (⫺0.77 to ⫺0.01) ml/min per month at the end of the study (Wilcoxon test p ⫽ 0.0009, fig. 1). In the linear regression model, the extrapolated median renal survival time was without therapy 7 (1–27) months (regression coefficient R ⫽ 0.88) and with therapy 72 (23–440) months (R ⫽ 0.72). Thus, renal survival time was

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GFR (ml/min per 1.73m2)

50

CyP

40 MPA 30 20 10 0 ⫺24

HD ⫺12

0 12 Months after MPA

24

36

Fig. 1. Estimated loss of renal function by linear regression analysis before and with induction therapy (cyclophosphamide (CyP) or steroid pulses) and while on mycophenolic acid (MPA) therapy in patients with progressive IgAN. The dotted line shows the extrapolated course of renal function without immunsuppressive therapy. HD ⫽ haemodialysis.

significantly prolonged by a median of 65 months (Wilcoxon test p ⫽ 0.0014, fig. 1). Proteinuria decreased significantly from 1.7 (0.1–5.6) g/l before therapy to 0.4 (0.1–3.2) g/l at the end of the study (Wilcoxon test p ⫽ 0.015). In the Cox regression model, only proteinuria (⬎1 g/l) was an independent and significant categorical variable tested for doubling of baseline creatinine before CyP-1 (p ⫽ 0.031, B ⫽ ⫺1.88; R ⫽ ⫺0.30, Exp B ⫽ 0.15). Body mass index (⬎25 kg/m2) was a significant but not independent categorical variable (p ⫽ 0.035; R ⫽ 0.29). The other categorical variables blood pressure (⬎140/90 mm Hg), age (⬎60 years), serum creatinine (⬎3.0 mg/dl), total cholesterol (⬎200 mg/dl), gender and smoking status were not significant in the Cox regression model (p ⬎ 0.05). No significant differences of the variables were found between the patients who received CyP and steroid pulse therapy (p ⬎ 0.05, Mann-Whitney U test). Blood pressure (median 130/80 mm Hg), serum protein, total cholesterol, hemoglobin, white blood cell and platelet count and body mass index were unchanged before and with MPA or CyP therapy (p ⬎ 0.5, Wilcoxon test). At the end of the study, the median daily dose was 1,000 mg and the median MPA 12-hour plasma concentration was 2.0 (0.1–7.0) ␮g/ml. One patient discontinued MPA due to severe gastrointestinal symptoms, and in two patients mild abdominal pain and diarrhea resolved after a MPA dose reduction. Severe myelotoxicity (WHO grade III) or infections were not observed during MPA therapy in any patient.

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Discussion

Sequential immunosuppressive therapy in progressive IgAN has only been studied in two protocols with azathioprin [5, 6], but the effect of the more specific inhibitor of the purine synthesis, MPA, is largely unknown in progressive IgAN as sequential maintenance therapy. We demonstrated in patients with progressive IgAN that sequential MPA therapy significantly reduces the loss of renal function from ⫺2.4 to ⫺0.12 ml/min per month and proteinuria from 1.4 to 0.4 g/l. Cyclophosphamide induces a depletion and inhibition of T and B lymphocytes. Consecutively, MPA inhibits further B and T cell proliferation and has been shown to suppress immunoglobulin and cytokine secretion of B cells [14]. In addition, MPA inhibits the migration of lymphocytes and the antigen presentation by dendritic cells [15], and induces apoptosis of activated T lymphocytes [16]. These effects may be responsible for a reduction of the proliferative lesions, and the arresting of glomerular sclerosis and tubular fibrosis in IgAN [17]. Our experiences with MPA demonstrate for the first time, even in patients who have passed serum creatinine higher than 3.0 mg/dl (the so called ‘point of no return’) [18–20], that immunosuppression with an pulsed induction and sequential maintenance protocol might be an effective long-term approach for inhibiting immune-mediated renal damage in IgAN. Larger randomized controlled trials will be required to confirm the therapeutic value and safety of sequential immunosuppression in these patients.

References 1 2 3 4

5 6 7 8

Rasche FM, Klotz CH, Czock D, et al: Cyclophosphamide pulse therapy in advanced progressive IgA nephropathy. Nephron Clin Pract 2003;93:131–136. Pozzi C, Bolasco PG, Fogazzi GB, et al: Corticosteroids in IgA nephropathy: a randomised controlled trial. Lancet 1999;353:883–887. Roccatello D, Ferro M, Cesano G, et al: Steroid and cyclophosphamide in IgA nephropathy. Nephrol Dial Transplant 2000;15:833–835. Tsuruya K, Harada A, Hirakata H, et al: Combination therapy using prednisolone and cyclophosphamide slows the progression of moderately advanced IgA nephropathy. Clin Nephrol 2000;53: 1–9. Ballardie FW, Roberts IS: Controlled prospective trial of prednisolone and cytotoxics in progressive IgA nephropathy. J Am Soc Nephrol 2002;13:142–148. Goumenos DS, Davlouros P, El Nahas AM, et al: Prednisolone and azathioprine in IgA nephropathy – a ten-year follow-up study. Nephron Clin Pract 2003;93:58–68. Tang S, Leung JC, Chan LY, et al: Mycophenolate mofetil alleviates persistent proteinuria in IgA nephropathy. Kidney Int 2005;68:802–812. Maes BD, Oyen R, Claes K, et al: Mycophenolate mofetil in IgA nephropathy: results of a 3-year prospective placebo-controlled randomized study. Kidney Int 2004;65:1842–1849.

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10 11

12 13

14

15

16 17 18

19 20

Frisch G, Lin J, Rosenstock J, et al: Mycophenolate mofetil (MMF) vs placebo in patients with moderately advanced IgA nephropathy: a double-blind randomized controlled trial. Nephrol Dial Transplant 2005;20:2139–2145. Rasche FM, Keller F, von Muller L, Sailer LK, Karges W, Czock D: Mycophenolic acid therapy after cyclophosphamide pulses in progressive IgA nephropathy. J Nephrol 2006;19:465–472. Lee HS, Lee MS, Lee SM, et al: Histological grading of IgA nephropathy predicting renal outcome: revisiting H.S. Lee’s glomerular grading system. Nephrol Dial Transplant 2005;20: 342–348. Haas M: Histologic subclassification of IgA nephropathy: a clinicopathologic study of 244 cases. Am J Kidney Dis 1997;29:829–842. Poggio ED, Wang X, Greene T, et al: Performance of the modification of diet in renal disease and Cockcroft-Gault equations in the estimation of GFR in health and in chronic kidney disease. J Am Soc Nephrol 2004;22:22. Jonsson CA, Carlsten H: Mycophenolic acid inhibits inosine 5⬘-monophosphate dehydrogenase and suppresses immunoglobulin and cytokine production of B cells. Int Immunopharmacol 2003;3: 31–37. Colic M, Stojic-Vukanic Z, Pavlovic B, et al: Mycophenolate mofetil inhibits differentiation, maturation and allostimulatory function of human monocyte-derived dendritic cells. Clin Exp Immunol 2003;134:63–69. Allison AC, Eugui EM: Mycophenolate mofetil and its mechanisms of action. Immunopharmacology 2000;47:85–118. Pozzi C, Andrulli S, Del Vecchio L, et al: Corticosteroid effectiveness in IgA nephropathy: longterm results of a randomized, controlled trial. J Am Soc Nephrol 2004;15:157–163. Komatsu H, Fujimoto S, Sato Y, et al: ‘Point of no return (PNR)’ in progressive IgA nephropathy: significance of blood pressure and proteinuria management up to PNR. J Nephrol 2005;18: 690–695. D’Amico G, Ragni A, Gandini E, et al: Typical and atypical natural history of IgA nephropathy in adult patients. Contrib Nephrol 1993;104:6–13. Scholl U, Wastl U, Risler T, et al: The ‘point of no return’ and the rate of progression in the natural history of IgA nephritis. Clin Nephrol 1999;52:285–292.

Franz Maximilian Rasche Division of Nephrology, Department of Internal Medicine I University Hospital of Ulm, Robert-Koch-Strasse 8 DE–89081 Ulm (Germany) Tel. ⫹49 731 500 44561, Fax ⫹49 731 500 44567 E-Mail [email protected]

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Prospective Trial of Combined Therapy with Heparin/Warfarin and Renin-Angiotensin System Inhibitors in Progressive IgA Nephropathy Takeo Ishii, Tetsuya Kawamura, Nobuo Tsuboi, Makoto Ogura, Yasunori Utsunomiya, Tatsuo Hosoya Division of Kidney and Hypertension, Department of Medicine, Jikei University School of Medicine, Tokyo, Japan

Abstract We previously reported that a combined therapy with heparin/warfarin and reninangiotensin system (RAS) inhibitors dramatically reduces proteinuria for prolonged periods in advanced IgA nephropathy (IgAN). In the present study, we prospectively analyzed whether the combined therapy can inhibit the progressive decline in renal function of patients with progressive IgAN. Patients who had a marked linearity of decline in loss of glomerular filtration rate (GFR), assessed by reciprocal serum creatinine plots vs. time for more than one year, were recruited in this study if they were histologically diagnosed as IgAN at this point of declining renal function. Twelve patients were eligible for trial entry; reciprocal serum creatinine plot suggested end-stage renal failure within 5 years. All patients were treated with continuous intravenous infusion of heparin for 8 weeks, followed by oral administration of warfarin, ACE inhibitors and/or angiotensin II receptor blockers and dypiridamole. Eight patients were further given corticosteroid for 2 years because of the presence of acute glomerular lesions such as cellular crescent or angionecrosis. All patients were followed-up for at least 12 months, and the mean follow-up period was 34 ⫾ 20 (range 12–79) months. After the combined therapy, urinary protein excretion was significantly reduced from 2.4 ⫾ 1.5 g/day at baseline to 0.7 ⫾ 0.5 g/day at final observation, while the mean serum creatinine was not significantly different. Of note, the mean slope of 1/serum creatinine significantly increased from ⫺0.009 to ⫹0.0002 dl/mg/week (p ⬍ 0.05). Moreover, histological analysis of a repeat kidney biopsy which was performed in 5 patients at 2 years after the institution of the combined therapy revealed that the percentage of cellular/fibrocellular crescent and the degree of mesangial matrix expansion were significantly attenuated (19→0.1% and 1.6→0.6 score, respectively) while the percentage of global sclerosis and tubulointerstitial lesion did not increase. These results indicate that our combined therapy with heparin/warfarin and RAS inhibitors can inhibit the progressive decline in renal function

of patients with progressive IgAN through its marked antiproteinuric and anti-inflammatory effects. Copyright © 2007 S. Karger AG, Basel

Introduction

Treatments with corticosteroid or renin-angiotensin system (RAS) inhibitors have been considered as potent therapeutic tools in patients with IgAN. It has been reported, however, that the treatment with steroid alone cannot prevent the progressive decline in renal function of IgAN patients with a GFR less than 70 ml/min. A recent randomized controlled trial by Praga et al. [1] clearly demonstrated that the GFR-preserving effect of ACE inhibition is conferred in the patients with serum creatinine of less than 1.5 mg/dl at baseline. Thus, in advanced IgAN patients, therapeutic efficiency of steroid or RAS inhibitors may not be necessarily high. We previously performed a retrospective analysis to examine the long-term prognosis of 30 patients with advanced IgAN [2]. In that study, thirty patients were divided into the three groups: the first group was treated with steroid ⫹ heparin/warfarin ⫹ ACE inhibitors ⫹ antiplatelets (Group A), the second with ACE inhibitors ⫹ antiplatelets (Group B) and the third with antiplatelets alone (Group C). Renal survival rate by the Kaplan-Meier method revealed a significantly prolonged renal survival in Group A compared with Groups B and C. Since the patients in Group A had several risk factors for poor prognosis, corticosteroid might not have had a central role in inhibiting the progression to endstage renal failure. Therefore, it is conceivable to speculate that co-administered heparin and warfarin may have played an important role for preservation of renal function in Group A. Thus, in the present study, we prospectively analyzed whether the combined therapy with heparin/warfarin and RAS inhibitors can inhibit the progressive decline in renal function of patients with progressive IgAN.

Patients and Methods Patients who had a marked linearity of decline in loss of GFR, assessed by reciprocal serum creatinine plots vs. time for more than one year, were recruited in this study if they were histologically diagnosed as IgAN at this point of declining renal function. Twelve patients (male 5, female 7, mean age 40.5, range 27–54 years) were eligible. All patients except one were previously diagnosed as IgAN by initial renal biopsy. All patients were treated with continuous intravenous infusion of heparin (10,000–15,000 IU/24 h) for eight weeks in hospital, followed by oral administration of warfarin (1–2 mg/day), ACE inhibitors

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and/or angiotensin II receptor blockers (ARBs) and dypiridamole. Eight patients were further given corticosteroid for 2 years because of the presence of acute glomerular lesions such as cellular crescent or angionecrosis.

Results

Clinical and pathological features at baseline were as follows: on average, the percentage of hypertensive patients, urinary protein excretion rate, creatinine clearance and serum creatinine were 33%, 2.4 g/day, 48 ml/min and 1.7 mg/dl, respectively. Mean values for global sclerosis, cellular and fibrocellular crescent and tubulointerstitial injury were 42, 16 and 46%, respectively. During one year before the combined therapy, urinary protein excretion gradually increased up to an average of 2.4 g/day at the time of the institution of the therapy. After the combined therapy, however, urinary protein excretion was significantly and dramatically reduced to an average of 0.2 g/day two months after the therapy. Although creatinine clearance progressively declined to an average of 48 ml/min at baseline and further decreased to 30 ml/min in two months after the therapy, it gradually increased after 4 months and was completely restored to the level at baseline 12 months after the therapy. Figure 1 shows the rate of decline in renal function before and after the combined therapy assessed by reciprocal serum creatinine plots versus time from individual patients. All of the patients were followed up for more than 12 months and the mean follow-up period was 34 months. As shown in the left half of the figure, all patients had a marked linearity of decline in loss of GFR before the combined therapy, suggesting that they would progress to end-stage renal failure within 5 years. Of note, as shown in the right half of the figure, the mean slope of reciprocal serum creatinine plots significantly increased from ⫺0.009 to ⫹0.0002 dl/mg/week after the therapy. The repeat kidney biopsy was performed in 5 patients two years after the institution of the combined therapy. The percentage of cellular/fibrocellular crescent and the degree of mesangial matrix expansion were significantly attenuated, whereas the percentage of global sclerosis and tubulointerstitial injury, which are known to be the most important histologic risk factors for the progression of IgAN, did not increase after the therapy.

Discussion

Our data suggested that combined therapy with heparin/warfarin and RAS inhibitors can improve renal survival of patients with advanced IgAN. Regarding the effects of heparin and warfarin on the kidney, there have been

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2.0

Before

After 1/Cr vs. time (dl/mg/week) Before: ⫺0.009 p⫽0.0022 After: ⫹0.0002

1/Cr (dl/mg)

1.5

1.0

0.5

⫺5 ⫺4 ⫺3

⫺2

⫺1

0

1

2

3

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Fig. 1. Reciprocal serum creatinine plots from 12 patients. The rate of decline in renal function before and after the combined therapy assessed by reciprocal serum creatinine plots versus time from individual patients. All of the patients had a marked linearity of decline in loss of GFR before the combined therapy. Of note, the mean slope of reciprocal serum creatinine plots significantly increased from ⫺0.009 to ⫹0.0002 dl/mg/week after the therapy.

many experimental and clinical reports [3–6]. In cultured mesangial cells, heparin has an antiproliferative effect and suppresses the gene expressions of MMP2 and type IV collagen [7]. In the anti-Thy-1 nephritis model, infusion of heparin significantly reduced expressions of basic FGF and PDGF protein, and expansion of extracellular matrices. Furthermore, it was reported that heparin attenuated spontaneous apoptosis of podocytes in explanted rat glomeruli [8] and prevented glomerular anionic charge reduction in streptozotocin-diabetic rats [9]. The antiproteinuric effect of heparin was demonstrated not only in 5/6 nephrectomized rats [10] but also in proteinuric patients with IDDM [11, 12]. Continuous intravenous infusion of heparin for eight weeks may conflict with recent medical trends to reduce hospitalization periods, and does not seem to be practical. On the other hand, these patients would progress to end-stage renal failure if they receive the conventional therapy. In our previous retrospective study, it was speculated that a marked reduction in proteinuria during 8 weeks’ heparin treatment played an important role in the preservation of renal function. Therefore, we positively selected the long-term infusion of heparin in hospital to improve renal prognosis of those patients in the poor prognostic group. More recently, warfarin has been demonstrated to inhibit Gas 6, a vitamin K-dependent growth factor for mesangial cells, and inhibit mesangial cell proliferation and the gene expression of PDGF-B in anti-Thy-1 nephritis [13, 14]. The anti-albuminuric effect of warfarin was reported in streptozotocin-induced diabetic nephropathy rats [15]. Moreover, the GFR-preserving effect of warfarin was demonstrated in IgAN [16–18].

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These lines of evidence strongly suggest that heparin and warfarin can confer many different renoprotective effects, such as anti-proliferative, antiinflammatory and antiproteinuric effects, through mechanisms independent of their anticoagulant action. A marked and rapid reduction in heavy proteinuria is the most important strategy for the preservation of renal function in our patients with advanced IgAN. This is the reason why we selected the long-term infusion of heparin in hospital as one of the first lines of therapy. From the previous reports, it is plausible to speculate that warfarin confers a GFR-preserving effect for long periods in IgAN. Treatment with warfarin should be one of the promising strategies in the chronic phase of advanced IgAN. In summary, the combined therapy with heparin/warfarin and RAS inhibitors inhibited the progressive decline in renal function of our patients with advanced IgAN, possibly through their marked antiproteinuric and/or anti-inflammatory effects.

References 1 2

3 4 5

6

7 8 9 10

11 12 13 14

Praga M, Gutierrez E, Gonzalez E, et al: Treatment of IgA nephropathy with ACE inhibitors: a randomized and controlled trial. J Am Soc Nephrol 2003;14:1578–1583. Ogura M, Ito Y, Utsunomiya Y, Kawamura T, et al: Combined therapy with steroid, ACE inhibitor and anticoagulant improved the long-term prognosis of advanced IgA nephropathy. J Am Soc Nephrol 2004;15:332A. Sarasin FP, Schifferli JA: Prophylactic oral anticoagulation in nephritic patients with idiopathic membranous nephropathy. Kidney Int 1994;45:578–585. Castellot JJ, Hoover RL, Harper PA, et al: Heparin and glomerular epithelial cell-secreted heparinlike species inhibit mesangial-cell proliferation. Am J Pathol 1985;120:427–435. Floege J, Eng E, Young BA, et al: Heparin suppresses mesangial cell proliferation and matrix expansion in experimental mesangioproliferative glomerulonephritis. Kidney Int 1993;43: 369–380. Burg M, Ostendorf T, Mooney A, et al: Treatment of experimental mesangioproliferative glomerulonephritis with non-anticoagulant heparin: therapeutic efficacy and safety. Lab Invest 1997;76: 505–516. Caenazzo C, Garbisa S, Onisto M, et al: Effect of glucose and heparin on mesangial (IV) COLL and MMP-2/TIMP-2 mRNA expression. Nephrol Dial Transplant 1997;12:443–448. Ishikawa Y, Kitamura M: Inhibition of glomerular cell apoptosis by heparin. Kidney Int 1999;56: 954–963. Gambaro G, Cavazzana AO, Luzi P, et al: Glycosaminoglycans prevent morphological renal alterations and albuminuria in diabetic rats. Kidney Int 1992;42:285–291. Benchetrit S, Mandelbaum A, Bernheim J, et al: Altered vascular reactivity following partial nephrectomy in the rat: a possible mechanism of the blood-pressure-lowering effect of heparin. Nephrol Dial Transplant 1999;14:64–69. Hansen PM, Jensen T, Kofoed-Enevoldsen A, et al: Effect of low-dose heparin on urinary albumin excretion in insulin-dependent diabetes mellitus. Lancet 1995;345:421–422. Van der Pijl JW, van der Woude FJ, Geelhoed-Duijvestijn PH, et al: Danaparoid sodium lowers proteinuria in diabetic nephropathy. J Am Soc Nephrol 1997;8:456–462. Yanagita M, Ishii K, Ozaki H, et al: Mechanism of inhibitory effect of warfarin on mesangial cell proliferation. J Am Soc Nephrol 1999;10:2503–2509. Yanagita M, Arai H, Ishii K, Nakano T, et al: Gas 6 regulates mesangial cell proliferation through Axl in experimental glomerulonephritis. Am J Pathol 2001;158:1423–1432.

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Nagai K, Arai H, Yanagita M, et al: Growth arrest-specific gene 6 is involved in glomerular hypertrophy in the early stage of diabetic nephropathy. J Biol Chem 2003;278:18229–18234. Walker RG, Yu SH, Oewn JE, et al: The treatment of mesangial IgA nephropathy with cyclophosphamide, dipyridamole and warfarin: a two-year prospective trial. Clin Nephrol 1990;34:103–107. Woo KT, Lee GSL, Lau YK, et al: Effects of triple therapy in IgA nephritis: a follow-up study 5 years later. Clin Nephrol 1991;36:60–66. Lee GSL, Choong HL, Chiang GSC, et al: Three-year randomized controlled trial of dipyridamole and low-dose warfarin in patients with IgA nephropathy and renal impairment. Nephrol 1997;3: 117–121.

Takeo Ishii, MD Division of Kidney and Hypertension Department of Medicine Jikei University School of Medicine Tokyo (Japan)

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Downregulation of the ␤1,3Galactosyltransferase Gene in Tonsillar B Lymphocytes and Aberrant Lectin Bindings to Tonsillar IgA as a Pathogenesis of IgA Nephropathy Tatsuyuki Inouea, Hitoshi Sugiyamaa, Yoko Kikumotoa, Naomi Fukuokaa, Yohei Maeshimaa, Hisashi Hattorib, Kunihiro Fukushimab, Kazunori Nishizakib, Yoshiyuki Hikic, Hirofumi Makinoa Departments of aMedicine and Clinical Science and bOtolaryngology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama; cDepartment of Medicine, Fujita Health University School of Medicine, Toyoake, Japan

Abstract IgA is a glycoprotein with multiple O-glycans. Under-O-glycosylation of the hinge in IgA in patients with IgA nephropathy (IgAN) is reported. The development of IgAN is frequently preceded by episodes of upper respiratory tract infections such as tonsillitis. Therefore, the tonsils may be related to the pathogenesis of IgAN. However, the mechanism of underglycosylation in tonsillar IgA has not yet been fully elucidated. Since O-glycans in IgA are produced by glycosyltransferases, we hypothesized that dysregulation of the enzymes is associated with underglycosylation. Copyright © 2007 S. Karger AG, Basel

Extracted tonsils were obtained from patients with IgAN (n ⫽ 11), and from patients with chronic tonsillitis (CT) (n ⫽ 9) and sleep apnea syndrome (SAS) (n ⫽ 5) as controls. Gene expressions of various glycosyltransferases were examined by real-time PCR in tonsillar CD19-positive B cells. Glycosylation levels in tonsillar IgA1 were determined by enzyme-linked lectin binding assay. Gene expression of ␤1,3-galactosyltransferase (␤3GalT), the enzyme responsible for O-galactosylation, was significantly decreased in tonsillar

B cells in IgAN compared with that in SAS (p ⬍ 0.05). VVL (Vicia villosa) and PNA (Peanut agglutinin) bindings to tonsillar IgA1 from IgAN significantly increased compared with those from SAS or CT (p ⬍ 0.05), suggesting the decrease of galactose as well as of sialic acids of IgA1 molecules. The level of renal dysfunction or increase in chronicity of renal biopsy findings was associated with down-regulation of ␤3GalT mRNA in tonsillar B cells of IgAN. This study reports for the first time that downregulation of the ␤3GalT gene in tonsillar B cells may be involved in under-O-glycosylation of IgA1 as a pathogenesis of IgAN.

Background

IgA is a glycoprotein containing multiple O-linked glycans as well as Nlinked carbohydrates [1]. Under-O-glycosylation of the hinge lesion in serum IgA and deposited IgA in glomeruli in patients with IgA nephropathy (IgAN) is reported [2–4]. The clinical development and exacerbation of IgA nephropathy are frequently preceded by episodes of upper respiratory tract infections such as tonsillitis. Therefore, tonsils, which represent the predominant immunocompetent tissue of the upper respiratory tract, may be related to the pathogenesis of IgAN [5]. However, the mechanism of production of underglycosylated IgA in tonsils has not yet been fully elucidated. Since O-glycans in IgA molecules are produced by glycosyltransferases [6], including galactosyltransferases and sialyltransferases, we hypothesized that dysregulation of the glycosyltransferases are associated with aberrant O-glycosylation in tonsillar IgA1 in IgAN.

Materials and Methods Tonsils were obtained from 11 patients with biopsy-proven IgAN. Nine patients with CT and 5 patients with sleep apnea syndrome (SAS) with normal urinalysis were examined. Tonsils were obtained after receiving informed consent, after approval by the Institutional Review Board of Okayama University. Tonsil tissues were cut into small pieces, and single-cell suspensions were obtained by gentle pressure with a glass homogenizer [7]. Mononuclear cells were separated by densitygradient centrifugation on LSM (density 1.077, Capple, Germany). Tonsillar B lymphocytes were isolated using CD19 microbead-associated cell sorting (Milteny Biotech, Germany) according to manufacturer’s protocol [8]. The mRNA expressions of various glycosyltransferases were examined by real-time PCR in tonsillar CD19-positive B cells. Oligonucleotides were used for real-time PCR to

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quantify the amounts of ␤1,3-galactosyltransferase (␤3GalT), core 1 ␤1,3-galactosyltransferasespecific molecular chaperone (Cosmc), UDP-N-acetyl-␣-D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 2 (pp-GalNAc-T2), ␣2,3-Gal-sialyltransferase (ST3Gal) and ␣2,6-GalNAc-sialyltransferase III (ST6GalNAc III). The amount of ␤-actin mRNA was measured as internal control in each sample [9]. A piece of the tonsils (IgAN: n ⫽ 16, CT: n ⫽ 15, SAS: n ⫽ 8) was homogenized, and the supernatant was centrifuged at 40,000 rpm for 70 min. The clear supernatant was used as tonsillar extract [4, 10]. Glycosylation levels in tonsillar IgA1 were determined by enzymelinked lectin binding assay using the following lectins: Jacaline, peanut agglutinin (PNA), Maackia amurensis (MAA), and Vicia villosa (VVL), each of which was bound to alternative forms of O-glycan moieties [11]. We then analyzed the relationships between the dysregulation of glycosyltransferases and clinical parameters or renal histopathology in IgAN.

Result

Gene expression of ␤3GalT, the enzyme responsible for O-galactosylation, was significantly decreased in tonsillar B cells in patients with IgAN compared with those in SAS (p ⬍ 0.05). VVL bindings to IgA1 in tonsillar extracts from IgAN were significantly increased compared with those from SAS (p ⬍ 0.05) or those from CT (p ⬍ 0.05). PNA bindings to IgA1 in tonsillar extracts from IgAN were significantly increased compared with those from SAS (p ⬍ 0.05) or those from CT (p ⬍ 0.05), suggesting the decrease of galactose as well as of sialic acids of IgA1 molecules. There were no significant differences in the of Jacaline and MAA lectin bindings between the three groups. Since we found a significant decrease in the gene expression of ␤3GalT in tonsillar B lymphocytes from patients with IgA nephropathy, we next investigated relationships between the level of ␤3GalT and clinical or pathological parameters. The decrease in ␤3GalT is significantly associated with a decrease in creatinine clearance and also associated with an increase in chronicity index of renal biopsy.

Discussion

Core 1 Gal␤1–3GalNAc␣1–3serine/threonine is the major constituent of O-glycan in IgA1 molecules. ␤3GalT transfers a galactose to a GalNAc residue with a ␤1,3-linkage. Recently, ␤3GalT gene had been cloned and been named as C1GALT1 [12, 13]. IgAN is one of the diseases reported to be related to C1GALTs. Allen et al. found a decreased synthesis activity of ␤1,3GT in

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the peripheral B lymphocyte lysate of IgAN patients [14]. They suspected that the decrease may result from lower gene expression or post-translational modifications. The increased binding of the tonsillar IgA1 to VVL suggested the decrease of the galactose content resulting in the increase of the GalNAc residue in the IgA1 hinge, because VV lectin binds to GalNAc but not to Gal residue. The galactose deficiency had been pointed out in IgAN [2, 15]. The increased binding of VVL to tonsillar IgA1 seems to be inconsistent with the results of PNA lectin. However, the phenomenon could occur because PNA lectin does not recognize sialylated-Gal␤1,3GalNAc, but does recognize the asialo-Gal␤1, 3GalNAc residue [3]. This study reports that in tonsillar B lymphocytes, downregulation of ␤3GalT may be involved in under-O-glycosylation of IgA1 in patients with IgAN and renal dysfunction, or chronicity of renal pathology, and thus may be involved in the pathogenesis of the disease.

References 1 2 3 4 5 6 7

8

9 10 11 12 13

Yoo EM, Morrison SL: IgA: an immune glycoprotein. Clin Immunol 2005;116:3–10. Allen AC, Harper SJ, Feehally J: Galactosylation of N- and O-linked carbohydrate moieties of IgA1 and IgG in IgA nephropathy. Clin Exp Immunol 1995;100:470–474. Hiki Y, Kokubo T, Iwase H, et al: Underglycosylation of IgA1 hinge plays a certain role for its glomerular deposition in IgA nephropathy. J Am Soc Nephrol 1999;10:760–769. Itoh A, Iwase H, Takatani T, et al: Tonsillar IgA1 as a possible source of hypoglycosylated IgA1 in the serum of IgA nephropathy patients. Nephrol Dial Transplant 2003;18:1108–1114. Horie A, Hiki Y, Odani H, et al: IgA1 molecules produced by tonsillar lymphocytes are under-Oglycosylated in IgA nephropathy. Am J Kidney Dis 2003;42:486–496. Ju T, Cummings RD: A unique molecular chaperone Cosmc required for activity of the mammalian core 1 beta 3-galactosyltransferase. Proc Natl Acad Sci USA 2002;99:16613–16618. Takahashi K, Asagoe K, Zaishun J, et al: Heterogeneity of dendritic cells in human superficial lymph node: in vitro maturation of immature dendritic cells into mature or activated interdigitating reticulum cells. Am J Pathol 1998;153:745–755. Yamana J, Yamamura M, Okamoto A, et al: Resistance to IL-10 inhibition of interferon gamma production and expression of suppressor of cytokine signaling 1 in CD4⫹ T cells from patients with rheumatoid arthritis. Arthritis Res Ther 2004;6:R567–R577. Kobayashi M, Sugiyama H, Wang DH, et al: Catalase deficiency renders remnant kidneys more susceptible to oxidant tissue injury and renal fibrosis in mice. Kidney Int 2005;68:1018–1031. Hiki Y, Odani H, Takahashi M, et al: Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy. Kidney Int 2001;59:1077–1085. Xu LX, Zhao MH: Aberrantly glycosylated serum IgA1 are closely associated with pathologic phenotypes of IgA nephropathy. Kidney Int 2005;68:167–172. Ju T, Brewer K, D’Souza A, et al: Cloning and expression of human core 1 beta1,3-galactosyltransferase. J Biol Chem 2002;277:178–186. Kudo T, Iwai T, Kubota T, et al: Molecular cloning and characterization of a novel UDPGal:GalNAc(alpha) peptide beta 1,3-galactosyltransferase (C1Gal-T2), an enzyme synthesizing a core 1 structure of O-glycan. J Biol Chem 2002;277:47724–47731.

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Allen AC, Topham PS, Harper SJ, et al: Leucocyte beta 1,3 galactosyltransferase activity in IgA nephropathy. Nephrol Dial Transplant 1997;12:701–706. Tomana M, Matousovic K, Julian BA, et al: Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG. Kidney Int 1997;52:509–516.

Tatsuyuki Inoue, MD Department of Medicine and Clinical Science Okayama University Graduate School of Medicine Dentistry and Pharmaceutical Sciences 2-5-1, shikata-cho, Okayama (Japan)

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Development of IgA Nephropathy-Like Disease with High Serum IgA Levels and Increased Proportion of Polymeric IgA in ␤-1,4-Galactosyltransferase-Deficient Mice Toshikazu Nishiea, Osamu Miyaishic, Haruhito Azumad, Akihiko Kameyamae, Chie Narusea, Noriyoshi Hashimotoa, Hitoshi Yokoyamab, Hisashi Narimatsue, Takashi Wadab, Masahide Asanoa a

Division of Transgenic Animal Science, Advanced Science Research Center, and Department of Disease Control and Homeostasis, Graduate School of Medical Science and Division of Blood Purification, Kanazawa University, Kanazawa, c Department of Pathology, Aichi Medical University School of Medicine, Nagakute, Aichi, dDepartment of Urology, Osaka Medical College, Takatsuki, Osaka, eGlycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), Umezono, Tsukuba, Ibaraki, Japan b

Abstract The glycosylation of glycoproteins is important for their biological activity, conformation and stability. Recent studies indicate that aberrant glycosylation causes various human disorders. Here we report that mice lacking ␤-1,4-galactosyltransferase-I (␤4GalT-I), which transfers galactose from UDP-Gal to terminal GlcNAc of N- and O-glycans in a ␤-1,4linkage, developed IgA nephropathy (IgAN)-like disease. Urinary albumin levels were significantly increased in the ␤4GalT-I-deficient mice. Hematuria was detected in some of the ␤4GalT-I-deficient mice, suggesting impaired renal function. Furthermore, histological and immunohistochemical examination showed expanded mesangial matrix, IgA deposition with mesangial pattern and electron-dense deposits in the paramesangial regions in the ␤4GalT-Ideficient mice. These results demonstrate that the ␤4GalT-I-deficient mice developed IgANlike disease. Furthermore, high serum IgA levels with increased polymeric forms were detected. In humans, serum IgA derived from patients with IgAN has aberrant ␤3-galactosylation and sialylation on its O-linked glycans of the hinge region. Mouse IgA does not have O-glycans of the hinge region and has several N-glycans. As expected, ␤4-galactosylation on the N-glycans of the serum IgA of the ␤4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. We suggest that aberrant ␤4-galactosylation of serum IgA participates in the

development of IgAN, including deposition of IgA, polymerization of IgA, and glomerular injury after IgA deposition. Copyright © 2007 S. Karger AG, Basel

The glycosylation of glycoproteins is important for their biological activity, conformation, stability, and so on. Recent studies indicate that aberrant glycosylation causes various human disorders, such as metastasis of tumor cells, muscular dystrophy, and dyserythropoietic anemia [1–3]. ␤-1,4-galactosyltransferases (␤4GalTs) transfer galactose (Gal) from UDPGal to terminal GlcNAc of N- and O-glycans in a ␤-1,4-linkage to synthesize the Gal␤1–4GlcNAc structures [4]. Previously, we have generated mice lacking ␤4GalT-I, a major ␤4GalTs among seven family members and expressed in almost all tissues except the brain, to investigate the roles of ␤4-galactosylation of glycoproteins in vivo. The ␤4GalT-I-deficient mice showed semi-lethality before weaning because of growth retardation, and reduced inflammation and delayed skin wound healing because of impaired leukocyte infiltration [5–7]. Here we report that the ␤4GalT-I-deficient mice developed an IgAN-like disease and showed increased polymeric serum IgA. The ␤4GalT-I-deficient mice normally grew after weaning, but began to die from around 10 weeks old. Autopsy indicated that the kidneys were small and pale. We determined the urinary albumin levels and hematuria. The urinary albumin levels in the ␤4GalTI-deficient mice were markedly higher than those in control mice. Hematuria was also detected in some of the ␤4GalT-I-deficient mice. These results demonstrated the impaired renal function in the ␤4GalT-I-deficient mice. Histological examination of kidney sections stained by PAS revealed glomerulomegaly and glomerular sclerosis in the ␤4GalT-I-deficient mice. In addition, Massontrichrome staining suggested the deposition of immune complexes in the mesangial region of the ␤4GalT-I-deficient mice. The ratio of segmental lesions, such as glomerular sclerosis, focal adhesion and small crescent formation, was less than 10% and slightly increased with age in control mice. In contrast, more than half of the glomeruli in the ␤4GalT-I-deficient mice were already affected even at a younger age, and the ratio of global lesions such as global mesangial expansion increased with age. IgA staining of frozen kidney sections revealed the IgA deposition with mesangial pattern in most glomeruli of the ␤4GalT-I-deficient mice. Electron microscopic analysis indicated electron-dense deposits in paramesangial areas in the ␤4GalT-I-deficient mice. These results indicate that the ␤4GalT-I-deficient mice developed IgAN-like glomerular lesions. In IgAN patients, high serum IgA levels are observed [8]. Therefore, we determined serum IgA levels, and demonstrated that serum IgA levels in the ␤4GalT-I-deficient mice were 10-fold higher than those in control mice. To

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Increased serum polymeric IgA

Participation of impaired ␤4-galactosylation of serum IgA molecule?

Glomerular IgA deposits

Glomerular injuries

IgA nephropathy

Fig. 1. Molecular mechanisms of IgAN-like disease development in the ␤4GalT-Ideficient mice. The most probable explanation is that IgA molecules with impaired ␤4galactosylation participate in increased serum polymeric IgA, deposition of IgA, and glomerular injury after IgA deposition. However, we cannot exclude the possibility that abnormality in other glycoproteins causes IgAN-like disease, because many glycoproteins were deficient in ␤4-galactosylation in the ␤4GalT-I-deficient mice.

characterize the size and molecular forms of the serum IgA, we carried out gel filtration assay and Western blot analysis under non-reducing conditions. Gel filtration assay indicated that IgA with higher molecular weight was increased in sera of the ␤4GalT-I-deficient mice. These higher-molecular-weight IgAs seemed to be dimeric and polymeric IgA. These results demonstrated that dimeric and polymeric IgA were markedly increased compared to monomeric IgA in sera of the ␤4GalT-I-deficient mice. In human IgAN, serum IgA1 has aberrant ␤3-galactosylation and sialylation on its O-linked glycans of the hinge region, that is suggested to participate in the pathogenesis of IgAN [9–12]. Since amino acid sequences of the hinge region of mouse IgA are different from those of human IgA, mouse IgA does not have O-glycans. On the other hand, IgA has two N-glycosylation sites both in mouse and human. We therefore examined structures of N-glycans of serum IgA in the ␤4GalT-I-deficient mice by MALDI-TOF MS. As expected, ␤4-galactosylation of N-glycans of serum IgA in the ␤4GalT-I-deficient mice was completely absent. Here we show that the ␤4GalT-I-deficient mice developed an IgAN-like disease with mesangial expansion, mesangial IgA deposition and paramesangial electron-dense deposits. Furthermore, polymeric serum IgA with aberrant ␤4galactosylation was significantly increased in the ␤4GalT-I-deficient mice. Our study demonstrates for the first time that genetic remodeling of glycosylation

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causes IgAN-like disease in mice [13]. We suggest that increased polymeric IgA with aberrant ␤4-galactosylation is the cause of IgAN-like disease in the ␤4GalT-I-deficient mice. Aberrant ␤4-galactosylation of serum IgA may participate in polymerization of IgA, deposition of IgA, and induction of glomerular injury after IgA deposition (fig. 1). Of interest, it has been reported that mutant IgA lacking N-glycosylation sites tends to form polymeric IgA that might occur in the ␤4GalT-I-deficient mice. However, we cannot exclude the possibility that abnormality in other glycoproteins causes IgAN-like disease, because many glycoproteins were deficient in ␤4-galactosylation in the ␤4GalT-I-deficient mice.

References 1 2 3 4 5

6 7 8 9 10 11

12

13

Granovsky M, Fata J, Pawling J, et al: Suppression of tumor growth and metastasis in Mgat5deficient mice. Nat Med 2000;6:306–312. Yoshida A, Kobayashi K, Manya H, et al: Muscular dystrophy and neuronal migration disorder caused by mutations in a glycosyltransferase, POMGnT1. Dev Cell 2001;1:717–724. Chui D, Oh-Eda M, Liao YF, et al: Alpha-mannosidase-II deficiency results in dyserythropoiesis and unveils an alternate pathway in oligosaccharide biosynthesis. Cell 1997;90:157–167. Hennet T: The galactosyltransferase family. Cell Mol Life Sci 2002;59:1081–1095. Asano M, Furukawa K, Kido M, et al: Growth retardation and early death of beta-1,4-galactosyltransferase knockout mice with augmented proliferation and abnormal differentiation of epithelial cells. Embo J 1997;16:1850–1857. Asano M, Nakae S, Kotani N, et al: Impaired selectin-ligand biosynthesis and reduced inflammatory responses in beta-1,4-galactosyltransferase-I-deficient mice. Blood 2003;102:1678–1685. Mori R, Kondo T, Nishie T, et al: Impairment of skin wound healing in beta-1,4-galactosyltransferasedeficient mice with reduced leukocyte recruitment. Am J Pathol 2004;164:1303–1314. Maeda A, Gohda T, Funabiki K, et al: Significance of serum IgA levels and serum IgA/C3 ratio in diagnostic analysis of patients with IgA nephropathy. J Clin Lab Anal 2003;17:73–76. Allen AC, Bailey EM, Barratt J, et al: Analysis of IgA1 O-glycans in IgA nephropathy by fluorophore-assisted carbohydrate electrophoresis. J Am Soc Nephrol 1999;10:1763–1771. Allen AC, Bailey EM, Brenchley PE, et al: Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients. Kidney Int 2001;60:969–973. Hiki Y, Odani H, Takahashi M, Yasuda Y, Nishimoto A, Iwase H, Shinzato T, Kobayashi Y, Maeda K: Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy. Kidney Int 2001;59:1077–1085. Odani H, Hiki Y, Takahashi M, Nishimoto A, Yasuda Y, Iwase H, Shinzato T, Maeda K: Direct evidence for decreased sialylation and galactosylation of human serum IgA1 Fc O-glycosylated hinge peptides in IgA nephropathy by mass spectrometry. Biochem Biophys Res Commun 2000;271: 268–274. Nishie T, Miyaishi O, Azuma H, Kameyama A, Naruse C, Hashimoto N, Yokoyama H, Narimatsu H, Wada T, Asano M: Development of Immunoglobulin A nephropathy-like disease in beta-1, 4-galactosyltransferase-I-deficient mice. Am J Pathol 2007;170:447–456.

Masahide Asano Division of Transgenic Animal Science Advanced Science Research Center Kanazawa University, 13-1 Takara-machi, Kanazawa 920–8640 (Japan) Tel. ⫹81 76 265 2460, Fax ⫹81 76 234 4240, E-Mail [email protected]

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IgA Nephropathy: Characterization of IgG Antibodies Specific for Galactose-Deficient IgA1 Hitoshi Suzukia,b, Zina Moldoveanua, Stacy Halla, Rhubell Browna, Bruce A. Juliana, Robert J. Wyattc, Milan Tomanaa, Yasuhiko Tominob, Jan Novaka, Jiri Mesteckya a

University of Alabama at Birmingham, Birmingham, Ala., USA; bDivision of Nephrology, Department of Internal Medicine, Juntendo University, School of Medicine, Tokyo, Japan; cUniversity of Tennessee Health Sciences Center, Memphis, Tenn., USA

Abstract The circulating immune complexes in IgA nephropathy (IgAN) are composed of galactose (Gal)-deficient IgA1 bound to IgG or IgA1 antibodies specific for hinge-region O-linked glycans of Gal-deficient IgA1. To analyze properties of the anti-glycan antibodies, we determined the binding of serum IgG and IgG secreted by Epstein-Barr virus (EBV)immortalized B cells from patients with biopsy-proven IgAN (n ⫽ 12) and healthy controls (n ⫽ 5) to a panel of antigens coated on ELISA plates. These antigens were: (1) enzymatically desialylated and degalactosylated IgA1 myeloma protein (dd-IgA1), (2) Fab fragment of Gal-deficient IgA1 containing part of the hinge region with O-glycans (Fab-IgA1), (3) synthetic hinge-region peptide linked to bovine albumin (HR-BSA), and (4) synthetic hingeregion glycopeptide with three GalNAc residues linked to BSA (HR-GalNAc-BSA). IgG-secreting EBV-immortalized cell lines were subcloned by limiting dilution. The concentration of total IgG and distribution of IgG subclasses were measured by ELISA. The levels of IgG in sera and supernatants directed against dd-IgA1 and Fab-IgA1 were significantly higher in IgAN patients than in controls (p ⬍ 0.01). IgG from IgAN patients exhibited strong reactivity with HR-GalNAc-BSA, but not with HR-BSA. The IgG-secreting cell lines produced antibodies specific to dd-IgA1; the antigen-specific IgG was most frequently of the IgG2 subclass. In summary, sera and supernatants from IgG-secreting cell lines from patients with IgAN were characterized by high levels of IgG antibodies with specificity to the Gal-deficient O-linked glycans of IgA1. The immortalized cell lines will provide a stable and convenient source of IgG for molecular studies of antibodies specific to the aberrant O-glycans in IgA1. Copyright © 2007 S. Karger AG, Basel

IgA nephropathy (IgAN) is characterized by IgA1-containing immune complexes in glomerular deposits and in the circulation [1, 2]. IgA1 in the circulation and glomerular deposits of IgAN patients is aberrantly glycosylated; the hinge-region O-linked glycans are Gal-deficient [3]. This aberrantly glycosylated IgA1 is recognized by N-acetylgalatosamine (GalNAc)-specific lectins as well as by IgG glycan-specific antibodies [4–6]. Although the origin of the aberrantly glycosylated IgA1 is unknown, the glycosylation aberrancy has been related to an altered expression of specific glycosyltransferases in circulating B lymphocytes [7, 8]. Our studies have confirmed these findings: clones of IgA1-producing Epstein-Barr virus (EBV)-immortalized B cell lines secreted Gal-deficient IgA1, as evidenced by its high reactivity with GalNAc-specific lectin isolated from Helix aspersa (HAA) [9]. Serum levels of Gal-deficient IgA1 of IgAN patients correlated with high HAA reactivity of IgA1 secreted by the corresponding cell lines. Western blotting under non-reducing conditions showed that IgA1 secreted by cell lines from IgAN patients is predominantly polymeric. In the present study, we subcloned IgG-producing cell lines from IgAN patients and healthy controls to generate stable clones producing antigen-specific (to Galdeficient IgA1) IgG antibodies and to characterize these antibodies at the molecular level. Together, our results showed that EBV-immortalized B-cell lines from IgAN patients and healthy controls offer a novel tool for studies of IgA1 glycosylation and characterization of anti-IgA1 antibodies.

Subcloning IgG-Secreting B-Cell Lines

Mononuclear cells from patients with IgAN and healthy controls were isolated from heparinized peripheral blood and immortalized by EBV transformation. The immortalized B cells from IgAN patients (n ⫽ 12) and healthy controls (n ⫽ 5) were further subcloned by limiting dilution to obtain IgGsecreting cell lines.

Glycan-Specific IgG Antibodies from IgAN Patients Recognize GalNAc on the IgA1 Hinge Region

To analyze the properties of the anti-glycan antibodies, we determined the binding of IgG from serum and of IgG secreted by EBV-immortalized B cells of 12 IgAN patients and five healthy controls to a panel of antigens coated on ELISA plates. These antigens were: (1) enzymatically desialylated and degalactosylated IgA1 (dd-IgA1), (2) Fab fragment of Gal-deficient IgA1 containing part of the hinge region with O-glycans (Fab-IgA1), (3) synthetic hinge-region

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dd-IgA1

2.8

2.5

2.4 2.0 1.6

1.0

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Fig. 1. a The levels of antigen-specific IgG from supernatants of cell lines was measured by capture ELISA, and expressed as optical density (OD) at 490 nm. Levels of IgG directed against dd-IgA1 and Fab-IgA1 were significantly higher in IgAN patients than in controls (*p ⬍ 0.01). b Serum levels of IgG directed against dd-IgA1 and Fab-IgA1 were significantly higher in IgAN patients than in controls (p ⬍ 0.01; Data are shown as individual values and mean ⫾ SD; *p ⬍ 0.01). c Western blot of Gal-deficient IgA1 (Mce) used as antigen demonstrated binding of IgG secreted by B-cell line from an IgAN patient, but not from a healthy control. Anti-human IgA (heavy-chain specific) western blot was used as positive control (far right; anti-IgA). Mce ⫽ Gal-deficient IgA1 myeloma protein.

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peptide linked to bovine albumin (HR-BSA), and (4) synthetic hinge-region glycopeptide with three GalNAc residues linked to BSA (HR-GalNAc-BSA). The amount of total IgG was normalized between samples. The levels of IgG secreted by B-cell lines directed against dd-IgA1 and Fab-IgA1 were significantly higher in IgAN patients than in the controls (p ⬍ 0.01) (fig. 1a). IgG from sera of IgAN patients had the same tendency to bind with dd-IgA1 and Fab-IgA1 (fig. 1b). IgG from IgAN patients exhibited reactivity with HRGalNAc-BSA, but not with HR-BSA. For confirmation of the ELISA results by Western blotting, reduced Gal-deficient IgA1 protein was used as the antigen after SDS-PAGE and blotting. IgG secreted by B-cell lines was the antibody source, and its binding to IgA component chains on the blot was detected with human IgG-specific antibody. The IgG from supernatants of cell lines from an IgAN patient bound to the heavy chain of Gal-deficient IgA1 (fig. 1c). To determine the dominant subclass of the antigen-specific IgG, dd-IgA1 was coated on ELISA plate and the binding of IgG subclasses was determined with HRP-conjugated mouse anti-human IgG1, IgG2, IgG3, and IgG4 antibodies. The antigen-specific IgG was predominantly of the IgG2 subclass, followed by IgG3 and IgG1. In summary, sera and supernatants from immortalized IgG-secreting cell lines from patients with IgAN contained higher levels of IgG antibodies with specificity for Gal-deficient O-linked glycans of IgA1 than those from healthy controls. IgG, mostly of the IgG2 subclass, was specific for GalNAc in the hinge region of IgA1. IgA1-secreting cell lines from IgAN patients produced aberrantly glycosylated IgA1. The immortalized cell lines from IgAN patients will provide a stable and convenient tool to facilitate studies of the molecular properties of IgG and IgA1 immunoglobulins in IgAN. Acknowledgments Supported in part by grants from the National Institutes of Health, PO1 DK61525 and DK64400, by the General Clinical Research Centers of the University of Alabama at Birmingham, M01 RR00032, and the University of Tennessee Health Sciences Center, M01 RR00211 (USA).

References 1 2 3

Novak J, Tomana M, Matousovic K, et al: IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells. Kidney Int 2005;67:504–513. Novak J, Vu HL, Novak L, et al: Interactions of human mesangial cells with IgA and IgA-containing immune complexes. Kidney Int 2002;62:465–475. Coppo R, Amore A: Aberrant glycosylation in IgA nephropathy (IgAN). Kidney Int 2004;65: 1544–1547.

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4

5 6 7 8 9

Schena FP, Pastore A, Ludovico N, et al: Increased serum levels of IgA1-IgG immune complexes and anti-F(ab⬘)2 antibodies in patients with primary IgA nephropathy. Clin Exp Immunol 1989;77: 15–20. Tomana M, Matousovic K, Julian BA, et al: Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG. Kidney Int 1997;52:509–516. Tomana M, Novak J, Julian BA, et al: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies. J Clin Invest 1999;104:73–81. Allen AC, Topham PS, Harper SJ, Feehally J: Leucocyte ␤1,3 galactosyltransferase activity in IgA nephropathy. Nephrol Dial Transplant 1997;12:701–706. Qin W, Zhou Q, Yang LC, et al: Peripheral B lymphocyte ␤1,3-galactosyltransferase and chaperone expression in immunoglobulin A nephropathy. J Intern Med 2005;258:467–477. Suzuki H, Moldoveanu Z, Vu HL, et al: Epstein-Barr Virus-immortalized B cells from patients with IgA nephropathy secrete IgA1 with galactose-deficient O-linked glycans. 11th International Symposium on IgA Nephropathy, abstract B-P-05, Tokyo, Japan, October 5–7, 2006.

Hitoshi Suzuki, MD, PhD University of Alabama at Birmingham, Department of Microbiology BBRB 754, 845 19th Street South Birmingham, AL 35294–2170 (USA) Tel. ⫹1 205 934 2233, Fax ⫹1 205 934 3894, E-Mail [email protected]

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IgA Nephropathy and Henoch-Schoenlein Purpura Nephritis: Aberrant Glycosylation of IgA1, Formation of IgA1-Containing Immune Complexes, and Activation of Mesangial Cells Jan Novaka, Zina Moldoveanua, Matthew B. Renfrowa, Takeshi Yanagiharaa, Hitoshi Suzukia, Milan Raskaa, Stacy Halla, Rhubell Browna, Wen-Qiang Huanga, Alice Goepferta, Mogens Kilianc, Knud Poulsenc, Milan Tomanaa, Robert J. Wyattb, Bruce A. Juliana, Jiri Mesteckya a

University of Alabama at Birmingham, Birmingham, Ala., University of Tennessee Health Sciences Center, Memphis, Tenn., USA; c Aarhus University, Aarhus, Denmark b

Abstract IgA1 in the circulation and glomerular deposits of patients with IgA nephropathy (IgAN) is aberrantly glycosylated; the hinge-region O-linked glycans are galactose-deficient. The circulating IgA1 of patients with Henoch-Schoenlein purpura nephritis (HSPN) has a similar defect. This aberrancy exposes N-acetylgalactosamine-containing neoepitopes recognized by naturally occurring IgG or IgA1 antibodies resulting in formation of immune complexes. IgA1 contains up to six O-glycosylation sites per heavy chain; it is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. We sought to define the aberrant glycosylation of a galactose-deficient IgA1 myeloma protein and analyze the formation of the immune complexes and their biological activities. Supplementation of serum or cord-blood serum with this IgA1 protein resulted in formation of new IgA1 complexes. These complexes stimulated proliferation of cultured human mesangial cells, as did the naturally-occurring IgA1-containing complexes from sera of patients with IgAN and HSPN. Uncomplexed IgA1 did not affect cellular proliferation. Using specific proteases, lectin Western blots, and mass spectrometry, we determined the O-glycosylation sites in the hinge region of the IgA1 myeloma protein and IgA1 proteins from sera of IgAN patients. The IgA1 myeloma protein had galactose-deficient sites at residues 228 and/or 230 and 232. These sites reacted with IgG specific to galactose-deficient IgA1. IgA1 from the IgAN patients had galactose-deficient O-glycans at the same residues. In summary, we identified the neoepitopes on IgA1 responsible for formation of the pathogenic immune complexes. These studies may lead to development of noninvasive diagnostic assays and future disease-specific therapy. Copyright © 2007 S. Karger AG, Basel

IgAN is characterized by mesangial immune deposits, likely derived from circulating immune complexes containing aberrantly glycosylated IgA1 [1–3]. The circulating IgA1 of patients with HSPN has a similar defect. These IgA1 molecules have incompletely galactosylated O-linked glycans in the hinge region that are recognized by anti-glycan IgG and IgA1 antibodies [4, 5]. The resultant complexes deposit in the mesangium and induce mesangial cellular proliferation and matrix expansion. Elucidation of the mechanisms of abnormal IgA1 glycosylation and immune complex formation is therefore important for understanding the disease pathogenesis. IgA1 hinge region has up to six O-linked glycans of variable composition; the prevailing forms include galactose (Gal)-N-acetylgalactosamine (GalNAc) disaccharide without sialic acid, or in mono- or di-sialylated form [6, 7]. A variant with terminal GalNAc or sialylated GalNAc is rare in normal serum IgA1 [6], but more common in the IgA1 of IgAN patients (for review, see [8, 9]). It is not known whether the Gal deficiency in IgAN and HSPN occurs randomly or preferentially at specific sites. To address this question, we used a naturally Gal-deficient IgA1 myeloma protein (Mce) digested with five bacterial IgA proteases (fig. 1a). To identify sites with terminal GalNAc and GalNAc⫹Gal residues, the resultant fragments were desialylated, separated by SDS-PAGE, blotted, and probed with O-glycan-specific probes, such as lectin from Helix aspersa (HAA) specific for GalNAc (fig. 1b). We found GalNAc- and GalNAc⫹ Gal-containing glycans between Thr228 and Thr236 and only GalNAc⫹Gal before Thr228 (fig. 1a). To further analyze this heterogeneity, hinge-region glycopeptides were generated by proteolytic digestion of the IgA1 protein with two proteases. Monosaccharide compositional analysis of the resultant preparation revealed GalNAc, Gal, and sialic acid; matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry indicated 3–6 O-glycans and a variable number of Gal residues. Mass spectra of desialylated hinge-region glycopeptides were acquired with a home-built 9.4 Tesla Fourier transform-ion cyclotron resonance (FT-ICR) mass spectrometer equipped with an external microelectrospray ion source [10] or with a 7 Tesla LTQ FT-ICR mass spectrometer (Thermo Finnigan). The spectra of the IgA1 (Mce) hinge region revealed a series of ions separated by m/z values corresponding to the presence of GalNAcand Gal⫹GalNAc-containing glycopeptide species. Hinge-region glycopeptides were fragmented by activated ion-electron capture dissociation (AI-ECD) without the loss of labile post-translational modifications. AI-ECD FT-ICR MS/MS spectra of the different glycosylated variants of the hinge region localized the sites of glycosylation and the sites deficient in Gal (fig. 1a). The IgA1 myeloma protein had Gal-deficient sites at residues 228 and/or 230 and 232. These three sites reacted with IgG specific to Gal-deficient IgA1. Additional analyses with several serum IgA1 proteins from IgAN patients indicated that the major

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Fd

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Fig. 1. a Hinge region of IgA1 and sites cleaved by IgA proteases with glycosylation sites determined by Western blotting and mass spectrometry in desialylated IgA1 myeloma preotein (Mce). Squares show N-acetylgalactosamine and circles show galactose. Saccharides present in only a fraction of IgA1 molecules are in brackets. b Western blot of IgA1 (Mce) digested by IgA proteases listed under numbers 1–5 in a. Arrows show positions of Fc and Fd fragments and the dashed rectangles show the positions of the Fc and Fd fragments not reacting with HAA lectin. c Proliferation of human mesangial cells with fractions of cord-blood serum supplemented with Gal-deficient IgA1 (circles) and IgA1 concentration in the same fractions (triangles).

Gal-deficient site(s) are at Thr228 and/or Ser230. This Gal-deficient IgA1 is also produced by Epstein-Barr virus-immortalized peripheral blood B cells from patients with IgAN (H. Suzuki et al., this volume) and by Dakiki cell line. This defect is apparently limited to only IgA1-producing B cell lineages, because IgD in the patients’ circulation has normal O-glycans [11]. To evaluate the biological activity of various forms of IgA1, we developed a model with cultured human mesangial cells. First, we assessed binding of IgA1, Gal-deficient IgA1, and IgA1 in immune complexes to these cells. Gal-deficient IgA1 bound better than did normally glycosylated IgA1. Furthermore, IgA1-containing immune complexes from patients with IgAN bound with higher affinity to mesangial cells than uncomplexed IgA1 [12]. The

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binding was mediated by a receptor different from CD89, asialoglycoprotein receptor, and polymeric immunoglobulin receptor [12–14]. Transferrin receptor (CD71) has been identified as a mesangial receptor binding polymeric IgA1 and complexes containing Gal-deficient IgA1 [15, 16]. To study the activation of cultured human mesangial cells by uncomplexed and complexed IgA1, we measured changes in cellular proliferation after stimulation of serum-starved cells. We used naturally occurring IgA1 complexes from sera of IgAN patients and healthy controls isolated by size-exclusion chromatography on a Superose 6 column. These IgA1-containing complexes stimulated mesangial cell proliferation [17–19]. Furthermore, we developed a new protocol to generate IgA1-complexes with Gal-deficient IgA1 in vitro. We used sera of IgAN patients and healthy controls and supplemented them with Gal-deficient IgA1; the resultant complexes stimulated proliferation of cultured human mesangial cells [19]. In contrast, uncomplexed IgA1 or IgA1-depleted fractions did not change cellular proliferation. We refined the process for formation of IgG-Gal-deficient IgA1 complexes by using cord-blood serum that contains maternal IgG with anti-IgA1 glycan specificity, but only trace amounts of IgA or IgM. Cord-blood serum, before or after supplementation with Gal-deficient IgA1, was fractionated by size-exclusion chromatography on a Superose 6 column. Immune complexes consisting of IgA and IgG were formed and detected by cross-capture ELISA; their biological activity was determined as cellular proliferation after adding the serum fractions to cultures of human mesangial cells. Cord-blood serum supplemented with Gal-deficient IgA1 contained high-molecular-weight complexes (⬎700 kDa) that stimulated cellular proliferation; these fractions contained IgA-IgG complexes that were absent from unsupplemented serum (fig. 1c). Cord-blood serum thus offers an excellent resource for the analysis of IgG binding to aberrantly glycosylated IgA1 because other immunoglobulin isotypes cannot participate in the formation of the complexes. In summary, we developed techniques that allow identification of the sites of attachment of the aberrant IgA1 O-glycans and studied mechanisms of immune complex formation and activation of human mesangial cells. These approaches will be useful for analysis of the role of IgA1 in the pathogenesis of IgAN.

Acknowledgments Supported in part by grants from the National Institutes of Health, PO1 DK61525 and DK64400 by the General Clinical Research Centers of the University of Alabama at Birmingham, M01 RR00032, and the University of Tennessee Health Sciences Center, M01 RR00211 (USA).

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References 1 2 3

4 5 6

7 8 9 10

11 12 13 14 15 16

17 18 19

Allen AC, Bailey EM, Brenchley PEC, et al: Mesangial IgA1 in IgA nephropathy exhibits aberrant O-glycosylation: observations in three patients. Kidney Int 2001;60:969–973. Hiki Y, Odani H, Takahashi M, et al: Mass spectrometry proves under-O-glycosylation of glomerular IgA1 in IgA nephropathy. Kidney Int 2001;59:1077–1085. Mestecky J, Tomana M, Crowley-Nowick PA, et al: Defective galactosylation and clearance of IgA1 molecules as a possible etiopathogenic factor in IgA nephropathy. Contrib Nephrol 1993;104: 172–182. Tomana M, Matousovic K, Julian BA, et al: Galactose-deficient IgA1 in sera of IgA nephropathy patients is present in complexes with IgG. Kidney Int 1997;52:509–516. Tomana M, Novak J, Julian BA, et al: Circulating immune complexes in IgA nephropathy consist of IgA1 with galactose-deficient hinge region and antiglycan antibodies. J Clin Invest 1999;104:73–81. Mattu TS, Pleass RJ, Willis AC, et al: The glycosylation and structure of human serum IgA1, Fab, and Fc regions and the role of N-glycosylation on Fc␣ receptor interactions. J Biol Chem 1998;273: 2260–2272. Novak J, Tomana M, Kilian M, et al: Heterogeneity of O-glycosylation in the hinge region of human IgA1. Mol Immunol 2000;37:1047–1056. Barratt J, Feehally J: IgA nephropathy. J Am Soc Nephrol 2005;16:2988–2097. Julian BA, Novak J: IgA nephropathy: an update. Curr Opin Nephrol Hypertens 2004;13:171–179. Renfrow MB, Cooper HJ, Tomana M, et al: Determination of aberrant O-glycosylation in the IgA1 hinge region by electron capture dissociation Fourier transform-ion cyclotron resonance mass spectrometry. J Biol Chem 2005;280:19136–19145. Smith AC, de Wolff JF, Molyneux K, et al: O-Glycosylation of serum IgD in IgA nephropathy. J Am Soc Nephrol 2006;17:1192–1199. Novak J, Vu HL, Novak L, et al: Interactions of human mesangial cells with IgA and IgAcontaining circulating immune complexes. Kidney Int 2002;62:465–475. Leung JCK, Tsang AWL, Chan LYY, et al: Size-dependent binding of IgA to HepG2, U937, and human mesangial cells. J Lab Clin Med 2002;140:398–406. Leung JCK, Tsang AWL, Chan DTM, et al: Absence of CD89, polymeric immunoglobulin receptor, and asialoglycoprotein receptor on human mesangial cells. J Am Soc Nephrol 2000;11:241–249. Moura IC, Arcos-Fajardo M, Sadaka C, et al: Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy. J Am Soc Nephrol 2004;15:622–634. Moura IC, Centelles MN, Arcos-Fajardo M, et al: Identification of the transferrin receptor as a novel immunoglobulin (Ig)A1 receptor and its enhanced expression on mesangial cells in IgA nephropathy. J Exp Med 2001;194:417–425. Amore A, Cirina P, Conti G, et al: Glycosylation of circulating IgA in patients with IgA nephropathy modulates proliferation and apoptosis of mesangial cells. J Am Soc Nephrol 2001;12:1862–1871. Coppo R, Amore A: Aberrant glycosylation in IgA nephropathy (IgAN). Kidney Int 2004;65: 1544–1547. Novak J, Tomana M, Matousovic K, et al: IgA1-containing immune complexes in IgA nephropathy differentially affect proliferation of mesangial cells. Kidney Int 2005;67:504–513.

Jan Novak University of Alabama at Birmingham, Department of Microbiology 845 19th Street S, BBRB 734 (Box 1) Birmingham, AL 35294 (USA) Tel. ⫹1 205 934 4480, Fax ⫹1 205 934 3894, E-Mail [email protected]

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Patients with IgA Nephropathy Respond Strongly Through Production of IgA with Low Avidity Against Staphylococcus aureus Yoshio Shimizua, Masanori Sekia, Shuzo Kanekoa, Masahiro Hagiwaraa, Keigyo Yoha, Kunihiro Yamagataa, Akio Koyamab a Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba; bIbaraki Prefectural University of Health Sciences, Ami, Japan

Abstract Background: Post-methicillin-resistant Staphylococcus aureus infection glomerulonephritis (post-MRSA infection GN) is seen after MRSA infection and is a similar disorder to IgA nephropathy (IgAN). We have found that immune complexes composed of staphylococcal cell membrane antigen and IgA are deposited in the glomerular mesangial areas in patients with post-MRSA infection GN and in more than 60% of patients with IgAN. Aim: To examine the characteristics of serum IgA obtained from patients with IgAN. Patients and Methods: Experiment 1. Serum samples were obtained from patients with IgAN (n ⫽ 16) and post-MRSA infection GN (n ⫽ 19), and from healthy donors (n ⫽ 13). Serum IgA and IgG titers against Staphylococcus aureus (S. aureus) were measured using an ELISA. Experiment 2. Relative antibody avidities of serum IgA and IgG against S. aureus from patients and healthy donors were determined in a dissociation assay using an ELISA. Results: Experiment 1. IgA class titers of anti-S. aureus antibodies in patients with IgAN and post-MRSA infection GN were significantly higher than in healthy controls, and IgG class titers of anti-S. aureus antibodies in patients with post-MRSA infection GN were significantly higher than those in IgAN patients and healthy donors. A significant correlation between IgG and IgA titers was found across all cases, and this was particularly evident in patients with IgAN. Experiment 2. No significant correlation was observed between avidity and titer of IgA class antibodies. The avidity of anti-S. aureus IgG did not differ significantly between IgAN patients, post-MRSA infection GN patients, and healthy donors, but the avidity of anti-S. aureus IgA from patients with IgAN was significantly lower than that from the other groups. There was a significant correlation between antibody titer and antibody avidity for the IgG class in patients with post-MRSA GN, but no significant correlation was observed for the IgA class. Conclusion: These results suggest that patients with IgAN have a strong response through production of IgA with low avidity against S. aureus. Copyright © 2007 S. Karger AG, Basel

Introduction

Although a large number of studies have examined the relationship between infectious diseases and glomerulonephritis (GN), definite antigens have yet to be found [3, 4]. IgA nephropathy (IgAN) is considered to be an infectionrelated GN because urinary findings worsen after upper respiratory tract infection. In 1995, we reported that rapidly progressive GN developed among patients infected by methicillin-resistant Staphylococcus aureus (MRSA) [5]. These patients showed a rapid loss in renal function with massive proteinuria and hematuria, and mesangial proliferative GN with IgA, IgG, and C3 deposition was observed in biopsy specimens. We designated this GN as post-MRSA infection GN. Post-MRSA infection GN serves as an appropriate model of bacterial infection-related IgA-type GN owing to glomerular IgA deposition, and follows an explicit clinical course. Although it has been suggested that hypercytokinemia and polyclonal immunoglobulin activation by superantigens derived from S. aureus play an important role in the pathogenesis of post-MRSA infection GN [5], we found a 35 kDa staphylococcal cell-membrane antigen in the glomeruli of the patients with post-MRSA infection GN [1]. Moreover, we also found this antigen in the glomeruli of ⬎60% patients with IgAN [1, 2]. We identified this staphylococcal cell-membrane antigen (GenBank and DDBJ: accession number; BAB 41819.1) by library screening with monoclonal anti-S. aureus antibody S1D6 [1]. Therefore, we speculated that this staphylococcal cell-membrane antigen was a possible antigen for IgAN. S. aureus is a common bacterium that is present in all humans. Hence, we developed a hypothesis that patients with IgAN show a specific response against S. aureus and aimed to characterize the serum IgA against S. aureus in patients with IgAN to prove our hypothesis.

Patients and Methods Sixteen patients with IgAN, 19 patients with post-MRSA GN, and 13 healthy donors were included in the study. First, the serum IgA and IgG titers against S. aureus were measured in IgA-type GN patients, using an ELISA. Crude staphylococcal cell-membrane antigens were prepared for ELISA by adsorption of protein A with human IgG-coated Sepharose 4B, as reported previously [6], and fixed to ELISA plates. Titers of anti-S. aureus IgA or IgG were measured using mouse anti-human IgA or IgG as the primary antibody and horseradish peroxidase-conjugated rabbit anti-mouse IgG as the second antibody. Next, we examined the avidity of IgA and IgG among the patients. The bound antibodies can be dissociated by sodium thiocyanate, and we measured the concentration of sodium thiocyanate that inhibited 50% of antibody binding, the so-called ID50 [7]. The resulting

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data were subjected to linear regression analysis, and the ID50 is referred to as the relative antibody avidity.

Results

IgG and IgA Titers Statistical analysis proved that patients with post-MRSA infection GN had higher titers than those with IgAN and healthy controls. The IgA titers were statistically different between the three groups. The highest titers were seen in patients with post-MRSA infection GN, and the titers for IgAN patients were higher than in healthy controls. The correlation coefficients between serum IgG and IgA titers were 0.427 for all patients and 0.425 for IgAN patients. These data suggested a moderate correlation between the IgG and IgA titers for all patients and for IgAN patients. Avidity of IgA and IgG There were no statistical differences in IgG avidity between the three groups. However, IgAN patients showed significantly lower IgA avidity than healthy controls, and also showed a tendency to lower IgA avidity compared to patients with post-MRSA infection GN (fig. 1). Patients with post-MRSA infection GN showed a strong positive correlation between antibody avidity and IgG titer. A similar analysis was performed for antibody avidity and IgA titer, but no significant correlation was found in any of the three groups. Moreover, no significant correlation between IgA and IgG antibody avidity was observed in any of the groups.

Discussion

Patients with post-MRSA infection GN produce more IgG than IgAN patients and healthy controls. The IgA titers were highest in patients with postMRSA infection GN, followed by those with IgAN. These two groups had IgA titers that were statistically higher than the IgA titers of healthy controls. A moderate correlation between IgA and IgG titers was observed for all patients and IgAN patients. The antibody avidity for IgG did not differ significantly between the three groups. However, patients with IgAN showed significantly lower IgA avidity than healthy controls. IgAN patients also showed a tendency to lower IgA avidity than that in patients with post-MRSA GN. For IgG, a strong correlation between antibody avidity and titer was observed in patients

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M 2.8 2.6 2.4

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0.0190*

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0.0641

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0.4696

Fig. 1. The avidity of anti-S. aureus IgA in patients with IgAN and post-MRSA infection GN and healthy donors. IgAN patients revealed significantly lower IgA avidity than healthy controls (*p ⫽ 0.0190), and showed a tendency to lower IgA avidity than patients with post-MRSA infection GN (p ⫽ 0.0641).

with post-MRSA infection GN, whereas IgA did not show such a correlation. Collectively, these results lead us to conclude that patients with IgAN respond strongly through production of IgA with low avidity against S. aureus. This conclusion leads to a new hypothesis, in which we propose that under conditions of an excess of antigens and antibodies with low avidity, small immune complexes are formed due to the low efficiency of antibody-antigen precipitation [8, 9]. These complexes then tend to be deposited in the glomeruli.

Acknowledgments This work was supported by research grants from the Japanese Ministry of Education, Culture, Sports, Science, and Technology, and from the University of Tsukuba. We are thankful to the organizing committee of the 11th International Symposium on IgA nephropathy and Professor Yasuhiko Tomino for giving us the opportunity to attend the symposium and to present our work.

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References 1 2 3 4 5 6

7 8

9

Koyama A, Sharmin S, Sakurai H, et al: Staphylococcus aureus cell envelope antigen is a new candidate for the induction of IgA nephropathy. Kidney Int 2004;66:121–132. Kai H, Shimizu Y, Hagiwara M, et al: Post-MRSA infection glomerulonephritis with marked Staphylococcus aureus cell envelope antigen deposition in glomeruli. J Nephrol 2006;19:215–219. Suzuki S, Nakatomi Y, Sato H, et al: Haemophilus parainfluenzae antigen and antibody in renal biopsy samples and serum of patients with IgA nephropathy. Lancet 1994;343:12–16. Iwama H, Horikoshi S, Shirato I, et al: Epstein-Barr virus detection in kidney biopsy specimens correlates with glomerular mesangial injury. Am J Kidney Dis 1998;32:785–793. Koyama A, Kobayashi M, Yamaguchi N, et al: Glomerulonephritis associated with MRSA infection: a possible role of bacterial superantigen. Kidney Int 1995;47:207–216. Arakawa Y, Shimizu Y, Sakurai H, et al: Polyclonal activation of IgA subclass against Staphylococcus aureus cell membrane antigen in post-methicillin-resistant S. aureus infection glomerulonephritis. Nephrol Dial Transplant 2006;21:1448–1449. Pullen GR, Fitzgerald MG, Hosking CS: Antibody avidity determination by ELISA using thiocyanate elution. J Immunol Methods 1986;86:83–87. Germuth FG Jr, Rodriguez E, Lorelle CA, et al: Passive immune complex glomerulonephritis in mice: models for various lesions found in human disease. II. Low avidity complexes and diffuse proliferative glomerulonephritis with subepithelial deposits. Lab Invest 1979;41:366–371. Koyama A, Inage H, Kobayashi M, et al: Role of antigenic charge and antibody avidity on the glomerular immune complex localization in serum sickness of mice. Clin Exp Immunol 1986;64: 606–614.

Yoshio Shimizu, MD, PhD Graduate School of Comprehensive Human Sciences University of Tsukuba, 1-1-1 Tennodai, Tsukuba Ibaraki 305–8575 (Japan) Tel. ⫹81 298 53 3202, Fax ⫹81 298 53 3202, E-Mail [email protected]

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Transferrin Receptor Engagement by Polymeric IgA1 Induces Receptor Expression and Mesangial Cell Proliferation: Role in IgA Nephropathy Houda Tamouza, Florence Vende, Meetu Tiwari, Michelle Arcos-Fajardo, François Vrtovsnik, Marc Benhamou, Renato C. Monteiro, Ivan C. Moura INSERM U699, Paris, and University of Paris 7, Denis Diderot, Paris, France

Abstract IgA nephropathy (IgAN) is characterized by IgA immune complex-mediated mesangial cell proliferation. We have previously identified the transferrin receptor (TfR) as an IgA1 receptor and found that, in kidney biopsies of patients with IgAN, TfR is overexpressed and co-localized with IgA1 mesangial deposits. We also showed that IgA1 binding to TfR was strikingly increased when IgA1 was hypogalactosylated and of high molecular weight, both features found in IgA from IgAN patients. More recently, we showed that purified polymeric IgA1 (pIgA1) is a major inducer of TfR expression (3-fold increase) in quiescent human mesangial cells (HMC). In addition, sera from IgAN patients upregulate TfR expression in cultured HMC in an IgA-dependent manner. IgA1-induced HMC proliferation is dependent on TfR engagement and can be inhibited by both TfR1 and TfR2 ectodomains as well as by the anti-TfR mAb A24. Finally, activation of mesangial cells through pIgA1 binding to TfR induced secretion of IL-6 and TGF-␤ from the cells, that could be involved, respectively, in the inflammatory and pro-fibrogenic events observed in IgAN. We propose that deposited pIgA1 or IgA immune complexes could initiate an auto-amplification process involving hyper-expression of TfR allowing increased IgA1 mesangial deposition. Altogether, these data unveil a functional cooperation between pIgA1 and TfR for IgA1 deposition and HMC proliferation, features which are commonly implicated in the chronic mesangial injuries observed in IgAN. Copyright © 2007 S. Karger AG, Basel

Several groups have proposed the existence of a mesangial IgA receptor in IgA nephropathy (IgAN) [1]. This was initially suggested by studies showing that the binding of IgA to human mesangial cells is dose-dependent. The interaction of IgA with its receptor appeared to be intermediary and the constant of

association was calculated by Scatchard analysis to be around 10–8 M [2]. The interaction of IgA with IgA receptor leads to functional consequences such as cell activation and mesangial cell proliferation. This was determined following several criteria such as the induction of protein-tyrosine phosphorylation, cytokine secretion and synthesis of extra-cellular matrix components [1, 3]. The IgA receptor family is composed of 6 members. It includes Fc␣RI (CD89), polymeric IgA receptor (pIgR), Fc␣/␮ receptor, asialoglycoprotein receptor (ASGP-R), ␤1–4 galactosyltransferase (␤1–4 GT) and transferrin receptor (TfR) [4]. Among all these members, the only one described so far that binds exclusively IgA is CD89. All other IgA receptors can bind other immunoglobulins (pIgR and Fc␣/␮ receptor) or other non-immunoglobulinrelated ligands. ASGP-R, for example, can bind asialoglycoproteins, and ␤1–4 GT binds IgA and to a lesser extent IgG, IgM and transferrin. We have previously demonstrated that TfR acts as a mesangial IgA receptor [5]. We have also shown that IgA1-TfR interaction is dependent on glycosylation and multimerization of IgA, and that TfR is highly expressed in the mesangium of IgAN patients and co-localizes with IgA1 deposits [1, 6, 7]. Recent data from our group show that polymeric IgA is also functionally associated with TfR [8]. TfR expression in mesangial cells can be induced by polymeric IgA but not by IgG. This induction of TfR expression is dose- and time-dependent, beginning from 2 hours of incubation of quiescent mesangial cells with IgA. TfR expression induced by polymeric IgA occurs at both transcriptional and translational levels. More interestingly, polymeric IgA1 induces a strong TfR expression of about 3 fold, compared to LPS and pro-inflammatory cytokines, that could only induce a maximum of 1.5-fold increase. We also investigated the induction of TfR expression in IgAN patients. Serum from patients strongly induces TfR expression. This induction is IgAspecific, since the depletion of IgA from serum inhibits its ability to up-regulate mesangial TfR expression. We recently characterized a new anti-transferrin receptor monoclonal antibody named A24 [9]. A24 competes with ferro-transferrin (Fe-Tf) for binding to TfR. Comparison of the binding of A24 and Fe-Tf by Biacore revealed that Fe-Tf has a higher affinity for TfR than A24. Biacore chips were coated with low- and high-density TfR. Surprisingly, under high-density TfR, A24 binds better than Fe-Tf itself. Thus, under physiologic circumstances, such as quiescent mesangial cells, Fe-Tf will bind to TfR but not to A24. However, in pathological situations, like in proliferative mesangial cells that express high levels of TfR, A24 binding will be favored. We used A24 to block mesangial cell activation. Quiescent mesangial cells incubated in the presence of IgA secrete both IL-6 and TGF-␤. The secretion of these cytokines by activated mesangial cells is blocked by A24. In addition, A24

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Hypogalactosylated IgA1 sFc␣Rl, fibronectin, etc Genetic factors

Serum IgA1 complexes

Proliferation

Mesangial IgA deposition

TfR

IgAN → Chronicity → Recurrence of IgA nephropathy in renal allografts

Fig. 1. Proposed model for TfR implication in IgAN.

and the bacullovirus-derived soluble forms of TfR1 and TfR2 were able to inhibit IgA-induced mesangial cell proliferation. This blockage was specific for IgA, since none of these agents could block pro-inflammatory or LPS-derived mesangial cell proliferation. In conclusion, polymeric IgA1 complexes from patients induce TfR hyperexpression on mesangial cells but also cytokine secretion and mesangial cell proliferation. We propose a model to explain the role of TfR in IgAN, in which hypogalactosylated IgA1 from patients complexed with serum proteins, are deposited in the mesangium through binding to TfR, inducing cell activation (secretion of pro-inflammatory and pro-fibrogenic cytokines) as well as receptor expression and mesangial cell proliferation. The induction of this autocrine loop could explain both the chronicity of the disease but also the recurrence of IgAN in renal allografts (fig. 1).

References 1 2

Monteiro RC, Moura IC, Launay P, et al: Pathogenic significance of IgA receptor interactions in IgA nephropathy. Trends Mol Med 2002;8:464–468. Gomez-Guerrero C, Gonzalez E, Egido J: Evidence for a specific IgA receptor in rat and human mesangial cells. J Immunol 1993;151:7172–7181.

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4 5

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Gomez-Guerrero C, Duque N, Egido J: Stimulation of Fc(alpha) receptors induces tyrosine phosphorylation of phospholipase C-gamma(1), phosphatidylinositol phosphate hydrolysis, and Ca2⫹ mobilization in rat and human mesangial cells. J Immunol 1996;156:4369–4376. Monteiro RC, Van De Winkel JG: IgA Fc receptors. Annu Rev Immunol 2003;21:177–204. Moura IC, Centelles MN, Arcos-Fajardo M, et al: Identification of the transferrin receptor as a novel immunoglobulin (Ig)A1 receptor and its enhanced expression on mesangial cells in IgA nephropathy. J Exp Med 2001;194:417–425. Haddad E, Moura IC, Arcos-Fajardo M, et al: Enhanced expression of the CD71 mesangial IgA1 receptor in Berger disease and Henoch-Schonlein nephritis: association between CD71 expression and IgA deposits. J Am Soc Nephrol 2003;14:327–337. Moura IC, Arcos-Fajardo M, Sadaka C, et al: Glycosylation and size of IgA1 are essential for interaction with mesangial transferrin receptor in IgA nephropathy. J Am Soc Nephrol 2004;15: 622–634. Moura IC, Arcos-Fajardo M, Gdoura A, et al: Engagement of transferrin receptor by polymeric IgA1: evidence for a positive feedback loop involving increased receptor expression and mesangial cell proliferation in IgA nephropathy. J Am Soc Nephrol 2005;16:2667–2676. Moura IC, Lepelletier Y, Arnulf B, et al: A neutralizing monoclonal antibody (mAb A24) directed against the transferrin receptor induces apoptosis of tumor T lymphocytes from ATL patients. Blood 2004;103:1838–1845.

Ivan Cruz Moura INSERM U699 Immunopathologie rénale, récepteurs et inflammation Faculté de Médicine Xavier Bichat 16, Rue Henri Huchard FR-75870 Paris Cedex 18 (France) Tel. ⫹33 1 44 85 62 81, Fax ⫹33 1 44 85 62 60, E-Mail [email protected]

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IgA Fc Receptor I Is a Molecular Switch that Determines IgA Activating or Inhibitory Functions Yutaka Kanamarua,b, Ulrich Blanka,b, Renato C. Monteiroa,b a

INSERM U699 and bMedical School of Denis Diderot University, Bichat Campus, Paris, France

Abstract IgA Fc receptor I, Fc␣RI or CD89, an ITAM-bearing receptor, has revealed unique genetic, structure and function features among immunoreceptors. While all genes encoding for the human Fc receptors are located in chromosome 1, the Fc␣RI gene has been found in a cluster in 19q13 that includes the killer inhibitory receptors (KIRs) and the leukocyteimmunoglobulin-like receptors (LIRs). Fc␣RI-IgA complexes display an original crystal structure and a 2:1 stoichiometry. Our results show that intrinsic signals elicited by Fc␣RI have outlined the particularity of this receptor, since it could function in two opposite ways: cell activation and inhibition of a heterologous FcR. Contrary to the dogma that receptor multimerization is necessary to ensure signaling, we demonstrated that monomeric targeting of Fc␣RI is sufficient to trigger a low-intensity signaling cascade that leads to cell desensitization by recruiting the tyrosine phosphatase SHP-1. By contrast, multimerization of Fc␣RI allows a high-intensity signaling pathway that leads to the recruitment of tyrosine kinase Syk and cell activation. Both types of signals require the FcR␥-ITAM motif. This dual function is unique among ITAM-bearing FcR and led us to postulate that ITAM motifs could deliver an unexpected array of signaling intensity. These findings redefine Fc␣RI as a molecular switch of the immune system that mediates both pro- and anti-inflammatory functions of IgA. Copyright © 2007 S. Karger AG, Basel

The maintenance of immune system homeostasis is essential for protecting the host against inflammation and autoimmunity. Control mechanisms involve humoral and cellular factors (e.g. cytokines, immunoglobulins, regulatory cells) as well as immune receptors [1, 2]. IgA, the second most abundant Ig isotype in serum, is usually not involved in humoral immune responses and does not activate complement, functions manifestly well fulfilled by IgM and IgG [3].

Rather, it has been considered as an anti-inflammatory isotype, although in some cases activation can occur after immune complex formation [4]. Serum IgA downregulates IgG-mediated phagocytosis, chemotaxis, bactericidal activity, oxidative burst, and cytokine release [5–10]. In agreement with these observations, patients with selective IgA deficiency show increased susceptibility to autoimmune and allergic disorders [11]. The myeloid Fc␣RI mediates cellular activation following triggering by IgA-containing immune complexes [4], but the molecular basis that underlies IgA inhibitory functions remains obscure. To establish whether Fc␣RI could mediate inhibitory functions such as IgG-mediated phagocytic activity of monocytes, we targeted Fc␣RI independently of IgG receptors, using Fab fragments of an anti-Fc␣RI mAb (clone A77) [12]. Preincubation of monocytes with A77 Fab inhibited IgG-mediated phagocytosis (⬎80%) compared to an irrelevant Fab fragment. Thus, Fc␣RI targeting can inhibit phagocytosis induced by a heterologous receptor. To characterize this inhibitory function, we produced Fc␣RI transfectants in the rat mast-cell line RBL-2H3, which constitutively expresses the highaffinity receptor for IgE (Fc␧RI) [12]. Preincubation with anti-Fc␣RI Fab markedly inhibited Fc␧RI-initiated degranulation (74%) and in two other transfectants, compared to an irrelevant Fab and non-transfected cells. We then examined how ligand valence influenced this negative signaling. Monovalent anti-Fc␣RI Fab had a stronger inhibitory effect than divalent F(ab’)2 fragments, whereas Fc␣RI crosslinking alone induced cell activation. No degranulation was observed with anti-Fc␣RI Fab or F(ab’)2 alone. We next examined the effect of Fc␣RI physiological ligand, IgA. Incubation of transfectants with serum IgA, but not with IgG, significantly inhibited IgE-dependent degranulation (43%). As observed for anti-Fc␣RI F(ab’)2 crosslinking, aggregation of Fc␣RI by IgA complexes resulted in degranulation. Thus, our data establish that the interactions of IgA with Fc␣RI in the absence of antigen cause a profound inhibition while aggregation by IgA complexes leads to cell activation. This is in agreement with moderately fast on- and off-rates of the Fc␣RI:IgA binding reaction: IgA binding is transient, whereas IgA immune complexes stabilize Fc␣RI aggregates [13, 14]. Thus, a low natural frequency of antigen-specific serum IgA antibodies would favor the physiological inhibitory function of Fc␣RI, and suggests that the main role of circulating IgA is to keep the immune system under tight steady-state control. Fc␣RI is devoid of an inhibitory signaling motif in its cytoplasmic tail, but can be expressed associated with the FcR␥-chain homodimer [15, 16], a subunit that contains an immunoreceptor tyrosine-based activation motif (ITAM) that allows activatory signaling [17, 18]. To explore whether FcR␥ could be involved

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IgA-IC

DNP-HSA IgA IgE

IgA Fc␣RI ␣

A77 (anti-Fc␣RI) Fab

␣ P P P P P P P

ITAM

␥

␤

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P ␥ SHP-1

P Syk

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P P P P P P P ␥2

ITAM

P Syk

a

Fc␣RI

Fc␧RI

P LAT

P ERK

Activation

b

P ERK

Inhibition

Fig. 1. Activation of Fc␣RI signal by aggregation of receptor. An Fc␣RI was introduced into the RBL mast cell line. IgA immune-complex-induced receptor aggregation results in ITAM phosphorylation, allowing recruitment and activation of the Syk family (a). Inhibition of Fc␣RI by monovalent targeting of receptor. The RBL transfectants expressing Fc␣RI were pretreated either with monomeric IgA or with a Fab fragment of a monoclonal antibody (A77) resulted in the inhibition of IgE-mediated degranulation through recruitment of SHP-1 phosphatase (b).

in the inhibitory signal, we first examined the ability of a ␥-less Fc␣RI to induce inhibition [12]. Arginine at position 209, within the Fc␣RI transmembrane domain, was replaced by leucine (R209L), which abolishes its association with the FcR␥ chain [16, 19] and transfected into RBL cells. This mutant receptor was unable to elicit either an inhibitory or activatory response, indicating that the intracellular tail of Fc␣RI did not contain the motif responsible for these functions. To directly assess the involvement of FcR␥, we constructed a chimeric receptor by fusing the extracellular and R209L transmembrane domains of Fc␣RI to the intracytoplasmic tail of human FcR␥. Binding of anti-Fc␣RI Fab to Fc␣RIR209L/␥ chimeric receptor transfectants restored the inhibitory effect on degranulation to an extent similar to that observed in wild-type transfectants (91% vs. 74%). Aggregation of Fc␣RIR209L/␥ chimeric receptor induced degranulation, demonstrating that, like wild-type Fc␣RI, it was able to mediate both activation and inhibition. As the FcR␥ chain does not contain any known inhibitory motif we asked whether the FcR␥ ITAM could also mediate the inhibitory effect. Single or double Y to F point-mutations within the ITAM of the chimeric receptor were made and stable transfectants established in RBL cells.

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All mutants were no longer able to mediate the inhibitory and the activatory response. Of note, all transfectants expressed significant levels of Fc␣RI at the cell surface and exhibited over 50% Fc␧RI-mediated degranulation. Thus, our data establish that the FcR␥ ITAM in the context of Fc␣RI mediates not only activatory but also inhibitory functions. Interestingly, in contrast to classic immunotyrosine-based inhibition motif (ITIM)-bearing inhibitory receptors that act through the co-aggregation with activatory receptors [2, 20], the Fc␣RIinhibitory mechanism does not require co-aggregation. Phosphatase recruitment to the Fc␣RI-associated FcR␥ was then investigated. SHP-1, but not SHIP, was recruited in a time-dependent manner to the Fc␣RI chimera. No association of Fc␣RI with SHP-2 was observed. As Fc␣RImediated inhibition does not require co-aggregation with the target receptor, we examined whether SHP-1 could also be recruited to engaged Fc␧RI. A77 Fab induced the association of SHP-1 with the heterologous receptor Fc␧RI, apparently with delayed kinetics compared to Fc␣RI. SHP-1 recruitment to Fc␧RI was dependent on Fc␣RI-associated FcR␥ ITAM, since dead ITAM mutant chimera were unable to recruit SHP-1 to Fc␧RI. SHP-1 is known to dephosphorylate different proteins of the activation pathway [21]. We therefore examined the phosphorylation state of crucial effectors involved in Fc␧RI signaling, such as Syk, LAT and ERK. Fc␣RI targeting by A77 Fab prevented phosphorylation of Syk, LAT and ERK induced by Fc␧RI signaling, in a time-dependent manner. These findings redefine Fc␣RI as a novel type of immune receptor that acts as a molecular switch that directs toward either an activatory or an inhibitory function, and provide a molecular basis for the previously observed anti-inflammatory role of IgA [5–10].

References 1 2 3 4 5 6

7 8 9

Van Parijs L, Abbas AK: Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science 1998;280:243–248. Ravetch JV, Lanier LL: Immune inhibitory receptors. Science 2000;290:84–89. Kerr MA: The structure and function of human IgA. Biochem J 1990;271:285–296. Monteiro RC, Van De Winkel JG: IgA Fc receptors. Annu Rev Immunol 2003;21:177–204. Wilton JM: Suppression by IgA of IgG-mediated phagocytosis by human polymorphonuclear leucocytes. Clin Exp Immunol 1978;34:423–428. Nikolova EB, Russell MW: Dual function of human IgA antibodies: inhibition of phagocytosis in circulating neutrophils and enhancement of responses in IL-8-stimulated cells. J Leukoc Biol 1995;57:875–882. Van Epps DE, Williams RC Jr: Suppression of leukocyte chemotaxis by human IgA myeloma components. J Exp Med 1976;144:1227–1242. Van Epps DE, Reed K, Williams RC Jr: Suppression of human PMN bactericidal activity by human IgA paraproteins. Cell Immunol 1978;36:363–376. Wolf HM, et al: Human serum IgA downregulates the release of inflammatory cytokines (tumor necrosis factor-alpha, interleukin-6) in human monocytes. Blood 1994;83:1278–1288.

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Wolf HM, et al: Inhibition of receptor-dependent and receptor-independent generation of the respiratory burst in human neutrophils and monocytes by human serum IgA. Pediatr Res 1994;36: 235–243. Schaffer FM, Monteiro RC, Volanakis JE, Cooper MD: IgA deficiency. Immunodefic Rev 1991;3: 15–44. Materials and methods are available as supporting material on Science Online. Wines BD, et al: Identification of residues in the first domain of human Fc alpha receptor essential for interaction with IgA. J Immunol 1999;162:2146–2153. Herr AB, Ballister ER, Bjorkman PJ: Insights into IgA-mediated immune responses from the crystal structures of human FcalphaRI and its complex with IgA1-Fc. Nature 2003;423:614–620. Pfefferkorn LC, Yeaman GR: Association of IgA-Fc receptors (Fc alpha R) with Fc epsilon RI gamma 2 subunits in U937 cells. Aggregation induces the tyrosine phosphorylation of gamma 2. J Immunol 1994;153:3228–3236. Launay P, et al: Alternative endocytic pathway for immunoglobulin A Fc receptors (CD89) depends on the lack of FcRgamma association and protects against degradation of bound ligand. J Biol Chem 1999;274:7216–7225. Reth M: Antigen receptor tail clue. Nature 1989;338:383–384. Kinet JP: The high-affinity IgE receptor (Fc epsilon RI): from physiology to pathology. Annu Rev Immunol 1999;17:931–972. Morton HC, et al: Functional association between the human myeloid immunoglobulin A Fc receptor (CD89) and FcR gamma chain. Molecular basis for CD89/FcR gamma chain association. J Biol Chem 1995;270:29781–29787. Long EO: Regulation of immune responses through inhibitory receptors. Annu Rev Immunol 1999;17:875–904. Veillette A, Latour S, Davidson D: Negative regulation of immunoreceptor signaling. Annu Rev Immunol 2002;20:669–707.

Renato C. Monteiro INSERM U699 and Medical School of Denis Diderot University Bichat Campus 16, rue Henri Huchard, BP416 FR–75870 Paris Cédex 18 (France) Tel. +33 1 4485 6261, Fax +33 1 4485 6260, E-Mail [email protected]

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The Role of PDGF-D in Mesangioproliferative Glomerulonephritis Jürgen Floege, Claudia van Roeyen, Peter Boor, Tammo Ostendorf Division of Nephrology and Immunology, RWTH University of Aachen, Aachen, Germany

Abstract In view of increasing numbers of patients with end-stage renal disease (ESRD), new approaches to common underlying diseases, such as mesangioproliferative glomerulonephritis, including IgA nephropathy, are urgently needed. Whereas the role of the platelet-derived growth factor (PDGF) B chain in mediating mesangioproliferative changes is well established, the role of the PDGF-D chain has only recently been elucidated. The PDGF-D chain, like PDGF-B, signals through the PDGF ␤-receptor and therefore shares a number of biological activities with PDGF-B. Recent studies have shown that PDGF-D induces mesangial cell proliferation in vitro and is overexpressed in mesangioproliferative glomerulonephritis in vivo. Hepatic transfection with an adenoviral vector expressing PDGF-D induced prominent mesangioproliferative nephritis in mice, whereas antagonism of PDGF-D in a rat model of mesangioproliferative disease ameliorated the renal changes. These four observations establish PDGF-D, like -B, as an important mediator of mesangioproliferative nephritis in vivo and suggest that it may be an attractive therapeutic target. In addition, first observations suggest that PDGF-D may also contribute to secondary renal changes that characterize progressive renal failure, i.e. tubulointerstitial fibrosis. Copyright © 2007 S. Karger AG, Basel

Introduction

For the majority of progressive renal diseases there is no curative therapy and treatment is usually targeted at delaying or even reversing renal damage. Since many renal diseases, including diabetic nephropathy, IgA nephropathy and proliferative lupus nephritis, are histologically characterized by mesangial changes, targeting key growth factors which mediate such changes may represent a novel therapeutic option. In this respect ligands of the PDGF receptor-␤ are probably the best characterized mediators up-to-date.

PDGF-CC

PDGF-AA

␣␣-Receptor

PDGF-AB

PDGF-BB

␣␤-Receptor

␤␤-Receptor

PDGF-DD

Fig. 1. PDGF isoforms and receptor binding characteristics.

The PDGF System

Until recently the PDGF system consisted of two PDGF chains, PDGF-A and -B, that are secreted as homo- or heterodimers and bind to dimeric PDGF receptors composed of ␣- and/or ␤-chains. Whereas PDGF-A binds to the ␣-chain only, PDGF-B is a ligand for all receptor types. Recently, two novel PDGF isoforms, designated PDGF-C and -D, were identified that are released as homodimers, PDGF-CC and -DD [1]. These two new isoforms are produced as latent factors. Proteolytic cleavage of a CUB-domain from each chain is then required for activation [1]. Following this proteolytic processing, the core domain of PDGF-CC appears to be largely a ligand for the PDGF ␣␣-receptor, while PDGF-DD binds predominantly to the PDGF ␤␤-receptor [1] (fig. 1). All four PDGF isoforms, as well as both receptor chains, are expressed in the kidney, albeit in distinct spatial arrangements [2–5]. Increased expression of PDGF in glomerular and/or interstitial locations has been documented in a large variety of renal diseases. In addition, increased expression of PDGF receptors occurs in experimental and human renal diseases [4–10]. Of relevance in the context of PDGF-D is the observation that the PDGF receptor ␤-subunit is constitutively expressed in mesangial and parietal glomerular epithelial cells,

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vascular smooth muscle cells and renal interstitial cells [6–8]. In view of the similarity of events occurring during ontogenesis and renal pathology, it is important that both the PDGF-B chain and the PDGF receptor ␤-subunit play a critical role in mesangial development [11, 12]. Ample evidence is now available to link the PDGF system, in particular the PDGF-B-chain, to mesangial proliferation and matrix expansion, given that (1) mesangial cells produce PDGF-B in vitro, and various growth factors induce mesangial proliferation via induction of auto- or paracrine PDGF-B-chain excretion [13, 14]; (2) the PDGF-B chain and its receptor are overexpressed in many glomerular diseases [5–8]; (3) infusion of PDGF-BB or glomerular transfection with PDGF-B-chain cDNA can induce selective mesangial cell proliferation and matrix accumulation in vivo [15, 16]; (4) PDGF-B-chain or ␤-receptor knock-out mice fail to develop a mesangium [11, 12], and (5) specific inhibition of PDGF-B using antibodies, aptamers, soluble PDGF receptors or PDGF ␤-receptor tyrosine kinase blockers not only results in a diminution of mesangioproliferative changes, but, more importantly, was also able to prevent longterm renal scarring [17–20]. Given that PDGF-D, like PDGF-B, signals through the PDGF ␤-receptor, overlapping biological activity was to be expected.

PDGF-D and Mesangioproliferative Glomerulonephritis

In order to ascribe a specific biologic effect to the action of an individual cytokine or growth factor, e.g. PDGF-D mediating mesangioproliferative disease, several requirements should be fulfilled: The Factor Exerts the Effect in vitro Incubation of growth-arrested cultured rat and human mesangial cells with PDGF-DD led to a dose-dependent increase in proliferation [21]. Independence of the mitogenic PDGF-DD activity from PDGF-BB was demonstrated by incubating the cells with antagonistic PDGF-BB aptamers simultaneously to PDGF-DD. While the aptamers blocked PDGF-BB-induced proliferation, they had no effect on the mitogenic potential of PDGF-DD. The Effect in vivo Is Associated with Overproduction or Release of the Factor By immunohistochemistry, PDGF-DD localization in normal rat kidney was confined to arterial and arteriolar vascular smooth muscle cells, whereas no immunoreactivity was noted in the glomeruli [21]. Following the induction of mesangioproliferative anti-Thy 1.1 nephritis in rats, there was prominent

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glomerular localization of PDGF-DD in the expanded mesangium. In addition, glomerular PDGF-D mRNA expression increased 2.4- and 2.9-fold at days 7 and 9, respectively, in comparison to non-nephritic rats [21]. The Effect Is Reproduced in vivo by Administration or Overexpression of the Factor In mice, hepatic viral overexpression of a human PDGF-D construct has been demonstrated to potently induce mesangioproliferative changes, characterized by enlarged glomeruli as well as marked increases in glomerular cellularity, cell proliferation, extracellular matrix accumulation and macrophage counts [22]. Similar changes were obtained with a PDGF-B construct [22]. The Effect Can be Abolished or Diminished in vivo by Specific Antagonism of the Factor Following the induction of mesangioproliferative anti-Thy 1.1 nephritis, glomerular cell proliferation, as assessed by counting the number of glomerular mitoses or BrdU-positive nuclei, was significantly reduced in a dose-dependent manner in rats receiving a specific neutralizing anti-PDGF-D monoclonal antibody [21]. Specific identification of proliferating mesangial cells on day 8 after disease induction showed that the anti-PDGF-D antibody, but not a control antibody, led to a 57% reduction in mesangial cell proliferation. In addition, treatment with the antibody reduced the glomerular de novo expression of ␣-smooth muscle actin, which is only expressed by activated mesangial cells [23]. Other effects of the anti-PDGF-D antibody included a reduction of glomerular fibronectin accumulation and a marked reduction of glomerular monocyte/ macrophage influx [21]. In addition, we recently described that antagonism of PDGF in a progressive anti-Thy 1.1 nephritis model also prevents long-term sequelae such as glomerular and tubulointerstitial scarring [24]. The above observations establish PDGF-D, like PDGF-B, as a mediator of mesangioproliferative disease in vivo.

A Role of PDGF-D in Secondary Renal Interstitial Fibrosis?

Nearly all progressive renal diseases that can lead to ESRD are characterized by a progressive renal tubulointerstitial fibrosis, which occurs secondary to glomerular damage. In a recent study by Taneda et al. [5] expression of PDGF-D rapidly increased in renal interstitial cells following unilateral ureteral obstruction. Areas of PDGF-D overexpression closely overlapped with regions of ␤-receptor upregulation, providing the basis for increased biological activity. Taneda et al. [5] then

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confirmed these mouse data in human renal biopsies of patients with chronic obstructive nephropathy. Very recently, using the neutralizing antibody to PDGF-D, we demonstrated that in progressive anti-Thy 1.1 nephritis, treatment with the antibody, even after the acute glomerular injury phase had subsided, led to long-term benefit in terms of renal function and better preservation of renal morphology [25]. Thus, antagonism of PDGF-D not only offers hope for the treatment of acute mesangioproliferative disease, but also for secondary events such as tubulointerstitial damage. The latter appears of particular clinical relevance given the oligosymptomatic nature in particular of IgA nephropathy, and the frequent late presentation of such patients.

References 1 2 3 4 5 6 7 8 9 10 11 12 13 14

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Li X, Eriksson U: Novel PDGF family members: PDGF-C and PDGF-D. Cytokine Growth Factor Rev 2003;14:91–98. Changsirikulchai S, Hudkins KL, Goodpaster TA, et al: Platelet-derived growth factor-D expression in developing and mature human kidneys. Kidney Int 2002;62:2043–2054. Eitner F, Ostendorf T, Van Roeyen C, et al: Expression of a novel PDGF isoform, PDGF-C, in normal and diseased rat kidney. J Am Soc Nephrol 2002;13:910–917. Eitner F, Ostendorf T, Kretzler M, et al: PDGF-C expression in the developing and normal adult human kidney and in glomerular diseases. J Am Soc Nephrol 2003;14:1145–1153. Taneda S, Hudkins KL, Topouzis S, et al: Obstructive uropathy in mice and humans: potential role for PDGF-D in the progression of tubulointerstitial injury. J Am Soc Nephrol 2003;14:2544–2555. Iida H, Seifert R, Alpers CE, et al: Platelet-derived growth factor (PDGF) and PDGF receptor are induced in mesangial proliferative nephritis in the rat. Proc Natl Acad Sci USA 1991;88:6560–6564. Seifert RA, Alpers CE, Bowen-Pope DF: Expression of platelet-derived growth factor and its receptors in the developing and adult mouse kidney. Kidney Int 1998;54:731–746. Alpers CE, Seifert RA, Hudkins KL, et al: Developmental patterns of PDGF B-chain, PDGFreceptor, and alpha-actin expression in human glomerulogenesis. Kidney Int 1992;42:390–399. Floege J, Hudkins KL, Davis CL, et al: Expression of PDGF alpha-receptor in renal arteriosclerosis and rejecting renal transplants. J Am Soc Nephrol 1998;9:211–223. Floege J, Hudkins KL, Seifert RA, et al: Localization of PDGF alpha-receptor in the developing and mature human kidney. Kidney Int 1997;51:1140–1150. Leveen P, Pekny M, Gebre-Medhin S, et al: Mice deficient for PDGF B show renal, cardiovascular, and hematological abnormalities. Genes Dev 1994;8:1875–1887. Soriano P: Abnormal kidney development and hematological disorders in PDGF beta-receptor mutant mice. Genes Dev 1994;8:1888–1896. Silver BJ, Jaffer FE, Abboud HE: Platelet-derived growth factor synthesis in mesangial cells: induction by multiple peptide mitogens. Proc Natl Acad Sci USA 1989;86:1056–1060. Floege J, Topley N, Hoppe J, et al: Mitogenic effect of platelet-derived growth factor in human glomerular mesangial cells: modulation and/or suppression by inflammatory cytokines. Clin Exp Immunol 1991;86:334–341. Floege J, Eng E, Young BA, et al: Infusion of platelet-derived growth factor or basic fibroblast growth factor induces selective glomerular mesangial cell proliferation and matrix accumulation in rats. J Clin Invest 1993;92:2952–2962. Isaka Y, Fujiwara Y, Ueda N, et al: Glomerulosclerosis induced by in vivo transfection of transforming growth factor-beta or platelet-derived growth factor gene into the rat kidney. J Clin Invest 1993;92:2597–2601.

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Floege J, Ostendorf T, Janssen U, et al: Novel approach to specific growth factor inhibition in vivo: antagonism of platelet-derived growth factor in glomerulonephritis by aptamers. Am J Pathol 1999;154:169–179. Gilbert RE, Kelly DJ, McKay T, et al: PDGF signal transduction inhibition ameliorates experimental mesangial proliferative glomerulonephritis. Kidney Int 2001;59:1324–1332. Nakamura H, Isaka Y, Tsujie M, et al: Electroporation-mediated PDGF receptor-IgG chimera gene transfer ameliorates experimental glomerulonephritis. Kidney Int 2001;59:2134–2145. Ostendorf T, Kunter U, Grone HJ, et al: Specific antagonism of PDGF prevents renal scarring in experimental glomerulonephritis. J Am Soc Nephrol 2001;12:909–918. Ostendorf T, Van Roeyen CR, Peterson JD, et al: A fully human monoclonal antibody (CR002) identifies PDGF-D as a novel mediator of mesangioproliferative glomerulonephritis. J Am Soc Nephrol 2003;14:2237–2247. Hudkins KL, Gilbertson DG, Carling M, et al: Exogenous PDGF-D is a potent mesangial cell mitogen and causes a severe mesangial proliferative glomerulopathy. J Am Soc Nephrol 2004;15: 286–298. Johnson RJ, Iida H, Alpers CE, et al: Expression of smooth muscle cell phenotype by rat mesangial cells in immune complex nephritis. Alpha-smooth muscle actin is a marker of mesangial cell proliferation. J Clin Invest 1991;87:847–858. Ostendorf T, Rong S, Boor P, et al: Antagonism of PDGF-D by human antibody CR002 prevents renal scarring in experimental glomerulonephritis. J Am Soc Nephrol 2006;17:1054–1062. Boor P, Konieczny A, Villa L, et al: PDGF-D inhibition by CR002 in late stages of experimental glomerulonephritis ameliorates tubulointerstitial fibrosis. Nephrol Dial Transplant 2007, doi: 10.1093/ndt/gfl691.

Jürgen Floege, MD Medizinische Klinik II, Klinikum der RWTH Pauwelsstrasse 30 DE–52074 Aachen (Germany) Tel. ⫹49 241 8089 530, Fax ⫹49 241 8082 446, E-Mail [email protected]

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Analysis of Innate Immune Responses in a Model of IgA Nephropathy Induced by Sendai Virus Michifumi Yamashitaa, Subba R. Chintalacharuvua, Noriyoshi Kobayashib, John G. Nedruda, Michael E. Lamma, Yasuhiko Tominob, Steven N. Emancipatora a

Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA; Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan b

Abstract In a model of IgA nephropathy (IgAN) induced by Sendai virus (SeV) without Th1/Th2 polarizing immunization, Th2-prone BALB/c mice develop more severe nephritis with acute renal insufficiency than Th1-prone C3H mice. To determine whether Th1 or Th2 predominance influences the severity of experimental IgAN in mice, we employed polarizing immunizations in a SeV-induced IgAN model in Th1-prone C57Bl/6 mice and Th2-prone BALB/c mice. C57Bl/6 mice, immunized with SeV
CFA or
IFA, showed: (1) clear cytokine polarity by splenocytes in recall assays. (2) Total serum IgA and especially SeV-specific IgA from the IFA group showed a selective defect in galactosylation, not seen in the CFA group, and (3) serum creatinine in the IFA group was higher than in the CFA group or nonimmune controls. However, BALB/c mice did not show clear cytokine polarity with CFA/IFA adjuvant. Moreover, spleen cells from naïve BALB/c mice produce IFN- (but not IL-2, -4, -5, or -13) upon stimulation with inactivated SeV in vitro. By flow cytometry, IFN- producing cells are CD3(), CD19(), CD49b(
) natural killer cells. IFN- production by naïve splenocytes is blocked partially by anti-IL12 blocking Abs, and completely by anti-IL18R blocking Abs. In conclusion, C57Bl/6 mice with polarizing priming with SeV showed clear cytokine polarity and distinct kidney injuries. However, BALB/c mice did not show clear cytokine polarity in the same immunizing system, presumably due to the effects of innate responses to SeV upon antigen-specific lymphocytes. Natural IFN- production may influence the risk of renal failure in IgAN. Copyright © 2007 S. Karger AG, Basel

The distinct functions of committed T cells in altered cytokine environments are a central issue in autoimmune and hypersensitivity diseases. The tendency of

CD4
T cells to differentiate to the Th1 or Th2 pathway under neutral conditions, i.e. without Th1 or Th2 priming or in the absence of exogenous cytokines, is genetically regulated in each mouse strain [1]. Most inbred mouse strains, including C57Bl/6 mice, show Th1-prone immunity, but BALB/c mice show a Th2prone immune response, and are susceptible to chronic infection, often with severe progressive lesions [2]. Yip et al. reported that complete Freund’s adjuvant (CFA) and incomplete Freund’s adjuvant (IFA) promote Th1 and Th2 cytokine bias, respectively, in response to 7 different antigens in a total of 6 mouse strains including BALB/c and C57Bl/6 mice [3]. Recently, we developed a murine model of IgA nephropathy (IgAN) induced by Sendai virus (SeV) [4]. In this experimental system without Th1- or Th2-polarizing immunization, BALB/c mice develop more severe nephritis with acute renal insufficiency than C3HeB mice, which rarely develop renal insufficiency [5]. To determine the effects of Th1- or Th2-priming on the severity of IgAN, we employed polarizing immunizations in a SeV-induced IgAN model in Th1-prone C57Bl/6 mice and Th2-prone BALB/c mice. As shown in figure 1, female C57Bl/6 or BALB/c mice, six weeks old, received priming immunization by intraperitoneal injection of 0.1ml of inactivated SeV (1 mg/ml) emulsified in 0.1 ml of either CFA or IFA; on days 14 and 28, mice were orally boosted with 0.5 ml of aqueous inactivated SeV (1.0 mg/ml) containing 0.02 mg/ml cholera holotoxin as adjuvant. Some mice were maintained as non-immunized controls. On day 34, mice were challenged with infectious SeV intranasally, or left unchallenged. Urine collected from individual mice was analyzed. At sacrifice on day 38, blood, spleen, and kidney were sampled.

Glomerular Immune Deposits and Renal Function

In both C57Bl/6 mice and BALB/c mice, granular mesangial deposits of mouse IgA, IgG and C3 were present in immunized mice. The intensity and frequency of the glomerular immune deposits was identical in immunized mice primed with CFA or IFA. There was no difference in hematuria between the CFA group and the IFA group. However, serum creatinine was significantly increased only in the IFA group in C57Bl/6 mice, but not in other groups.

Glycosylation of SeV-Specific IgA in C57Bl/6 Mice

In C57Bl/6 mice, anti-SeV IgA antibody isolated by negative selection from the sera of mice from the IFA group has 72% less terminal sialic acid by lectin ELISA than comparable IgA antibody isolated from mice in the CFA

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0.01mg of cholera toxin Virus challenge: 1  108 pfu of infectious SeV, nasally

Fig. 1. The protocol for inducing IgAN model with SeV. Female C57Bl/6 or BALB/c mice, six weeks old, received priming immunization by intraperitoneal injection of 0.1 ml of inactivated SeV (1 mg/ml) emulsified in 0.1 ml of either CFA or IFA; on days 14 and 28, mice were orally boosted with 0.5 ml of aqueous inactivated SeV (1.0 mg/ml) containing 0.02 mg/ml cholera holotoxin as adjuvant. Some mice were maintained as non-immunized controls. On day 34, mice were challenged with infectious SeV intranasally, or left unchallenged. Urine collected from individual mice was analyzed. At sacrifice on day 38, blood, spleen, and kidney were sampled. iSeV  inactivated SeV.

group. Moreover, penultimate galactose on anti-SeV IgA antibody from mice in the IFA group is present at only 64% of the level associated with IgA antibody from mice in the CFA group.

Cytokine Profiles

In C57Bl/6 mice, priming with CFA evokes significantly higher production of IL-2 (3,896  645 pg/ml) and IFN- (5,150  681 pg/ml) in recall assays of splenocytes. Although priming with IFA elicits IL-2 (1,334  316 pg/ml) and IFN- (1,372  173 pg/ml) well in excess of the background levels produced by splenocytes from non-immune mice (28.3  11.6 pg/ml and 110  20 pg/ml, respectively), these levels are significantly lower than those observed in mice primed with CFA. Conversely, priming with IFA instigates responses rich in IL-4 (93.7  40.8 pg/ml) and IL-5 (860  109 pg/ml), that far exceed the levels produced by splenocytes from mice primed with CFA (0.34  0.24 pg/ml and 320  95.7 pg/ml, respectively). On the other hand, in BALB/c mice, surprisingly, Th1 cytokines such as IL-2 and IFN- were not suppressed in the IFA group (IL-2: 59.38  11.59 pg/ml, IFN-: 1,432  265.9 pg/ml) relative to the CFA group (IL-2: 73.0  9.08 pg/ml,

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IFN-: 1,444  214.8 pg/ml). Th2 cytokines such as IL-4 and IL-5 were not suppressed in the CFA group (IL-4: 12.37  0.508 pg/ml, IL-5: 10.77  3.44 pg/ml) relative to the IFA group (IL-4: 10.66  1.15 pg/ml, IL-5: 18.47  5.87 pg/ml). In addition, spleen cells from non-immunized BALB/c produced IFN-, but not the other cytokines evaluated, in response to inactivated SeV.

IFN-␥ Production in Naïve BALB/c and C57Bl/6 Mice

To confirm the unexpected IFN- production by splenocytes from nonimmunized BALB/c mice, we stimulated spleen cells from naïve BALB/c mice or naïve C57Bl/6 mice with inactivated SeV (50 g/ml) for 24 h. Splenocytes from naïve BALB/c (1,140  148.6 pg/ml) but not C57Bl/6 (15.54  9.521 pg/ml) mice produced significant amounts of IFN- (p  0.001) in response to inactivated SeV.

Source of IFN-␥ Production in BALB/c Mice

To identify the source of IFN- in splenocytes from naïve BALB/c mice, we incubated splenocytes with inactivated SeV for 24 h, followed by flow cytometry. We detected cell surface CD3, CD19 and CD49b, and intracellular IFN- using four-color immunostaining (with appropriate isotype control antibodies applied in parallel). IFN- positive cells were CD3 negative, CD19 negative and CD49b positive cells, a phenotype of murine natural killer (NK) cells. Furthermore, whereas only 1.528  0.5841% of total NK cells showed cytoplasmic IFN- after culture without inactivated SeV, 14.35  1.942% of total NK cells were IFN- positive after incubation with inactivated SeV (p  0.001).

IL-12 and IL-18 Receptor Blocking Study

Based upon the importance of IL-12 and IL-18 for IFN- production [6, 7], we tried to block IFN- production using blocking antibodies specific for IL-12 (20 g/ml) or IL-18 receptor (1 g/ml). IFN- production (2,488  323.6 pg/ml) in naïve BALB/c mice in response to inactivated SeV ex vivo was blocked partially by anti-IL-12 blocking antibody (1,546  179.8 pg/ml) and completely by anti-IL-18 receptor blocking antibody (72.56  28.60 pg/ml). In summary, C57Bl/6 mice with polarizing priming with SeV showed clear cytokine polarity and distinct kidney injuries. However, BALB/c mice did not

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show clear cytokine polarity in the same immunizing system, presumably due to the effects of innate responses to SeV upon antigen-specific lymphocytes. Natural IFN- production may influence the risk of renal failure in IgAN.

References 1 2 3 4

5 6

7

Hsieh CS, Macatonia SE, O’Garra A, et al: T cell genetic background determines default T helper phenotype development in vitro. J Exp Med 1995;18:713–721. Reiner SL, Locksley RM: The regulation of immunity to Leishmania major. Annu Rev Immunol 1995;13:151–177. Yip HC, Karulin AY, Tary-Lehmann M, et al: Adjuvant-guided type-1 and type-2 immunity: infectious/noninfectious dichotomy defines the class of response. J Immunol 1999;162:3942–3949. Jessen RH, Emancipator SN, Jacobs GH, et al: Experimental IgA-IgG nephropathy induced by a viral respiratory pathogen. Dependence on antigen form and immune status. Lab Invest 1992;67: 379–386. Chintalacharuvu SR, Nagy NU, Sigmund N, et al: T cell cytokines determine the severity of experimental IgA nephropathy by regulating IgA glycosylation. Clin Exp Immunol 2001;126:326–333. Kobayashi M, Fitz L, Ryan M, et al: Identification and purification of natural killer cell stimulatory factor (NKSF), a cytokine with multiple biologic effects on human lymphocytes. J Exp Med 1989;170:827–845. Okamura H, Tsutsi H, Komatsu T, et al: Cloning of a new cytokine that induces IFN- production by T cells. Nature 1995;378:88–91.

Michifumi Yamashita, MD, PhD Department of Pathology Case Western Reserve University 2103 Cornell Road, Wolstein Building 5204 Cleveland, OH 44106–7288 (USA) Tel.
1 216 368 3306, Fax
1 216 368 1357, E-Mail [email protected]

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Roles of Bone Marrow, Mucosa and Lymphoid Tissues in Pathogenesis of Murine IgA Nephropathy Masashi Aizawa, Yusuke Suzuki, Hitoshi Suzuki, Huihua Pang, Masao Kihara, Kenji Yamaji, Satoshi Horikoshi, Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan

Abstract Previous studies indicated that bone marrow cells may contribute to the pathogenesis of IgA nephropathy (IgAN). However, the cell types and mechanisms responsible remain unclear. Our recent study showed that ‘grouped ddY mice’ is a useful model to approach the pathogenesis of IgAN. Moreover, we also reported that bone marrow transplantation (BMT) from the onset mice of this model reconstituted IgAN in healthy recipients with strong Th1-polarization. We aimed to examine the roles of bone marrow (BM) cells, mucosa and lymphoid tissues in IgAN. We employed onset ddY mice and mutant mice lacking all systemic lymph nodes, Payer’s patch, isolated lymphoid follicles in the lamina propria (LP) and IgA producing cells. BM cells from the onset mice were transplanted into the mutant and wild type (C57BL/6:B6). After BMT, serum elevation of IgA and IgA⫹B220⫺ plasma cells in BM, but not IgA producing cells in LP, were observed in the mutant mice. Although both transplanted mice showed mesangial IgA deposition, glomerular lesions with IgG2a co-deposition were detected only in B6 mice. The present results suggest that glomerular IgA deposition, but not glomerular damage, can be induced by BM-derived or -primed IgA-producing cells independently of priming in mucosa and secondary lymphoid tissues. Copyright © 2007 S. Karger AG, Basel

Introduction

Previous studies suggested that mesangial IgA in IgA nephropathy (IgAN) may be produced in bone marrow (BM) [1–3]. A clinical study showed that bone marrow transplantation (BMT) in patients with leukemia and IgAN

resulted in cure of IgAN as well as of leukemia [4]. These findings suggest that bone marrow cells (BMC) contribute to the pathogenesis of IgAN. The ddY mouse is known as a spontaneous IgAN model. However, one of the major problems in this model is the high degree of variability in onset. We found that ddY mice could be divided by serial biopsy into 3 groups: earlyonset, late-onset and quiescent groups [5]. An association study between the onset and quiescent mice showed that one of the susceptibility loci of murine IgAN is syntenic to that in human familial IgAN (IGAN1) [5]. These findings suggest that the grouped ddY mice model can be a useful model for studying the pathogenic mechanisms of IgAN. We also reported that BMT from earlyonset ddY mice induced IgAN with Th1 polarization, and serum levels of the IgA/IgG2a complex correlated with severity of glomerular injury [6]. However, the underlying mechanism in which BMC induce IgAN remains unclear. It is still unknown whether BMC directly produce nephritogenic IgA or require additional encounters to antigen in lymphoid tissues. To approach this question, we employed alymphoplasia mice (aly/aly) lacking Peyer’s patches (PP), all lymph nodes and IgA-producing cells, due to a point mutation in the NF-␬Binducing kinase gene on a C57BL/6 background [7]. A recent study showed that injection of PP cells of normal mice into aly/aly mice could rescue gut IgA and PP cells migrated to the lamina propria (LP), and generated IgA plasma cells in aly/aly mice. On the other hand, transplantation of normal BMC into aly/aly mice failed to rescue gut IgA, in spite of the complete recovery of serum IgA, and presence of IgA-positive B cells and plasma cells [8]. The aim of the present study using aly/aly mice was to investigate whether induction of IgAN by BMC requires additional priming in mucosa or secondary lymphoid tissues.

Materials and Methods 9-week-old Nsc/Jcl-aly/aly(aly/aly) and B6 (control) (CLEA, Japan) mice were used as recipients, and 20-week-old early-onset ddY mice (animal facility of Juntendo University) as donors. 1 ⫻ 107 PP cells from the onset mice were injected into aly/aly mice and control mice. The recipient mice were sacrificed at 4 weeks after injection. 1 ⫻ 107 BMC from the onset mice were transplanted into irradiated aly/aly and control mice. The recipients were sacrificed at 12 or 24 weeks after BMT. For histopathological examination, we used light microscopy, immunofluorescence and electron microscopy. Serum immunoglobulin was measured by single radioimmunodiffusion (SRL, Japan). Serum IgA-IgG2a immune complex and urinary albumin were measured by ELISA. Polarization was determined by FACS analyses with intracellular staining for anti-mouse IFN-␥ and IL-4, and surface staining for anti-mouse CD4 (BD Pharmingen, San Diego, Calif., USA). IgA-producing cells in BM were detected by FACS analysis staining for anti-mouse IgA, CD138 and B220 (BD Pharmingen).

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LP Adoptive transfer

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Control

b Fig. 1. Histopathological examination of LP and glomeruli. a After adoptive transfer of PP cells: PP cells migrated into LP in both mice, but this transfer did not reconstitute glomerular IgA deposition in either recipient. b After BMT: BMC could reconstitute glomerular IgA in both mice, but did not migrate into LP in aly/aly mice.

Results

Adoptive Transfer of PP Cells Transferred PP cells migrated into LP in both kinds of recipients. However, the adoptive transfer did not reconstitute glomerular IgA deposition in either recipient (fig. 1a). Bone Marrow Transplantation Levels of serum IgA and numbers of IgA⫹B220⫺CD138⫹ plasma cells were elevated at 12 weeks after BMT in aly/aly mice and control mice. BMC from ddY mice induced glomerular IgA deposition, but not migration of IgApositive cells in the lamina propria at 12 weeks after BMT (fig. 1b). Transplanted aly/aly and control mice showed paramesangial electron-dense deposits that were similar to the lesions seen in early-onset ddY mice. However, transplanted

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aly/aly mice did not show glomerular lesions or albuminuria at 24 weeks after BMT. Moreover, elevation of serum IgA-IgG2a complex and glomerular IgA/ IgG2a co-deposition were absent in these mice. IFN-␥-producing cells in CD4⫹ spleen cells were not elevated after BMT in aly/aly mice. These results demonstrated that systemic Th1 polarization was absent in the transplanted aly/aly mice.

Discussion

Reconstitution of glomerular IgA deposition in the transplanted aly/aly mice indicates that secondary lymphoid tissues may be not required for this process. In addition, lack of migration of IgA⫹ B cells into the mucosa suggests that the homing process of B cells into the mucosa may be also not necessary. Accordingly, glomerular IgA may be directly induced by the IgA-producing cells in BM. However, disease progression after glomerular IgA deposition did not occur in transplanted aly/aly mice. This finding suggests that disease progression may require secondary lymphoid tissues. Furthermore, transplanted aly/aly mice did not show elevation of the serum IgA/IgG2a complex, glomerular IgA-IgG2a co-deposition or Th1 polarization, suggesting that an additional process in lymphoid tissue after IgA deposition, such as antigen presentation, may be necessary for the progression of this disease. BMC of murine IgAN seem to induce glomerular IgA deposition independent of homing to mucosa and secondary lymphoid tissues. However, secondary lymphoid tissues may be required for full progression of the glomerular injury after IgA deposition.

References 1 2

3

4 5

6

Barratt J, Feehally J, Smith AC: Pathogenesis of IgA nephropathy. Semin Nephrol 2004;24: 197–217. van den Wall Bake AW, Daha MR, van Es LA, et al: Serum IgA and the production of IgA by peripheral blood and bone marrow lymphocytes in patients with primary IgA nephropathy: evidence for the bone marrow as the source of mesangial IgA. Am J Kidney Dis 1988;12:410–414. Harper SJ, Allen AC, Feehally J, et al: Increased dimeric IgA producing B cells in the bone marrow in IgA nephropathy determined by in situ hybridisation for J chain mRNA. J Clin Pathol 1996;49:38–42. Sakai O: IgA nephropathy: current concepts and future trends. Nephrology 1997;3:2–3. Suzuki H, Suzuki Y, Tomino Y, et al: Genome-wide scan in a novel IgA nephropathy model identifies a susceptibility locus on murine chromosome 10, in a region syntenic to human IGAN1 on chromosome 6q22–23. J Am Soc Nephrol 2005;16:1289–1299. Suzuki H, Suzuki Y, Tomino Y, et al: Bone marrow derived cell dependent IgA-IgG2a immune complex is involved in the pathogenesis of murine IgA nephropathy. Reported at the annual meeting of the American Society of Nephrology, St Louis, USA, 2004.

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Shinkura R, Kitada K, Honjo T, et al: Alymphoplasia is caused by a point mutation in the mouse gene encoding Nf-kappa b-inducing kinase. Nat Genet 1999;22:74–77. Suzuki K, Meek B, Fagarasan S, et al: Two distinctive pathways for recruitment of naive and primed IgM⫹ B cells to the gut lamina propria. Proc Natl Acad Sci USA 2005;102:2482–2486.

Yasuhiko Tomino Division of Nephrology Department of Internal Medicine, Juntendo University School of Medicine Hongo 2-1-1, Bunkyo-ku 113–8421 Tokyo (Japan) Tel. ⫹81 3 5802 1065, Fax ⫹81 3 3813 1183, E-Mail [email protected]

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Concluding Remarks Richard J. Glassock The David Geffen School of Medicine at UCLA, Los Angeles, Calif., USA

For almost a quarter of a century I have had the good fortune to be a spectator but not a combatant in the struggle against IgA nephropathy. This position has afforded me some measure of perspective, both historical and scientific. The International Symposia dedicated to IgA nephropathy are always eagerly anticipated events. The prospect of hearing the latest research findings from the most advanced and sophisticated investigative groups in the world raises expectations to an impossibly high level. Previous Symposia have seldom disappointed and have even provided paradigm-shifting insights into this enigmatic disease. While true epiphanies have been uncommon, steady progress towards unraveling the complexities of this common affliction of man have been evident at this Symposium and all its predecessors. I must begin by congratulating Professor Tomino and his colleagues for organizing a superb meeting which flowed smoothly and invigorated the mind, and also to thank all of the presenters for sharing their important discoveries with us in such a clear and understandable fashion. Fortunately, the organizers of the 11th Symposium have wisely entitled this final presentation as ‘Concluding Remarks’ which provides me some much appreciated latitude with respect to content. I believe that it would be incredibly dull and even arrogant to try to summarize the presentations we have heard and seen over the last two days. Instead, I would like to focus on what mysteries remain and on the gaps which continue to hamper our full understanding of the processes underlying IgA nephropathy. Such an exercise is not entirely a futile one, since we now have tools at our disposal which allow many questions to be addressed that were hitherto unapproachable. In no particular order of importance, I can summarize these remarks in a few paragraphs. First, I believe that human studies must take precedence over animal models of disease for real progress to occur. This may seem strange coming from the

mouth of someone who began his research career exploiting animal models of disease in the field of experimental immunopathology. There is little doubt that animal models of IgA nephropathy have potential explanatory power, and all of the Symposia since the first one in 1982 in Milano have had at least one presentation of this genre. Nevertheless, the biological gap between man and mouse or rat can be formidable, and findings in murine species cannot be readily transferred to man. The goals of animal investigation in the field of IgA nephropathy, at least in this spectator’s opinion, should be expressly limited, and their inherent weakness clearly acknowledged. Second, if IgA deposition in the glomerular mesangium is as common as it is believed to be in the normal healthy population (estimated to be about 1 in 40), we need to understand why so few individuals ever express overt clinical signs of disease we call IgA nephropathy. Both environmental triggers (such as an S. aureus infection) or a genetic mutation may be the ‘second hit’ which brings the ‘disorder’ (if we can call asymptomatic mesangial IgA deposition a ‘disorder’) to full expression as a clinically recognizable disease. Clearly, the common finding of lanthanic IgA deposition in the ‘normal’ population means that genetic studies of susceptibility are in reality studies of the genetic basis of expression of disease. Third, at present the only reliable way to ‘diagnose’ IgA nephropathy is to perform a renal biopsy and examine the specimen by immunohistochemical methods. This is an unsatisfactory position since it hampers large scale epidemiological studies, leads to ascertainment bias in descriptive and therapeutic studies, and magnifies errors in the search for causative factors by limiting the sample population to those patients with clinically expressed disease. An accurate, simple and reliable non-invasive means of identifying patients with IgA nephropathy would be warmly welcomed. At the moment, urinary proteomics and serum profiling for aberrantly glycosylated IgA1 or antibodies to this abnormal plasma protein appear to be strong candidates for such a test, but much remains to be done in larger and more heterogenous populations. Fourth, the extraordinary diversity of renal pathological findings in IgA nephropathy is a given. Yet, even after decades of work, we still are only at the beginning stages of having a uniform, generally applicable and widely accepted approach to classifying and categorizing these pathological alterations. The efforts of the International IgA Network, the Renal Pathology Society and the International Society of Nephrology to create such an internationally accepted classification and nomenclature are essential if progress is to be made. In my opinion, one of the first orders of business after this classification schema is adopted is to establish beyond any reasonable doubt that pathological findings are of value in prognostication over and above the simple and readily available clinical parameters of anthropometrics (obesity), magnitude and quality of

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proteinuria, renal function, biochemical assessment (e.g. uric acid and triglycerides) and blood pressure. Such a confirmation of the supplemental value of renal pathology in prognostic stratification would have the side benefit of allowing for more homogenous groups for studies of potentially effective therapeutic regimens. Implicit in this focus on renal pathology is the notion that novel approaches to defining prognostic groupings by application of biological principles should be incorporated into pathologic analysis and interpretation. Examples might include the use of immuno-histochemical analysis of fibroblast specific protein in the interstitium, electron microscopic analysis of basement membrane changes, transcriptosomic analysis of glomerular and interstitial cellular traffic, and assessment of podocyte differentiation and apoptosis. Of course, the final goal of prognosis evaluation is to identify markers which accurately predict outcome in individual patients. While many such evaluation tools already exist, they explain only about 50–60% of the variability in prognosis in groups of patients. Fifth, the search for pathogenetic mechanisms and mediator systems operative in both the familial and sporadic forms of IgA nephropathy must continue and even intensify. Currently the focus is on abnormalities of IgA1 galactosylation and sialylation. Germ-line as well as acquired abnormalities in enzymes involved in this process appear to be at work, and sorting out the control of these critical enzymatic functions at the level of individual clones of B cells will likely be the next stage of research. Immortalization of B cells from patients with IgA nephropathy that exhibit the glycosylation defect is a major advance. It is my guess that studies of the genetic basis of IgA nephropathy (both familial and sporadic) will likely converge on the studies of the cellular basis of defective glycosylation and the receptor-mediated localization of aberrant IgA1 in the mesangium. These studies show incredible promise in elucidating the common pathways involved in IgA mesangial deposition, and it is easy to see how they may lead directly to novel pathogenesis-specific approaches to treatment. Finally, we are forced to maintain our efforts to design effective treatment strategies, even if they are heavily based on empiricism, since no viable method of prevention of IgA nephropathy has yet appeared on the horizon. One of the goals of investigation of therapeutics in IgA nephropathy should be to identify patients at clear and unmistakable danger for future progression. Currently our tools for such identification are rather crude and our treatments require rather long periods of observation to convincingly demonstrate efficacy. Surrogate markers of therapeutic effectiveness will have to be validated with long-term observations. Difficult as they are to design and execute, adequately powered, randomized controlled trials of new treatment regimens will remain as the ‘gold standard’. Refinements in pathological classification may permit more

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‘economical’ trial design with a narrower focus on specific variants (such as IgA nephropathy with focal and segmental necrotizing ‘capillaritis’). Based on current data one can foresee difficulties in future therapeutic trials arising from changes in what represents the ‘community’ standard of care (e.g. will dual blockade of angiotensin II with combinations of angiotensinconverting enzyme inhibitors and angiotensin receptor blockers become the standard of care for patient with IgA nephropathy and proteinuria?). With new paradigms of management, what will be the anticipated effect size needed to justify use of a new therapeutic agent? Can adequately powered randomized controlled trials be executed recognizing the limitations of numbers of qualified patients and the resources available for the conduct of long-term trials? The resolution of the current uncertainties regarding the overall efficacy of combined tonsillectomy and steroid treatment of IgA nephropathy should be a high priority. Until a well-powered randomized controlled trial of such therapy demonstrates improved efficacy for important end-points (e.g. slowing progression of renal disease or prevention of ESRD) as well as safety compared to ‘standard’ therapy plus steroids alone, the general application of this approach to therapy should be regarded as experimental, at least in my view. One of the key unresolved questions in the therapeutic realm is when should therapy be started. Current, admittedly incomplete, information suggests that there is a ‘window’ of opportunity for optimal treatment of IgA nephropathy. The closure of this ‘window’ is easier to identify than when the ‘window’ opens, but more and more data are accumulating that the time of opening of the ‘window’ may be much earlier that originally thought. This has profound implications for the use of, and indications for, renal biopsy in patients believed to possibly have IgA nephropathy. My own suspicion is that more patients with microscopic hematuria and minimal or no overt proteinuria (or even those with just microalbuminuria) should undergo diagnostic renal biopsy, especially in the United States. Unfortunately we have almost no data bearing on the ‘cost-effectiveness’ of this approach and purported ‘benefits’ will need to take into account lead-time and ascertainment biases. The development of an efficient and accurate non-invasive method of ‘diagnosing’ IgA nephropathy would change these circumstances dramatically. Most therapeutic trials in IgA nephropathy have studied the effects of agents already approved for other indications (e.g. steroids, cyclophosphamide, azathioprine, mycophenolate mofetil, ACE inhibitors, and angiotensin receptor blockers). I would predict that by the 12th International Symposium on IgA Nephropathy we will be learning about preliminary and pilot applications of novel agents for treatment. At last some of these agents will be based on a more complete understanding of the fundamental pathogenetic mechanisms (e.g. promoters of O-linked galactosylation of IgA1, inhibitors of IgA1 containing

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immune complex formation or receptor mediated deposition of polymeric IgA) and mediator pathways (e.g. PDGF inhibitors, complement inhibitors) involved in IgA nephropathy. The trail of discoveries in IgA nephropathy these past almost 40 years could be regarded as epitomizing the progress of medical research in renal diseases in general; starting with description and cataloguing of the clinical spectrum of disease and its ‘natural’ history; moving to the experimental laboratory with increasing sophistication of methods and study design; returning to the clinic to test hypotheses and apply promising approaches to empiric treatment; and finally developing rational techniques of diagnosis and therapy based on understanding of fundamental mechanisms and molecular genetics of disease. Hopefully progress will continue at a brisk pace. The millions of individuals with mesangial IgA deposits eagerly await the results of our inquiries. Richard J. Glassock, MD, MACP 8 Bethany Laguna Niguel, CA 92677 (USA) E-Mail [email protected]

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Abstracts Tomino Y (ed): IgA Nephropathy Today. Contrib Nephrol. Basel, Karger, 2007, vol 157, pp 174–243

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International IgA Nephropathy Network Clinico-Pathological Classification of IgA Nephropathy John Feehally Leicester General Hospital, Leicester, UK A number of different pathological classifications of IgA nephropathy (IgAN) have been proposed over the last 25 years. Each classification identifies histopathologic features associated with eventual poor prognosis. Comparative studies do not indicate that one classification should be preferred. Also well described are clinical features at presentation associated with a poor prognosis include proteinuria and hypertension. It is not yet clear whether clinical and pathological features are additive in improving prognostic accuracy. Global agreement on a pathological classification with proven prognostic value would be a major step forward in the management of IgAN. Not only would it improve prognostic advice for individual patients, it would improve opportunities for international comparisons of patient cohorts, and would refine entry into clinical trials, allowing smaller trials of new treatments to be informative. The International IgA Nephropathy Network working with the Renal Pathology Society (and supported by the International Society of Nephrology) has established a Consensus Group to develop agreement on the classification of IgAN. The work of this Consensus Group has developed as follows: • Nephrologists and pathologists from Europe, North America, Asia, and Australasia are collaborating closely. • Agreement has been reached on the range of histopathologic features on light microscopy which may be of prognostic significance and need to be assessed and scored. • Pathologists have worked collaboratively to achieve reproducible scoring systems. • Substantial numbers of cases have been identified from all collaborating centres, both adults and children. • These cases have sufficient biopsy material and sequential clinical information sufficient to allow analysis on the contribution of a wide range of pathological features to prognosis. It is expected that a new, globally acceptable classification will soon emerge from the work of the group.

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Clinical Advances since the 10th International Symposium on IgA Nephropathy Bruce A. Julian, Jan Novak University of Alabama at Birmingham, Birmingham, Ala., USA In the 18 months since meeting in St. Etienne, France, our understanding of the clinical aspects of IgAN has improved in several areas. An analysis of outcome has shown that proteinuria and mean arterial blood pressure more

accurately predicted prognosis when measured during follow-up than if considered only at the time of diagnosis. We also have learned that patients with familial disease exhibit outcomes similar to those of patients with sporadic disease. While the identity and function of the IgAN1 gene have remained elusive, the list of genetically determined factors that influence the clinical expression of IgAN continues to lengthen. Several new laboratory assays have important implications for development of a noninvasive diagnosis IgAN. Lectin-based ELISAs have shown increased serum levels of galactose-deficient IgA1 in patients with IgAN. Because galactose-deficient IgA1 forms immune complexes in the circulation that activate mesangial cells, it will be clinically relevant to measure their levels as well. Also, mass spectrometry studies have described disease-specific biomarkers in the complex urinary proteome. If confirmed, these findings will clarify the pathogenetic pathways and may provide better tools to detect subclinical disease. Such tests will improve phenotypic classification of members of multiplex pedigrees and thereby aid the search for the gene(s) of familial IgAN. Furthermore, tailoring of therapy and monitoring the response will be easier. For the present, treatment of most patients with IgAN remains nonspecific. A recent report of increased angiotensinogen in the proximal tubular cells supports the current approach to suppress angiotensin II by two-drug therapy, angiotensin converting enzyme inhibitors and angiotensin receptor type 1 blockers. Tonsillectomy, advocated by some investigators as a means to dampen the glomerular injury by reducing serum levels galactosedeficient IgA1, is now being tested in randomized trials. Recurrence of IgAN in the renal allograft remains a substantial concern. To date, there is no agreement as to how to prevent or ameliorate this complication.

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Angiotensin Antagonists to Treat IgA Nephropathy Rosanna Coppo, Alessandro Amore, Licia Peruzzi Nephrology, Dialysis and Transplantation, Regina Margherita University Hospital, Turin, Italy The renin-angiotensin system (RAS) plays a pivotal role in the progression of renal diseases, promoting both intra-glomerular and systemic hypertension, and acting on angiotensin receptors of mesangial and tubular cells triggering progression of interstitial fibrosis. On the other hand, IgA deposits may activate mesangial cells leading to production of biological effects which contribute to amplification of the angiotensin II induced damage, suggesting a unique role for RAS in IgAN. We previously demonstrated that in IgAN, particularly in proteinuric cases, there was a local RAS hyperreactivity; precocious activation of the RAS was also recently reported. Over the last decades, ACE-inhibitors (ACE-I) and angiotensin receptor blockers (ARB) have shown beneficial effects in hampering the process of renal sclerosis, particularly in proteinuric nephropathies: the benefits of the two drugs resulted comparable on renal survival and proteinuria. However, the sub-analyses of IgAN patients enrolled in these large studies, failed to prove a specific benefit for IgAN, while a favorable effect of ACE-I in IgAN was reported in some retrospective studies, particularly in hypertensive patients, and the anti-proteinuric effect has been thoroughly demonstrated. In 2003 the first RCT of ACE-I in IgAN was published by Praga et al, but it was not placebo-controlled, and enrolled a limited number of patients (44 subjects) from only one Center with wide range of proteinuria (from 0.5 to 5 g/day) and various degrees of renal function impairment, hence with likely variable prognosis. After a mean follow-up of 75 months, the proportion of patients developing the primary endpoint (50% increase of baseline plasma creatinine) was significantly lower in the treated group than in the control group (12 vs. 57%, respectively). In 1995 we designed a double-blind placebo RCT, since at that moment there was a definite expectation for a trial on ACE-I in IgAN, when the effect of ACE-I on progression of chronic nephropathies was just proved, but not yet for IgAN, and it was supported by the European Community Concerted Action of Biomedicine and Health. This is the first multicenter, double-blind, placebo-controlled RCT, investigating, in a selected cohort of IgAN, the effects of ACE-I on renal function decline and proteinuria. This trial strictly selected young patients (3–35 years old)

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of a very constant level of moderate proteinuria (
1  3.5 g/day/1.73 m2 over the 3 months before enrolment) and normal or moderately reduced renal function (creatinine clearance, CrCl,
50 ml/min/1.73 m2). Fifty-seven patients, average age 19.9 years (range 9–35 years), randomized to receive Benazepril 0.2 mg/kg/day (ACE-I) or placebo (PL), completed the trial (median follow-up 42 months). The primary outcome was progression of kidney disease, defined as
30% decrease of baseline CrCl and/or worsening of proteinuria until 3.5 g/day/1.73 m2. Secondary outcome was proteinuria partial (0.5 g/day/1.73 m2) or total remission (160 mg/day/1.73 m2) for
6 months. The survival to the events was evaluated by univariate (Kaplan-Meier, log-rank test) and Cox multivariate analysis. A single patient (4.3%) in the ACE-I group and 5 (14.7%) in the PL group showed a worsening of CrCl
30%. No patient on ACE-I developed nephrotic syndrome, versus 7 (20.6%) on PL. The primary outcome of renal disease progression resulted significantly different between the two groups (log-rank p  0.035). A stable partial remission of proteinuria was observed in 13/23 (56.5%) ACE-I patients versus 3/34 (8.8%) PL patients (log-rank p  0.033), with total remission in 17.4% of ACE-I treated patients and in none of PL ones (log-rank p  0.029). The multivariate Cox analysis showed that treatment with ACE-I was the independent predictor of prognosis, while no influence on the progression of renal damage was found for gender, age, baseline CrCl, systolic or diastolic blood pressure, mean arterial pressure, and proteinuria. In conclusion, angiotensin antagonism is successful in limiting progression of renal damage in young IgAN patients with proteinuria between 1 and 3.5 g/day. A new trial is ongoing in collaboration with Pozzi et al., to test whether the angiotensin inhibition by both ACE-I and ARB may decrease the risk of progression in patients with IgAN so far considered benign (proteinuria 0.5 g/day). Such inhibition will be at first achieved with a unique pharmacological class (ACE-I or ARB), then shifting to the association of the two classes as soon as the inhibition with one drug becomes ineffective.

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Treatment of IgA Nephropathy: Corticosteroids, Tonsillectomy, Mycophenolate Mofetil Tetsuya Kawamura Department of Medicine, Division of Kidney and Hypertension, Jikei University School of Medicine, Tokyo, Japan Many previous studies have examined the renoprotective effects of corticosteroids in patients with IgA nephropathy (IgAN). An early retrospective study by Kobayashi et al. demonstrated the potential of glucocorticoid therapy on proteinuria and renal survival of IgAN patients especially in those with an initial creatinine clearance (Ccr) of 70 ml/min or more, but not with that of 70 ml/min [1]. The same authors also demonstrated that steroid therapy for an average period of 18 months in IgAN patients with normal renal function and moderate proteinuria produced a better renal survival rate 10 years after therapy when compared with an untreated group (80 vs. 34%) [2]. On the basis of these findings, corticosteroid therapy is recommended if IgAN patients show a moderate degree of proteinuria and their Ccr exceeds 70 ml/min in ‘Clinical guidelines for IgA nephropathy in Japan’. However, the previous studies, most of which are not prospective or randomized, have not provided conclusive results. Recently, Pozzi et al. reported that treatment with glucocorticoids for 6 months significantly improved renal survival and proteinuria for a long-term follow-up [3, 4]. IgAN patients with serum creatinine 1.5 mg/dl and 1–3.5 g/day proteinuria were recruited and randomized to receive ‘pulse’ methylprednisolone (1 g/day for 3 days at the beginning of months 1, 3, and 5) followed by alternate day oral prednisolone (0.5 mg/kg), or placebo for 6 months. Cox regression analysis showed that steroid treatment correlated with a 50% increase of serum creatinine [3]. After 10 years of follow-up, the renal survival was significantly better in the steroid than in the placebo

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group (97 vs. 53%) with improvement favoring steroids regardless of the severity in histologic changes [4]. This randomized, controlled trial (RCT) demonstrated that a relatively short course of glucocorticoids protects against deterioration in renal function in IgAN with no notable adverse effects. A recent meta-analysis (13 trials, 623 patients) supports the use of corticosteroids in reducing proteinuria (weighted mean difference –0.49 g/day), and preventing progression to ESRD (RR 0.44) [5]. Increasing attention has been drawn to the role of tonsillectomy in the long-term prognosis of IgAN. The notion that tonsillectomy not only helps to prevent episodic macroscopic hematuria in the short term but also gives long-term renal protection in IgAN is supported by two large retrospective studies from Japan. A retrospective study of 329 patients with IgAN by Hotta et al. found that tonsillectomy plus high-dose methylprednisolone was identified as one of the independent variables in predicting remission of clinical findings and lack of renal progression [6]. Moreover, Xie et al. compared long-term renal survival in 48 patients with IgAN who underwent tonsillectomy and 70 patients who did not undergo the procedure. After 240 months of follow-up, renal survival was significantly better in patients who underwent tonsillectomy (90 vs. 64%), which had a significant impact on renal outcome by multivariate analysis [7]. However, the role of tonsillectomy in the long-term prognosis of IgAN remains unclear since it has not yet been tested in a controlled randomized trial. In this regard, a multicenter, randomized, controlled trial of steroid pulses with or without tonsillectomy is in progress in Japan. The design of the study will be presented in the lecture. The role of mycophenolate mofetil (MMF) in IgAN has been examined in four major trials. Two prospective randomized studies report no benefit from MMF (2 g/day) in patients at risk of progression (a mean serum creatinine of 1.4 mg/dl and urinary protein excretion of 1.6 g/day) [8], or those at more advanced phase (a mean serum creatinine of 2.4 mg/dl and greater than 2.5 g/day proteinuria on average) [9]. The remaining two studies showed a greater reduction of proteinuria in patients treated with MMF (1–2 g/day) compared to prednisone [10] or placebo [11]. In both studies, however, MMF did not effectively modify the progressive course of the disease. Thus, despite promising results in large RCTs in lupus nephritis, the evidence for the use of MMF in IgAN is inconclusive.

References 1 Kobayashi Y, Fujii K, Hiki Y, et al: Steroid therapy in IgA nephropathy: retrospective study in heavy proteinuric cases. Nephron 1988;48:12–17. 2 Kobayashi Y, Hiki Y, Kokubo T, et al: Steroid therapy during the early stage of progressive IgA nephropathy. A 10-year followup study. Nephron 1996;72:237–242. 3 Pozzi C, Bolasco P, Fogazzi G, et al: Corticosteroids in IgA nephropathy: a randomised controlled trial. Lancet 1999;353: 883–887. 4 Pozzi C, Andrulli S, Del Vecchio L, et al: Corticosteroid effectiveness in IgA nephropathy: long-term results of a randomized, controlled trial. J Am Soc Nephrol 2004;15:157–163. 5 Samuels JA, Strippoli GF, Craig JC, et al: Immunosuppressive treatments for immunoglobulin A nephropathy: a meta-analysis of randomized controlled trials. Nephrology (Carlton) 2004;9:177–185. 6 Hotta O, Miyazaki M, Furuta T, et al: Tonsillectomy and steroid pulse therapy significantly impact on clinical remission in patients with IgA nephropathy. Am J Kidney Dis 2001;38:736–743. 7 Xie Y, Nishi S, Ueno M, et al: The efficacy of tonsillectomy on long-term renal survival in patients with IgA nephropathy. Kidney Int 2003;63:1861–1867. 8 Maes BD, Oyen R, Claes K, et al: Mycophenolate mofetil in IgA nephropathy: results of a 3-year prospective placebocontrolled randomized study. Kidney Int 2004;65:1842–1849. 9 Frisch G, Lin J, Rosenstock J, et al: Mycophenolate mofetil (MMF) vs. placebo in patients with moderately advanced IgA nephropathy: a double-blind randomized controlled trial. Nephrol Dial Transplant 2005;20:2139–2145. 10 Chen X, Chen P, Cai G, et al: A randomized control trial of mycophenolate mofeil treatment in severe IgA nephropathy. Zhonghua Yi Xue Za Zhi 2002;82:796–801. 11 Tang S, Leung JC, Chan LY, et al: Mycophenolate mofetil alleviates persistent proteinuria in IgA nephropathy. Kidney Int 2005;68:802–812.

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Treatment of IgA Nephropathy in Children Norishige Yoshikawa Department of Pediatrics, Wakayama Medical University, Wakayama, Japan IgA nephropathy is a leading cause of chronic renal disease and end-stage renal disease in adult patients, and recent long-term studies assessing the prognosis in children have challenged earlier views that the condition represents a benign disorder. Thus IgA nephropathy presents a therapeutic challenge in both adults and children. Because of the variable rate of progression to renal failure, and because of the probable multifactorial pathogenesis of the disease, the effectiveness of any treatment can only be properly evaluated by means of a controlled trial. When considering treatment protocols, an issue of great importance is the selection of appropriate patients in whom the treatment is to be evaluated. Patients with most severe glomerular lesions on renal biopsy appear to be at greatest risk of progressive renal deterioration and, therefore, the most appropriate candidates for specific therapeutic interventions. Controlled Trial by the Japanese Pediatric IgA Nephropathy Treatment Study Group 1. Combined therapy for severe childhood IgA nephropathy (J Am Soc Nephrol 1999;10:101–109) Seventy-eight children with newly diagnosed IgA nephropathy showing diffuse mesangial proliferation were randomly assigned to receive either the combined therapy of prednisolone, azathioprine, heparin-warfarin, and dipyridamole for two years (group 1), or the combination of heparin-warfarin and dipyridamole for two years (group 2). Urinary protein excretion was significantly reduced in group 1 patients, but remained unchanged in group 2 patients. Serum IgA concentration was also significantly reduced in group 1 patients, but was unchanged in group 2 patients. Blood pressure and creatinine clearance were normal at the end of the trial in all but one group 2 patient, who developed chronic renal insufficiency. The percentage of glomeruli showing sclerosis was unchanged in group 1 patients, but significantly increased in group 2 patients. The intensity of mesangial IgA deposits significantly decreased in group 1 patients, but remained unchanged in group 2 patients. The beneficial effects of the prednisolone, azathioprine, heparin-warfarin and dipyridamole treatment were accompanied by relatively few serious side effects specifically attributable to the drugs. 2. Steroid treatment for severe childhood IgA nephropathy (Clin J Am Soc Nephrol 2006, in press) In this study we compared the effects of prednisolone, azathioprine, warfarin, and dipyridamole (combination) with those of prednisolone alone in 80 children with newly diagnosed IgA nephropathy showing diffuse mesangial proliferation. Patients were randomly assigned to receive either the combination or prednisolone alone for two years. The primary endpoint was the disappearance of proteinuria, defined as urinary protein excretion 0.1 g/m2/day, and the secondary endpoints were urinary protein excretion at the end of treatment, the change in the percentage of sclerosed glomeruli during the trial and side-effects. The two study groups were similar in terms of baseline characteristics. Thirty-nine of the 40 patients receiving the combination and 39 of the 40 receiving prednisolone completed the trial. Thirty-six of the 39 patients (92.3%) receiving the combination and 29 of the 39 (74.4%) receiving prednisolone reached the primary endpoint by the two-year follow-up point (p  0.007 logrank). The percentage of sclerosed glomeruli was unchanged in the patients receiving the combination, but increased from 3.1  4.8% to 14.6  15.2% in the prednisolone group (p  0.0003). The frequency of sideeffects was similar in the two groups. We conclude that combination treatment may be better for severe IgA nephropathy than treatment with prednisolone alone.

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Interpretation of Renal Biopsies in IgA Nephropathy Terence Cook Department of Histopathology, Imperial College, London, UK IgA nephropathy is defined by the presence of dominant or co-dominant mesangial deposits of IgA. Lupus glomerulonephritis with extensive IgA is excluded. It may be primary, part of the syndrome of Henoch-Schonlein purpura, or secondary to a number of other extra-renal conditions, particularly diseases of the liver or gastrointestinal tract. It is the most prevalent form of glomerulonephritis worldwide. Frequency of biopsy diagnosis depends both on policies for screening of asymptomatic individuals and criteria for renal biopsy particularly in patients with isolated haematuria. By light microscopy the glomeruli may show any of the morphologic manifestations of immune complex glomerulonephritis from normal, though mesangial proliferation, endocapillary proliferation and segmental necrosis to severe crescentic glomerulonephritis. Segmental proliferation and segmental scars are common. Glomerular scarring is associated with tubular atrophy and interstitial fibrosis. By immunohistochemistry IgA is the sole immunoglobulin in 26% of biopsies. 25% have IgA, IgG and IgM. C3 is present in 95%. C1q is present in only 12% of biopsies and if prominent should raise the possibility of SLE. Relationship of Clinical and Biopsy Features to Presentation and Outcome 1. At the initial biopsy, serum creatinine is correlated with interstitial fibrosis and globally sclerosed glomeruli. Haematuria and proteinuria are strongly correlated with crescents. Proteinuria is correlated with the amount of mesangial proliferation. 2. Predictors of progression to ESRD. a. Clinical – strong predictors are elevated creatinine at presentation, severe proteinuria and hypertension. Weaker predictors are absence of a history of macroscopic haematuria, male sex and older age at presentation. b. Pathological – strong predictors are severe glomerular and tubulointerstitial scarring. Weaker predictors are crescents, degree of mesangial proliferation, capillary wall deposits of IgA and marked arteriolar hyalinosis. These are not independent predictors in multivariate analysis. Histological grading systems have been used with some success to give an indication of likelihood of progression to ESRD. One example is the Haas classification. 3. Rate of decline of renal function is associated with proteinuria, blood pressure and histological grading but only blood pressure and proteinuria during follow-up are independent predictors. In patients with no, or only mild, abnormalities of renal function at presentation the amount of glomerular sclerosis is an independent predictor of renal outcome. Further studies will be necessary to determine whether histological features can predict response to treatment. Several studies have examined the effect of immunosuppressive treatment on renal morphology in repeat renal biopsies. Most show that treatment is associated with a reduction in mesangial proliferation, mesangial matrix accumulation and cellular crescents. In one there was also a reduction in segmental glomerulosclerosis and tubulointerstitial volume.

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Basic Reviews of IgA Nephropathy

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Innate Responses Modulate IgA Immunity Steven N. Emancipator, Michifumi Yamashita, Marc Buren Department of Pathology, Case Western Reserve University, Cleveland, Ohio, USA Defective humoral immune responses to mucosal immunogens, but intact responses to systemic or parenteral immunogens, are increasingly recognized in patients with IgA nephropathy (IgAN). Deficiency in mucosal immunity in IgAN is associated with reduced expression of J chain in gut lamina propria cells producing IgA, and with collateral increases in J chain expression in marrow cells producing IgA. Thus, there seems to be derangement within a ‘mucosa – marrow axis’ in IgAN patients. Recent evidence indicates that the localization of B cells into respiratory and gastrointestinal lamina propria is governed by the expression of chemokine receptors, in addition to the more established role of integrins (e.g. those of the 7 family). Particularly, airway mucosa expresses the chemokine CC9 whereas CXC28 is preferentially produced in the gut. Accordingly, there is reciprocal differential distribution of B cells that express the cognate receptors for these chemokines, and consequently subcompartmentalization of the mucosal immune system. Similarly, B cell commitment to IgA synthesis, expression of J chain and terminal differentiation to plasma cells are processes regulated by autocrine and paracrine factors. In turn, many of these factors are themselves induced by specific (adaptive) immune responses as well as by innate responses of lymphocytes, accessory cells and the mucosal epithelium to pathogen associated molecular patterns, and in particular engagement of toll-like receptor 3 and the helicases RIG-I and MDA-5 by viruses. These ‘viral sensors’ evoke intersecting activation of several key transcriptional regulators, notably IRF-7, NFB, IL-6, c-jun and smads 4 and 6. The potential contribution of virally induced innate responses to defective mucosal immunity and overproduction of oligomeric (J chain-containing) IgA in the marrow will be reviewed. In addition to modulation of the afferent limb of mucosal and systemic IgA responses, these same factors powerfully influence aspects of the efferent limb of IgA effectors. The level of IgA antibody in secretions, and therefore IgA agglutinating ability, is dependent on the expression of J chain by mucosal B cells and plasma cells, and of polymeric Ig receptor (pIgR) by mucosal epithelium. Likewise, the capacity for IgA to support its functions in intracellular neutralization of pathogens and excretion of antigens from within the mucosal tissue into the secretions depends on J chain and pIgR synthesis. IgA-mediated uptake and catabolism of antigens requires surface expression of CD89 on phagocytes, and is inhibited by free soluble CD89 as a competitor with cell bound CD89. Innate responses to viruses, and perhaps other pathogens, can reduce J chain, pIgR and CD89 expression on the relevant cell populations and increase the ratio of (inhibitory) soluble CD89 to cell surface CD89. All of these effects can simultaneously and synergistically impair IgA-mediated protection of mucosal surfaces while promoting access of antigen to the systemic immune system and deposition of IgA complexes in glomeruli. The ramifications of and evidence for these effects will be summarized.

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IgA Nephropathy (IgAN): Current Views of Immune Complex Formation Jiri Mesteckya, Hitoshi Suzukia,b, Takeshi Yanagiharaa,c, Zina Moldoveanua, Milan Tomanaa, Karel Matousovica,d, Bruce Juliana, Jan Novaka University of Alabama at Birmingham, Birmingham, Ala., USA; bJuntendo University, cNippon Medical School, Tokyo, Japan; dUniversity Hospital Motol, Prague, Czech Republic

a

Characteristic features of IgAN include the presence of IgA1-containing immune complexes in the circulation, urine, and renal mesangium. Several studies indicate that IgAN is an autoimmune disease. Immune complexes contain IgA1 deficient in hinge region-associated galactose (Gal) and antibodies specific for antigenic determinants present on the hinge region. Nevertheless, the biological effects of immune complexes are primarily related to their molecular size and composition: when added to cultured human mesangial cells, large immune complexes exhibit a proliferative effect while small complexes are inhibitory. These activities have been observed using immune complexes obtained from sera of IgAN patients as well as complexes generated in vitro. Specifically, various preparations of human IgA1 with modified glycan moieties formed immune complexes in vitro when incubated with IgG from sera of IgAN patients, healthy individuals, cord blood, or tissue culture supernatants of EBV-immortalized peripheral blood B cells. Depending on their molecular size and amount, these complexes stimulated cultured human mesangial cells to proliferate. Interestingly, IgG antibodies specific for human IgA1 Gal-deficient hinge region are commonly found in sera of hominoid as well as non-hominoid primates and many vertebrate species, and suggest the evolutionary uniqueness of the human IgA1 hinge region. Because of the molecular defect in IgA1 glycosylation and its subsequent recognition by naturally-occurring antibodies, experimental approaches that would interfere with or prevent formation of large immunostimulatory complexes should be further explored to decrease the molecular size of immune complexes. The generation of monovalent single-chain antibodies specific for antigenic determinants on IgA1 hinge region, or small peptides containing a single, glycan-associated antigenic determinant, may represent a potential direction for future studies. Supported by NIH grant 5 P01 DK061525.

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IgA Receptors Renato C. Monteiro INSERM U699 and Bichat Medical School, Paris, France The IgA receptor family includes the polymeric Ig receptor involved in trans-epithelial transport of IgA/IgM, the myeloid specific IgA Fc receptor (FcRI or CD89), Fc/ R and at least two alternative IgA receptors, asialoglycoprotein receptor and transferrin receptor (TfR) [1]. Two of these receptors are directly involved in the pathogenesis of IgA nephropathy (IgAN), FcRI and TfR. The FcRI is expressed on myeloid cells but not on mesangial cells [1]. It binds both IgA subclasses with low affinity. FcRI functions as a molecular switch that mediates both pro- and anti-inflammatory functions through the ITAM motif of the common FcR signalling subunit [2]. Anti-inflammatory signaling is triggered by monomeric targeting of FcRI and involves SHP-1 tyrosine phosphatase. In patients with IgAN, a reduced expression of FcRI is observed on the surface of circulating monocytes [3]. This may be due to shedding of a 50–70 kDa FcRI which is increased in serum of patients [4]. This soluble FcRI complexed with IgA is pathogenic in vivo in human FcRI transgenic mice that spontaneously develop IgAN [4]. However, another form of soluble FcRI (30 kDa) has been described which is not involved in IgAN [5]. Although transmembrane FcRI expression is

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decreased on blood phagocytes of patients, increased levels of IgA bound to these receptors are linked to the appearance of glomerulosclerosis and mesangial proliferation [3]. Recently, we demonstrated that membrane FcRI mediates cell infiltration in kidney and IgAN progression. To analyze the involvement of FcRI activation in nephritis development, we generated transgenic mice expressing a mutated, signaling incompetent, human FcRIR209L that cannot associate with FcR , and compared them to mice expressing wild-type FcRI in spontaneous FcRI-mediated IgAN model. Activation of FcRI resulted in marked inflammation with severe proteinuria and leukocyte infiltration indicating that it is crucial in the progression of kidney disease. The molecular mechanism involves activation of a cytokine/chemokine cascade that promotes leukocyte recruitment and kidney damage. The potential relevance in IgAN is highlighted by the capacity of IgA-IC from patients to activate FcRI and induce TNF production. TfR is the only IgA receptor described on mesangial cells. It binds IgA1, but not IgA2 [6]. Its expression is enhanced in the mesangium of IgAN patients and co-localizes with IgA1 deposits [6]. Thus, it may participate in the selective deposition of IgA1 complexes. Our recent studies indicate that abnormally glycosylated IgA1 and IgA1 complexes may favor the interaction with TfR as observed on cultured normal human mesangial cells [7]. Furthermore, IgA1 polymers can induce TfR expression, cytokine release and mesangial cell proliferation, all of which could in part be responsible for the observed injury and recurrence of deposits after transplantation [8].

References 1 2 3 4 5 6 7 8

Monteiro RC, Van De Winkel JG: Annu Rev Immunol 2003;21:177. Pasquier B, et al: Immunity 2005;22:31. Grossetete B, et al: Kidney Int 1998;53:1321. Launay P, et al: J Exp Med 2000;191:1999. Van Der Boog PJ, et al: Kidney Int 2003;63:514. Moura IC, et al: J Exp Med 2001;194:417. Moura IC, et al: J Am Soc Nephrol 2004;15:622. Moura IC, et al: J Am Soc Nephrol 2005;16:2667.

BS-04

Bone Marrow and Mucosa Axis in IgA Nephropathy Yusuke Suzuki, Yasuhiko Tomino Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, Tokyo, Japan Large numbers of studies have demonstrated that abnormal IgA/IgA immune complexes (IC) including aberrantly glycosylated IgA or polymeric IgA IC are detected in patients with IgA nephropathy (IgAN), suggesting that humoral immune dysfunction may contribute to the pathogenesis of this disease. On the other hand, abnormal cellular immune responses in IgAN have also been described in many previous papers. For example, some groups recently proposed an idea that a hygiene hypothesis may be adapted to explain the regional deviation of IgAN, suggesting that Th2 bias may exist in this disease. Although several responsible cell types have been suggested, there has been no clear explanation associating the two abnormalities in humoral and cellular immunity. Clinical evidence such as recurrence of IgAN after kidney transplantation indicates that the primary pathogenesis of IgAN may occur outside the kidneys. Remission or reconstitution of IgAN by bone marrow transplantation (BMT) in humans or experimental animal models further suggests that BM may play a key role in the pathogenesis. However, deterioration of this disease after upper respiratory tract infection and satisfactory therapeutic outcome

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by tonsillectomy with steroid therapy in Japan indicate involvement of mucosal immunity in the pathogenesis. Therefore, both BM and mucosa may be key participants in the pathogenesis, but the underlying mechanism linking both sites is still unknown. Appropriate models are required to approach those missing links. Our recent study showed that the ‘grouped ddY mouse’ is a useful model for the pathogenesis of IgAN (J Am Soc Nephrol 2005;16:1289). The onset ddY mice show mesangial proliferative glomerulonephritis with glomerular IgA and IgG co-deposition as in human IgAN. Moreover, an association study between onset and quiescent ddY mice revealed that susceptible genes of murine IgAN largely overlap those of human IgAN including IGAN1 and selectin, suggesting that human and murine IgAN may be partially regulated by the same genes. These findings indicate that certain common mechanisms involved may be approached by this model. Using this model and combination models, we are investigating underlying mechanisms by which BM and mucosal cells interact and finally induce this disease. To date, these studies have revealed that (1) Th polarity shift regulated by BM is closely linked to the onset of this disease. (2) Th2 bias in mucosal site may play an important role in pathogenic IgA production. (3) BM from onset animals can reconstitute glomerular IgA deposition independently of secondary lymph nodes (LN) including Payer’s patches, but disease progression may require secondary LN. (4) Innate immunity, especially TLR9 activation, may be involved in the disease progression. Based on these findings, we also clinically examined the therapeutic rationale of tonsillectomy with steroid therapy. Clinical analysis demonstrated that tonsils may be one of the critical mucosal sites for IgAIC formation and progression, partly via TLR9 activation.

LS-02

Searching for IgA Nephropathy Candidate Genes. Genetic Studies Combined to a High Throughput Innovative Investigation Francesco P. Schenaa, Giuseppina Cerulloa, Diletta D. Torresa, Gianluigi Zazaa, Sharon Natasha Coxa, Luigi Biscegliab, Francesco Scolaric, Antonio Amorosod, on behalf of the European IgA Nephropathy Consortium a Renal Unit, University of Bari, Bari, bMedical Genetic Service, IRCCS-Casa Sollievo della Sofferenza, San Giovanni Rotondo, Foggia, cRenal Unit, Spedali Civili, Brescia, dGenetic Unit, University of Torino, Torino, Italy

IgA nephropathy (IgAN) is the most common biopsy-proven glomerulonephritis worldwide with pathogenesis still poorly understood. Striking variation in prevalence among different ethnic groups, and familial aggregation, together with an increased familial risk, suggest important genetic influences in the pathogenesis of the disease. Two independent linkage studies have demonstrated the existence of at least three different genetic loci for familial IgAN, called IGAN1 on chromosome 6q22–23 (6,5cM), IGAN2 on chromosome 4q26–31 (21cM), IGAN3 on chromosome 17q12–22 (15cM). Linkage to multiple chromosomal regions is consistent both with an oligo/polygenic and a multiple susceptibility gene model with small/moderate effects in determining the pathological phenotype for familial IgAN. The gene(s) associated with onset and/or progression of IgAN has not yet been identified. The chromosomal gene map of the National Centre for Biotechnology Information (http://www.ncbi.nlm.nih.gov) includes 38, 97 and 347 known genes in these three loci, respectively. Different approaches may be taken into consideration for further investigations. First of all we evaluated those genes possibly involved in the main pathogenetic mechanisms of IgAN, searching for potential candidate genes in the available chromosomal map. We identified the following genes of major interest located in IGAN1 (serine/threonine protein kinase– SGK; vanin 3– VNN3); IGAN2 (transient receptor potential channel 3– TRPC3; interleukin 2–IL-2; interleukin 12–IL-12) and IGAN3 (histone deacetylase 5– HD5; granulin–GRN; T-box 21–TBX21). These genes, for their function, could

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be responsible for immune-response deregulation or could cause abnormal synthesis of deglycosylated IgA1. Case-control and family-based association studies evaluating the distribution of these candidate gene polymorphisms are in progress. These studies have been organized by the European IgAN Consortium (URL: http://www.igan.net) which has constituted a biobank collecting large sample size of biological specimens from well characterized IgAN patients, their relatives, and healthy subjects, resulting in an invaluable source for genetic and genomics studies of this complex disease. The other approach to gain information on genes responsible for IgAN is represented by global gene expression analysis using microarray technology. Gene expression analysis in leukocytes and renal tissue could define a set of genes whose expression level portray unequivocally the onset or progression of the disease. Subsequently, the genetic and genomics studies combine sequence polymorphisms with variation in expression level. These merged high throughput technologies aim to explain the pathogenesis of IgAN to provide potential targets for screening, prevention and early diagnosis of the disease and a more appropriate and effective treatment.

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Clinical Advances (Update)

C-O-01

Long-Term Survival in IgA Nephropathy Using Lee’s Glomerular Grading System Ladislava Grcevska, Sonja Dzikova, Vesna Ristovska, Maja M. Popovska, Momir Polenakovic Department of Nephrology, Clinical Center, Skopje, R. Macedonia H.S. Lee’s grading system for IgA nephropathy (IgAN) was defined as follows: grade I (normal or focal mesangial cell proliferation), grade II (diffuse mesangial cell proliferation or 25% of glomeruli with crescents (cr)/segmental sclerosis (ss)/global sclerosis (gs)), grade III (25–49% of glomeruli with cr/ss/gs), grade IV (50–75% of glomeruli with cr/ss/gs) and grade V (
75% of glomeruli with cr/ss/gs). We classified 81 patients with IgAN according to H.S. Lee’s system in order to present renal survival in different histological grades. Grade I: 31 patients, survival rate for the first 5 years was 100%, 6/31 experienced end-stage renal failure between 5 and 10 years and one after 12 years (total 7/31, 22.6%). 10/31 (32.6%) were followed at least 15 years, with normal renal function. Grade II: 32 patients, one of them started dialysis after 4 years, 6 cases between 5 and 10 years (total 7/32, 21.9%). Two patients were followed at least 15 years; they are with normal renal function. Grade III: 12 patients, 4 (33.3%) started dialysis before 5 years of follow-up, 3 (25%) between 5 and 10 years, and 5 patients survived 10 years with normal renal function (41.5%). Grade IV: 3 patients, all with end-stage renal failure within 5 years. Grade V: 3 patients, all with end-stage renal failure within 2 years. We can conclude that renal survival in IgAN in our patients correlated with the degree of the histological impairment of the kidney, but long-term survival in grade II (15 years) and grade III (10 years) presents the slow evolution of glomerular changes (crescents, sclerosis) found on biopsy. This phenomenon is not common for other forms of glomerulonephritis.

C-O-02

Major Risk Factors for Progression to End-Stage Renal Failure (ESRF) Revisited and Simplified in Primary IgA Nephropathy (IGAN): The Absolute Renal Risk (ARR) Evaluation François C. Berthoux, Aida Afiani, Blandine Laurent, Christophe Mariat, Hesham Mohey NDT Department, University North Hospital, Saint-Etienne, France In IgAN, the three major and consensus risk factors (RF) present at diagnosis and known to predict progression to chronic renal failure (CRF, defined as eGFR below 60 ml/min) and to end-stage renal failure (ESRF, defined as eGFR below 15 ml/min with the need for dialysis) are arterial hypertension (HT); the amount of proteinuria and severity of optical renal lesions (such as glomeruli with obsolescence and/or hyalinosis and/or crescents and others) together with at least 1 mesangial deposit of dominant or codominant IgA on immunofluorescence.

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In our prospective cohort of 333 cases (242 males  72%; mean age  35.6 years and median age  34.4 years at onset) of primary IGAN diagnosed at our institution from 1/01/1990 to 31/12/1999, we have reevaluated these 3 RF as independent predictors for both CRF and ESRF/dialysis and as part of an absolute renal risk (ARR) of the individual patient with 0RF, 1RF, 2RF and 3RF at time of diagnosis. HTA was defined as BP up to 140/90 mm Hg with the need for antihypertensive medication including diuretics (yes or no). 24-hour proteinuria was expressed in g/day and classified as follows: class 0  absent  below 0.30 g/day; class 1  0.30–0.99 g/day; 2  1–2.99 g/day; and 3  massive over 3 g/day (proteinuria up to 1 g/day; yes or no). The renal pathology global optical score (GOS1) was the sum of glomerular (0–6), vascular/arteriolar (0–5), interstitial (0–5) and tubular (0–4) elementary lesions as already described. The ROC analysis gave a threshold value of GOS up to 8 units (yes or no). The mean and median follow-up times were respectively 12.2 and 10.4 years. We used the Cox regression model and the Kaplan-Meier survival curves with time zero as onset of the disease, ESRF/dialysis (n  25) as the primary end-point, and CRF (n  84) as the secondary end-point. It was confirmed that these RF were highly predictive for ESRF-Dialysis: RR (95% CI)  0.06 (0.01–0.28); p  0.0001 for absence of HT at diagnosis; RR  1.82 (1.54–2.16); p  0.0001 for each g/day of proteinuria; and RR  1.47 (1.32–1.64) for each unit of GOS1. The distribution of ARR at diagnosis was 155 patients (46%) with ORF (ARR  0); 69 (21%) with 1 RF (ARR  1); 66 (20%) with 2 RF (ARR  2); and 43 patients (13%) with 3 RF (ARR  3). In fact at 20 years from onset, the percentage of patients with ESRF/dialysis was 46% for ARR  3; 7% for ARR  2; 5% for ARR  1 and 0% for ARR  0 (p  0.0001). At 10 years from onset, the % of patients with CRF was 54% with ARR3 (high), 37% with ARR2 (medium); 16% with ARR1 (moderate) and only 4% with ARR0 (very low/nil); p  0.0001. We confirmed that the absolute renal risk evaluation at diagnosis, based on the presence of the following RF: HT (yes/no); Proteinuria up to 1 g/day (yes/no) and GOS1 up to 8 (yes/no) is simple and sufficient to predict accurately the ultimate progression to CRF and then to ESRF/dialysis. Patients with HIGH ARR (3 RF) should be specifically targeted for the proper evaluation of specific treatment in IgA nephropathy.

C-O-03

Establishing a Simple Semiquantitative Histological Scoring System for IgA Nephropathy Lei Jiang, Ji-Cheng Lv, Wenfang Chen, Suxia Wang, Gang Liu, Wanzhong Zou, Hong Zhang, Hai-Yan Wang Renal Division of Peking University First Hospital, Peking University Institute of Nephrology, Beijing, China IgA nephropathy (IgAN) is a common and progressive disease. The aim of this study was to establish a less complicated semiquantitative histological scoring system that can differentiate active lesions from chronic ones, containing only the lesions most correlating with renal outcome. One hundred and fifty five cases diagnosed as primary IgA nephropathy with more than 2 years follow-up information were enrolled into our study, 25 (16.13%) of whom progressed into irreversible end-stage renal disease (ESRD) during follow-up (69.07  28.66 months, range 10–170 months). Serum creatinine at biopsy was 112.18  83.13 mol/l. The histological variables initially included: (1) extracapillary glomerular activity index (exGAI); (2) endothelial proliferative index (endoI); (3) mesangial hypercellularity index (MsAI); (4) glomerular chronicity index (GCI); (5) widening of mesangial area (MsCI); (6) tubular atrophy and interstitial fibrosis (TCI), and (7) vascular chronicity index (VCI). In multivariate analysis using the Cox proportional hazard model, MsCI, MsAI and VCI were found to have poor correlation to renal survival rate, with p values of 0.907, 0.529 and 0.446, respectively (enter method). GCI and TCI were added to form the chronicity index (CI). CI and exGAI were both proved ‘independent’ factors in predicting renal outcome (p  0.05, RR  1.694 and 1.377, respectively). The reproducibility of the scoring system was proved acceptable (inter- and intra-observers’ kappa values
0.4). This relatively concise histological scoring system containing exGAI and CI had good reproducibility and correlates well with the renal survival rate of IgAN. It can be used in further clinical and research work.

Clinical Advances (Update)

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C-O-04

Glomerular Basement Membrane (GBM) Thickness as an Additional Risk Factor for Progression Towards End-Stage Renal Failure in Primary IgA Nephropathy (IGAN): A Long-Term Prospective Study François C. Berthoux, Carole Deprele, Blandine Laurent, Christophe Mariat NDT Department, University North Hospital, Saint-Etienne, France In 1990, we started a prospective systematic measurement of GBM thickness in all incident cases of IGAN diagnosed between 1 Jan 1990 and 31 Dec 1999. This was done by electron microscopy (EM) with an automatic image analyzer at 8,000 magnification with at least 100 different measurements for each biopsy. The normal range for GBM thickness in normal kidneys was mean (SD)  371 (48) nm in males and 350 (44) nm in females. Thin GBM was defined as mean below 275 nm for males and 265 for females; Thick GBM was defined as mean over 465 nm for males and 436 nm for females; Normal GBM was in between. Finally out of 333 diagnostic biopsies, we achieved successful measurement in 270 cases (81%) [195 males (72.2%); mean age at onset  34.7 (15.1) years] with normal GBM in 174 patients (142 males  81.6%), thin GBM in 80 patients (40 males  50.0%), and thick GBM in only 16 patients (13 males  81.3%). Thin GBM was clearly more frequent in females:

2  28.0; p  0.0001. The overall exposure time from onset was mean (SD)  12.3 (9.2) years. At time of diagnosis, the three major risk factors for progression were distributed as follows: arterial hypertension (HT) was present respectively in 20.0% (16/80), 34.5% (60/174), and 68.8% (11/16) in IGAN patients with thin or normal or thick GBM. Proteinuria over 1 g/day was present respectively in 18.8% (15/80), 29.3% (51/174), and 50% (8/16) in patients with thin or normal or thick-GBM. Renal pathology disclosed a global optical score (GOS1)
8 (ROC curve) in 21.3% (17/80), 36.8% (64/174), and in 56.3% (9/16) with thin or normal or thick GBM. The % of patients with all 3 RF present at diagnosis (highest absolute renal risk) was respectively 8.9% (7/80), 9.2% (16/174), and 50.0% (8/16) in patients with thin or normal or thick GBM. At last follow-up (FU) progression to CRF (eGFR  60 ml/min) was observed in 10.0% (8/80), 18.3% (31/174), and 53.3% (9/16) respectively in patients with thin or normal or thick-GBM. At last FU, progression to ESRF/Dialysis was noticed in 3.8% (3/80), 5.2% (9/174), and 43.8% (7/16) respectively in patients with thin or normal or thick GBM. Cox regression analyses confirmed that both the mean GBM thickness was associated with an increased risk for ESRF/dialysis: B/SE  3.65; p  0.0003 and RR  1.008 (1.004–1.013) for each nanometer increment and that thick GBM is deleterious: B/SE  2.92; p  0.0035; RR  4.65 (1.66–13.05). In conclusion: Renal pathology should include, beside immunofluorescence and optical microscopy examinations, GBM thickness measurement which appears as an additional risk factor for progression towards CRF and ESRF.

C-O-05

Aberrant Sialylation of Serum IgA1 Is a Predictor of Poor Prognosis in IgA Nephropathy Jia-Xiang Ding, Li-Xia Xu, Ji-Cheng Lv, Ming-Hui Zhao, Hai-Yan Wang, Hong Zhang Renal Division of Peking University First Hospital, and Peking University Institute of Nephrology, Beijing, China Aberrant glycosylation of serum IgA1 might play an important role in the pathogenesis of IgAN. Our previously work demonstrated that deglycosylation of serum IgA1 was correlated with pathological lesions of patients with IgAN. The aim of this study was to investigate whether asialylation of serum IgA1 was associated with renal

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survival in IgAN. 127 patients with biopsy-proven IgAN and followed up to 8 years were enrolled. 79 healthy persons and 42 patients with non-IgAN renal disease were selected as controls. The level of 2,6 sialic acid (SA) in serum IgA1 was measured by sandwich ELISA. Renal survival was estimated by the Kaplan-Meier method. The 2,6 SA level in patients with IgAN was lower than that in normal controls (p  0.001). These patients were further stratified as two groups: Group I (2,6 SA level  0.78) and Group II (2,6 SA level
0.78). There were no significant differences in age, gender, hypertension, serum creatinine, or proteinuria excretion between the two groups. However, the renal function evaluated by eGFR was worse in Group I than in Group II (p  0.03). However the SA level was not associated with the renal function injury in the case controls. After the eight-year follow-up, renal survival was 53.3% in Group I and 83.5% in Group II (p  0.0008). Also, 2,6 SA level of serum IgA1 was associated with the prognosis of patients with IgAN and could serve as a predictor of poor prognosis of IgAN.

C-O-06

Clinicopathological Influence of Obesity in IgA Nephropathy: Comparative Study of 74 Patients Mari Tanakaa, Tomomi Tsujiia, Toshiyuki Komiyaa, Satomi Yonemotoa, Tatsuo Tsukamotoa, Takeshi Sugishitaa, Yukako Iwasakia, Satoshi Fukuib, Akimasa Takasub, Eri Musoa Nephrology and bPathology, Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka, Japan

a

The pathological role of obesity in the progression of glomerular lesion has been suggested in obesity-related nephropathy, but rarely been studied in primary glomerular diseases. The purpose of this study is to examine the clinicopathological influence of excessive body weight in IgA nephropathy. 74 patients with renal biopsy-proven IgA nephropathy in our institute from October 2000 to January 2004 were retrospectively divided into two groups according to body mass index (BMI); non-obese group (group N) with BMI 25 kg/m2 and obese group (group O) with BMI 25 kg/m2. Patients with diabetes mellitus or autoimmune diseases were excluded. There were 50 patients in group N (17 males and 33 females) and 24 patients in group O (18 males and 6 females). Clinical analysis showed no significant difference between these two groups in blood pressure, serum cholesterol, albumin, IgA, creatinine clearances and the grade of hematuria. However, 24-hour urinary protein excretion and serum creatinine were significantly greater in group O compared to group N (0.74  0.83 vs. 0.43  0.50 g; p  0.026, 0.97  0.44 vs. 0.73  0.23 mg/dl; p  0.001). Although semiquantitative analysis of light microscopical findings showed no significant difference of the severity of mesangial proliferation, matrix expansion, glomerulosclerosis and the crescent formation, computer-aided image analysis showed that total glomerular area and tuft area were significantly larger in group O. In addition, ultrastructural study revealed significantly higher mean glomerular basement membrane thickness in group O (402  100 vs. 322  82 nm; p  0.001). On the other hand, immunopathological study revealed that the percentage of -SMA positive area per tuft area was similar between two groups. 62 patients (46 patients, group N, 16 patients, group O) were followed in our institute for one year. Urinary protein was significantly decreased only in patients who received steroid in both groups. Although administration of ACE inhibitor or ARB tended to decrease urinary protein in group O, the change was not statistically significant (p  0.068). Our findings indicate that obesity may accelerate the increase of proteinuria in IgA nephropathy through matrix expansion especially with ultrastructural modification of glomerular basement membrane.

Clinical Advances (Update)

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C-O-07

The Deletion Allele of Angiotensin-Converting Enzyme Gene Is Closely Related to the Prognosis of IgA Nephropathy Shuwen Liu, Rui Ding, Xiangmei Chen, Jie Wu, Lijun Shen, Yang Lu, Pu Chen Department of Nephrology, General Hospital of PLA, PLA Research Institute of Kidney Disease, Beijing, P.R. China Objective: Being the most common glomerulonephritis worldwide, IgA nephropathy (IgAN) accounts for 30–40% of the primary glomerulonephritis cases in China. 30–40% of the patients progressed to end stage renal failure in twenty years followed renal biopsy. The present study was designed to investigate the role of angiotensinconverting enzyme (ACE) insertion/deletion polymorphism in the pathogenesis and progression of IgAN. Patients and Methods: Six hundred and forty-one patients with biopsy-proven IgAN were enrolled. We observed the blood pressure, and determined urinary protein excretion, serum creatinine and creatinine clearance, etc. The renal pathological changes were evaluated with Katafuchi Scoring System. Two stepwise polymerase chain reactions were performed to determine ACE genotypes, and SPSS software was used for statistical analysis. Results: (1) There was no significant difference in ACE genotype frequency and allele frequency between IgAN patients and normal controls (p
0.05). (2) Creatinine clearance (Ccr) was markedly lower in DD and ID groups than that in II group (p  0.01). (3) There was no significant difference of ACE genotype frequency and allele frequency between patients with or without gross hematuria (p  0.210), hypertension (p  0.127) and urinary protein excretion (UPE) no less than 2.0 g/day (p  0.188). (4) In the group with normal serum creatinine (SCr), or Ccr 60 ml/min, or normal blood pressure accompanied with both UPE 2.0 g/day and Ccr 60 ml/min, or in the group with mild renal injury, or mild glomerular injury, or mild tubulointerstitial lesions or less tuft adhesion, there was a higher II genotype frequency and I-allele frequency as compared with its respective group (p  0.05). (5) The glomerular score was significantly higher in DD and ID groups than that in II group (p  0.05). The total Katafuchi score was also higher in ID group than that in II group (p  0.017). Conclusion: Although no association was found between the ACE insertion/deletion polymorphism and the onset of IgA nephropathy, homozygote DD was closely associated with early renal insufficiency, moderate to severe glomerular and tubulointerstitial lesions, implying that DD genotype might be a genetic marker of poor prognosis in IgA nephropathy.

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C-O-08

A Multicenter Prospective Cohort Study of Tonsillectomy and Steroid Pulse in Japanese Patients with IgA Nephropathy: A 5 Year Report Mariko Miyazakia, Atsushi Komatsudab, Tatsuya Shojic, Chikao Yasunagad, Shigeru Nakaie, Osamu Hottaa, Hirokazu Imaif, Yoshio Tagumaa, Japanese Multicenter Study Group on the Treatment of IgA Nephropathy (JST-IgAN) a

Department of Nephrology, Sendai Shakaihoken Hospital, Sendai, Miyagi, bThird Department of Internal Medicine, Akita University School of Medicine, Akita, cDepartment of Nephrology, Osaka General Medical Center, Osaka, dThe Kidney Center, Saiseikai Yahata Hospital, Kitakyushu, e Department of In-Home Medicine, Nagoya University School of Medicine, Nagoya, fDivision of Nephrology and Rheumatology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Japan Although tonsillectomy combined with corticosteroids has became popular for the treatment of IgA nephropathy in Japan, there was no prospective study including these combined treatment. We have conducted a multicenter prospective cohort study and presented 2-year interim report in ASN’s 37th meeting. We herein report the latest clinical data. Eighty-seven cases were observed for 5 years, 24 cases were observed for 4 years. Subjects were graded as follows; proteinuria(UP) was excreted below 0.5 g/day (Stage 1), 0.5–1.0 g/day (Stage 2), more than 1.0 g/day and serum creatinine less than 1.5 mg/dl (Stage 3). Tonsillectomy and steroid therapy were performed in 37 (Stage 1), 32 (Stage 2), and 17 (Stage 3) patients. The number of subjects with steroid monotherapy were 1 (Stage 1), 6 (Stage 2), and 6 (Stage 3). Two cases treated with combined methods and 1 treated with monotherapy showed 50% or higher value of serum creatinine compared with baseline. The primary endpoint in this study was the normalized urinalysis as clinical remission. The remission rate treated with combined therapy was 83.8% in Stage 1, 65.6% in Stage 2, and 41.1% in Stage 3. On the other hand, it was 50% in Stage 2, and 33% in Stage 3 of monotherapy subjects. We concluded that the tonsillectomy combined steroid therapy was effective in early stage to release from the disease. Furthermore, the combined therapy is able to intercept from a progression of disease in higher proteinuria group.

C-O-09

The Efficacy of Steroid Pulse Therapy in IgA Nephropathy: A Multivariate Analysis Using Cox’s Proportional Hazard Model Ritsuko Katafuchia, Toshiharu Ninomiyab, Tohru Mizumasaa, Kiyoshi Ikedaa, Harumitsu Kumagaia, Hideki Hirakatab Kidney Unit, Fukuoka Red-Cross Hospital (previous office), Koga, and bThe Department of Medicine and Clinical Science, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan

a

The ideal steroid protocol for IgA nephropathy has not been established yet. In the present study, the influence of clinical parameters, histological grade, and treatment on kidney survival was retrospectively examined by multivariate analysis using the Cox proportional hazards model in 702 patients with IgA nephropathy. Histological grade was determined as follows: grade I; glomerular score 1 or 2, grade II; glomerular score 3 or 4, grade III; glomerular score 5 or 6, grade IV; glomerular score 7 or 8, grade V; glomerular score over 9. Glomerular score, ranging from 1 to 12, was calculated as the sum of indices of the following three glomerular lesions: (1) hypercellularity

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(mesangial and endocapillary), (2) segmental lesions such as crescent, tuft necrosis, tuft adhesion, and segmental sclerosis, and (3) global sclerosis. There were 295 men and 407 women. The mean follow-up period was 6.2  4.0 years. The mean urinary protein-creatinine excretion ratio (UP-UCR) was 1.5  1.9 and the mean degree of hematuria was 2.2  0.9. The mean serum creatinine level was 0.98  0.58 mg/dl. Two hundred and twenty-seven patients were treated with steroids. Methylprednisolone (M-PSL) pulse therapy was undertaken in 33, and oral prednisolone (PSL) therapy was given in 194 patients. Two hundred forty-one were treated with angiotensinconverting enzyme inhibitor (ACE-I) or angiotensin II receptor antagonist (ARB). Twenty-eight patients had tonsillectomy. During the follow-up, eighty-five patients developed end-stage renal failure. In multivariate analysis, UP-UCR, serum creatinine and histological grade significantly increased the risk of development of ESRF (p  0.001). Steroid treatment, use of ACE-I or ARB, and tonsillectomy significantly decreased the risk of renal death. Multivariate-adjusted hazard ratio for kidney death in grade III showed a tendency to increase compared to that of grade I. The hazard ratios for development of ESRF in grade IV and V were significantly higher than grade I. When the hazard ratio for ESRF in the steroid-untreated patients with grade I or II was estimated 1.00 as control, in histological grade III, the hazard ratio for ESRF in steroid-untreated patients was significantly higher than in control, and, in steroid-treated patients the hazard ratio for ESRF was not significantly different from that of control. In histological grade IV and V, the hazard ratios for ESRF in both steroid-treated and steroid-untreated patients were significantly higher than in control. However, the hazard ratios in steroid-treated patients was about half or one-fifth of the hazard ratio in steroid-untreated patients in grade IV and V, respectively. As for the method of steroid treatment, the hazard ratio in patients with steroid pulse therapy was significantly lower than that in steroid-untreated patients. The hazard ratio in patients with oral steroid therapy was not significantly different from that in steroiduntreated patients. In conclusion, the patients with IgA nephropathy with moderate to severe histology, glomerular score over 5, should be treated by steroid pulse therapy.

C-O-10

Combination Therapy of Tonsillectomy and Corticosteroid Pulse to IgA Nephropathy Hirotsugu Iwatania, Ryohei Yamamotoa, Yasuyuki Nagasawaa, Takahito Itoa, Arata Horiib, Manabu Tamurab, Takeshi Kubob, Enyu Imaia Department of Nephrology and bDepartment of Otolaryngology, Osaka University School of Medicine, Suita, Osaka, Japan

a

Background: IgA nephropathy is the most common form of primary glomerulonephritis and its prognosis had turned out to be poor. Although antiplatelet agent, fish oil, angiotensin converting enzyme inhibitor, angiotensin receptor blocker and glucocorticoid have been put to clinical use, the effect has been unsatisfactory. Recently, tonsillectomy combined with corticosteroid pulse therapy has been shown to exert a long-lasting reno-protective effect to IgA nephropathy by retrospective study. Object: We investigated the short-term effect of the combination therapy of tonsillectomy and corticosteroid pulse to IgA nephropathy patients in Osaka University Hospital. Methods: In this study, the subjects were chosen as follows; biopsy-proven IgA nephropathy patients whose renal fibrosis was not severe and who could be followed as outpatients in Osaka university hospital. Severe renal dysfunction cases were excluded therefore, only cases with serum creatinine level at tonsillectomy is equal to or less than 1.3 mg/dl were enrolled. During the hospitalization in our hospital, tonsillectomy was performed and subsequently glucocorticoid pulse therapy (0.5 g/day, 3 consecutive days at 1 course, 2 courses in total) was started. One year after the tonsillectomy, urinary protein, urinary occult blood and serum creatinine were analyzed. The control patients were chosen from biopsy-proven IgA nephropathy patients who could be followed for at least one

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year as outpatients in Osaka university hospital and who met the following criteria: a) patients who were treated with glucocorticoid without tonsillectomy (glucocorticoid group) and b) patients who were treated without glucocorticoid and tonsillectomy (non-glucocorticoid group). The both group were chosen whose serum creatinine levels at the start of the evaluation were equal to or less than 1.3 mg/dl. The start of the evaluation period was assigned to the date of tonsillectomy for combination group, the date of the start of glucocorticoid administration for glucocorticoid group and the date of renal biopsy for non-glucocorticoid group. The combination group consisted of 22 patients, the glucocorticoid group 22, the non-glucocorticoid group 32. Results: When urine dipstick test () and () are defined as negative, the negative rate for urinary protein at one year follow up were 77.3%, 63.6% and 58.1% for the combination group, the glucocorticoid group, the nonglucocorticoid group, respectively. The negative rate for urinary occult blood at one year follow up were 54.5%, 22.7% and 35.5% for the combination group, the glucocorticoid group, the non-glucocorticoid group, respectively. Double negative rate at one year follow up were 40.9% 20.8% and 32.3% for the combination group, the glucocorticoid group, the non-glucocorticoid group, respectively. When serum creatinine was evaluated in terms of non-elevation (meaning the same level or decrease), the non-elevation rate for serum creatinine at one year follow up was 71.4%, 68.4% and 50% for the combination group, the glucocorticoid group, the non-glucocorticoid group, respectively. Conclusions: In terms of the urinary findings and the renal function, the combination therapy of tonsillectomy and corticosteroid pulse brought about better results compared to the glucocorticoid group and the nonglucocorticoid group.

C-O-11

Combination of Tonsillectomy and Corticosteroid Therapy with Concomitant Methylprednisolone Pulse Therapy for the Treatment of Patients with IgA Nephropathy Suwabe Tatsuyaa, Ubara Yoshifumia, Sogawa Yokoa, Nomura Kazufumia, Higa Yasushia, Nakanishi Shoheia, Hoshino Jyunichia, Sawa Naokia, Katori Hideyukia, Takemoto Fumia, Hara Shigekob, Takaichi Kenmeia Nephrology Center and bHealth Managing Center, Toranomon Hospital, Tokyo, Japan

a

Background: IgA nephropathy is the most common chronic kidney diseases in Japan, but the optimum treatment remains controversial. Methods: The combination of tonsillectomy and corticosteroid therapy with concomitant methylprednisone pulse therapy was evaluated in 91 patients, 42 men and 49 women aged from 19 to 56 years (33.8  10.0 years), with diagnoses of IgA nephropathy based on renal biopsy selected using Hotta’s regimen. Serum creatinine level ranged from 0.5 to 3.4 mg/dl (1.08  0.55 mg/dl), and proteinuria ranged from 0.14 to 5.49 g/dl (1.39  1.17 g/dl). These patients were divided into 4 groups according to the initial Ccr calculated by the Cockcroft-Gault formula:
90 ml/min (n  63), 60–90 ml/min (n  6), 30–60 ml/min (n  20), 30 ml/min (n  2). Mean observation period after tonsillectomy was 10.9  5.8 months. Results: In all patients, proteinuria decreased from 1.39 g/day to 0.47 g/day, and hematuria (urine erythrocyte sediment/HPF) improved from Grade 3.77 to Grade 2.30. Serum creatinine level unchanged. In patients with Ccr
90 ml/min, proteinuria decreased from 1.00 g/day to 0.51 g/day. In patients with Ccr 60–90 ml/min, proteinuria decreased from 1.82 g/day to 1.27 g/day. In patients with Ccr 30–60 ml/min, proteinuria decreased from 2.24 g/day to 1.02 g/day. In patients with Ccr 30 ml/min, proteinuria decreased from 1.53 g/day to 1.02 g/day. In all groups, hematuria improved equally. Conclusions: This therapy was effective for patients with Ccr
60 ml/min, but only partially effective for patients with Ccr 60 ml/min.

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C-O-12

Impact of Annual Urine Health Check-Up System to Obtain Clinical Remission in Patients with IgA Nephropathy Norio Ieiri, Yoshio Taguma, Osamu Hotta Department of Nephrology, Sendai Shakaihoken Hospital, Miyagi, Japan Purpose: In Japan, the annual urine health check-up system is well developed, while the value of this system is now questionable based on its cost-effectiveness in Europe and US. Recently we reported a significant impact of tonsillectomy and steroid pulse therapy on clinical remission in our IgA nephropathy patients especially those in relative early stage of nephropathy and indicated that clinical remission could terminate the progressive deterioration in renal function. We surveyed whether early detection of urinary abnormalities by annual urine health check-ups contribute to clinical remission in IgA nephropathy patients treated with tonsillectomy and steroid pulse therapy. Methods: We investigated our 380 IgA nephropathy patients in whom the onset year could be identified by annual urine check-ups documented in medical records. Group A consisted of 264 patients in whom treatment intervention was initiated within 3 years after the first appearance of urinary abnormalities, and group B consisted of 116 patients who were diagnosed after more than 3 years duration of urinary abnormalities. We also classified our 380 patients by the degree of glomerular lesions; 233 patients in mild glomerular lesions, 83 in moderate, and 64 in severe. All patients were treated with tonsillectomy and steroid pulse therapy in our renal unit, and their follow-up periods were at least 5 years. Results: The clinical remission rate of group A was 87.1%, while that of group B was 54.3%. The glomerular filtration rate of group B at the time of diagnosis showed significant deterioration as compared with that of group A, and glomerular lesions were significantly more severe in group B. In mild glomerular lesion group, group B had significantly lower remission rate compared with group A. Even in severe glomerular lesion group, the remission rate of group A was significantly higher than that of group B. Conclusion: Our results indicate the annual health check-up system to be very useful for achieving clinical remission in IgA nephropathy patients, if they are treated with tonsillectomy and steroid pulse therapy.

C-O-13

Mycophenolic Acid Therapy after Cyclophosphamide Pulses in Progressive IgA Nephropathy Franz M. Raschea, David Czockb, Wolfram Kargesa, Lutz von Müllerb, Frieder Kellera Department of Internal Medicine I and bDepartment of Microbiology and Virology, University Hospital of Ulm, Ulm, Germany

a

Backgrounds: In progressive IgA nephropathy (IgAN), cyclophosphamide or steroids have been used to reduce the loss of renal function, but disease progression may occur after the end of treatment. The value of mycophenolic acid (MPA) maintenance therapy following initial immunosuppression in progressive IgAN is largely unknown. Methods: In a prospective single center trial, twenty patients with advanced IgAN (median GFR, 22 ml/min) and disease progression after cyclophosphamide (CyP; n  18) or steroid pulse therapy (n  2) were treated with MPA for a median of 27 months. MPA doses (initially mycophenolate mofetil 500 mg twice daily) were adjusted

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according to predose plasma concentrations (target concentrations 1.5–4 g/ml). The course of renal function was assessed by linear regression of glomerular filtration rates. Results: Median loss of renal function per month was significantly reduced from 0.8 ml/min to 0.03 ml/min per month after 6 months, to 0.05 ml/min per month after 12 months, and to 0.12 ml/min per month at the end of the study after median 27 months (p  0.05). An improved or stable GFR was observed in 16 of 20 patients during the first 12 months, and sustained in 10 patients during 24 months of follow-up. Proteinuria decreased significantly from 1.1 g/l to 0.4 g/l during MPA treatment (p  0.018). Conclusion: Our results indicate that MPA may be beneficial to slow down the loss of renal function in patients with progressive IgAN after previous immunosuppressive treatment.

C-O-14

Prospective Trial of Combined Therapy with Heparin/Warfarin and Renin-Angiotensin System (RAS) Inhibitors in Progressive IgA Nephropathy (IgAN) Takeo Ishii, Tetsuya Kawamura, Makoto Ogura, Yasunori Utsunomiya, Tatsuo Hosoya Department of Nephrology and Hypertension, Jikei University School of Medicine, Tokyo, Japan We previously reported that a combined therapy with heparin/warfarin and RAS inhibitors dramatically reduces proteinuria for a long-term in advanced IgAN (JASN 2002). In this study, we prospectively analyzed whether the combined therapy can inhibit the progressive decline in renal function of patients with progressive IgAN. Patients who had a marked linearity of decline in loss of GFR, assessed by reciprocal serum creatinine plots vs. time for more than one year were recruited in this study if they were histologically diagnosed as IgAN at this point of declining renal function. Twelve patients (male 5, female 7, mean age 40.5, range 27–54 years) were eligible for trial entry; reciprocal serum creatinine plot suggested end-stage renal failure within 5 years. All patients were treated with continuous intravenous infusion of heparin (10,000–15,000 unit/24 h) for 8 weeks, followed by oral administration of warfarin (1–2 mg/day), ACE inhibitors and/or angiotensin II receptor blockers and dypiridamole. Eight patients were further given corticosteroid for 2 years because of the presence of acute glomerular lesions such as cellular crescent or angionecrosis. All patients were followed for at least 12 months and the mean follow-up period was 34  20 (range 12–79) months. After the combined therapy, UprV was significantly reduced from 2.4  1.5 g/day at baseline to 0.7  0.5 g/day at final observation, while the mean serum creatinine was not significantly different (1.7  0.8 vs. 1.7  0.6 mg/dl, respectively). Of note, the mean slope of 1/serum creatinine significantly increased from –0.009 to 0.0002 mg/dl/week (p  0.005). Moreover, histological analysis of a repeat kidney biopsy which was performed in 5 patients at 2 years after the institution of the combined therapy revealed that the percentage of cellular/fibrocellular crescent and the degree of mesangial matrix expansion were significantly attenuated (from 19 to 0.1% and from 1.6 to 0.6 score, respectively) while the percentage of global sclerosis and tubulo-interstitial lesion did not increase (from 49 to 50% and from 37 to 36%, respectively). These results indicate that our combined therapy including heparin/warfarin and RAS inhibitors can inhibit the progressive decline in renal function of patients with progressive IgAN through its marked antiproteinuric and antiinflammatory effects.

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C-P-01

A Scoring System to Predict Renal Outcome in IgA Nephropathy: From Nationwide Prospective Study in Japan Masayuki Endoha, Kenji Wakaib, Takashi Kawamurac, Yasuhiko Tominod a

Department of Internal Medicine, Tokai University School of Medicine, Isehara, Kanagawa, Division of Epidemiology and Prevention, Aichi Cancer Center, Nagoya, cKyoto University Health Service, Kyoto University, Kyoto, dDepartment of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan b

Aim: IgA nephropathy (IgAN) is the most common form of glomerulonephritis in the world and considerable number of patients develop end-stage renal disease (ESRD). Although several prognostic indicators are proposed, it remains difficult to predict outcome in individual patients. In order to evaluate Japanese status of IgAN patients and develop a prognostic score, we performed large-scale, nationwide prospective study. Method: A prospective cohort study was conducted in 97 clinical units in Japan from 1995 to 2002. We analyzed the data from 2,269 patients with IgAN using proportional hazards models to determine the predictors of ESRD in IgAN. We also developed a scoring system to estimate ESRD risk of patients. Results: During the follow-up period, 207 patients developed ESRD. Systolic hypertension, proteinuria, hypoproteinemia, azotemia, and high grade histological damage at initial renal biopsy were independently associated with the risk of ESRD. Using a proportional hazard model, a scoring system was established to estimate the seven-year ESRD risk from eight clinical and pathological variables. This score was verified by the repeated derivation-validation method. Conclusions: Our scoring system will serve as a useful clinical tool to predict a risk of ESRD in clinical practice for patients with IgAN.

C-P-02

Histological Scoring Predicts Progression of Renal Failure in Adult IgA Nephropathy Patients without Steroid Therapy Kensuke Joh, Kentaro Koike, Hiroshi Kitamura, Toshiyuki Imasawa, Takeshi Nakazato Division of Immunopathology, Clinical Research Center, Chiba-East National Hospital, Chiba, Japan The purpose of this study was to investigate how the histological lesions in IgA Nephropathy (IgAN) can predict the clinical prognosis of renal functional impairment (RFI) in adult IgA nephropathy (IgAN). The 354 adult patients without steroid therapy, who provided more than 8 glomeruli in a renal biopsy and more than 2 years’ clinical observation period after a renal biopsy, were analyzed. RFI was defined as duration of months to reach an endpoint of serum Cr 2 mg/dl. An extent of the active (A) and chronic (C) lesions (8 parameters) including glomerular mesangial cell proliferation (AGm), intracapillary hypercellularity (AGi), active extracapillary lesions (cellular and fibrocellular crescent) (AGe), interstitial inflammatory cell infiltration (Ai), global sclerosis (CGs), segmental sclerosis/hyalinosis or collapsing lesions (CGi), chronic extracapillary lesions (fibrous crescent or adhesion) (CGe) and tubulointerstitial fibrosis (Ci), were expressed as a percent on the basis of a new histological scoring system (Oxford, 2005). For the scoring, the histogram of each graded histological lesion was first produced and then divided equally into 4 or 3 categories. Score 0, 1, 2, or 3 was given to each of those categories, respectively. Uni- and multivariate (non-categorized or categorized) analysis was attempt for the analysis. The

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prognostic significance of each histological variable was determined by Cox proportional hazards model. Odds ratio (OR) and 95% CI (confidence interval) of each variable were calculated using SPSS System (SPSS Institute, Carry, NC, USA). Significance was considered to be present of confidence intervals either at
1 or 1 in all analysis. In the categorized univariate analysis, significant and critical grade, which can influence the prognosis of RFI was estimated as 43% for AGm, 13% for AGi, 11% for AGe, 11% for Ai, 25% for CGs, 20% for CGi, 22% for CGe, and 16% for Ci. To determine the independent histological predictors, the non-categorized multivariate model was refined until it included 3 predictors such as AGi with 0.5 (0.3 – 0.8), Ai with 1.7(1.1 – 2.7) and Ci with 2.0 (1.5 – 2.8). In the categorized multivariate analysis using the selected and adjusted odds ratios of each categorized histological lesion, equation predicting RFI was produced as follows; logit p  fAGi (x)  fAi (x)  fCi (x)  constant. (fAGi (1)  0, fAGi (2)  1, fAGi (3)  5, fAi (1)  0, fAi (2)  0, fAi (3)  3, fCi (1)  0, fCi (2)  1, fCi (3)  3, fCi (4)  7). Eight Kaplan Meier survival curves were obtained for each eight losit scores (1–8) by substituting the individual histological scores of 354 patients into the predicting equation. These prediction curves are useful for a guideline of treatment, because a patient with losit score 8, for example, can be predicted by this curve with 50% probability to reach serum Cr 2 mg/dl within 33 months. In conclusion, systematical scoring of active and chronic histological parameters in the renal biopsy is useful to predict the progression renal failure and for a choice of therapy in the IgAN patients.

C-P-03

Receiver Operating Characteristic (ROC) Curve Analysis Selects Histological Parameters and Determins Their Critical Severity for Renal Functional Impairment of IgA Nephropathy Kentaro Koike, Takeshi Nakazato, Toshiyuki Imasawa, Hiroshi Kitamura, Kensuke Joh Division of Immunopathology, Department of Clinical Research Center, Chiba-East National Hospital, Chiba, Japan Background/Purpose: Renal biopsy plays a role not only for diagnosis but also for predicting a clinical prognosis in the IgA nephropathy (IgAN). However, there has been no appropriate statistical method how to select histological parameters and how to determine the cutoff value for predicting clinical prognosis. The purpose of this study is to propose a novel statistical method to select histological parameters and evaluate their severity, which affect the renal functional impairment of IgAN patients. Method: The 210 adult IgAN patients, who have more than 8 glomeruli in renal biopsy specimen between 1981 and 1998, and have been observed more than 2 years, were analyzed. The patients were divided into two groups consisting of a group with steroid therapy and a group without steroid therapy. The numbers of the patients of a group with steroid therapy and of a group without steroid therapy were 160 and 50, respectively. A distribution of the glomeruli, which involved mesangial hypercellularity (GM), intracapillary hypercelluarity (GI), extracapillary lesions (GE), global sclerosis (GS), adhesion (AD), as well as an extent of interstitial inflammations (II) and interstitial fibrosis (IF) were expressed as a percent. Clinical prognosis was designated as renal functional impairment, which attained an endpoint of serum creatinine (sCr) as 2 mg/dl or showed sCr level more than twice of initial value within 5 years. ROC curve analysis was applied to select histological parameters for RFI by showing more than 0.7 of under ROC curve area. The cut off values for the parameters was calculated by a discrimination analysis to identify optimal threshold values for renal functional impairment respecting each parameter. Result: In a whole group, which consists of a group with steroid therapy and a group without steroid therapy, GS and IF were selected as the parameters which discriminated the patients with progression to renal functional impairment or without progression. The cutoff values of GS and IF were 15.0% (sensitivity; 79.8%, specificity; 58.0%) and 16.2% (sensitivity; 86.3%, specificity; 60.0%), respectively. In a group without steroid therapy, GS, AD,

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II and IF were selected and the each cutoff values of GS, AD, II and IF were 15.5% (sensitivity; 81.3%, specificity; 62.5%), 8.0% (sensitivity; 79.7%, specificity; 61.3%), 5.5% (sensitivity; 75.8%, specificity; 65.5%), and 16.6% (sensitivity; 86.7%, specificity; 65.6%), respectively. In a group with steroid therapy, only IF was selected and its cutoff value was 15.7% (sensitivity; 80.6%, specificity; 64.3%). These results suggest that, regardless steroid therapy, IF was a reliable parameter to discriminate the patients who progress to renal functional impairment of our definition. Conclusion: We present a novel statistical method to select histological parameters and determine cut off values for predicting clinical prognosis. These concrete data concerning critical severity of the selected histological parameters for renal functional impairment can be noticed to assess the renal biopsy.

C-P-04

Clinical and Immunological Study of the Tonsils in IgA Nephropathy Hanako Takechia, Hiroshi Nagurab, Kouji Houzawac, Mika Adachic, Osamu Hottaa Departments of aNephrology, bPathology and cOtolaryngology, Sendai Shakaihoken Hospital, Sendai, Miyagi, Japan The relationship between immunoglobulin A (IgA) nephropathy and infection of tonsils attracts attention in recent years. Tonsillectomy and steroid pulse therapy for IgA nephropathy is being established. There are many reports suggesting the relationship between the immune response of tonsils and the pathogenesis of IgA nephropathy from both clinical and experimental aspects. However, the immunological abnormality of tonsils in IgA nephropathy has not been fully revealed. The purpose of this research is to clarify the pathological change of tonsils and the immunological abnormality which is specific for IgA nephropathy and to investigate the mechanism of tonsillectomy and steroid pulse therapy. 209 patients with IgA nephropathy (aged 4–71 years) and 10 patients with chronic tonsillitis (aged 11–36 years) participated in this study. Tonsillectomy was performed in all of those patients from January 2004 to August 2005. 80 of these patients of IgA nephropathy had tonsillectomy before steroid pulse therapy. Others had tonsillectomy after steroid pulse therapy. We examined histological change and immunohistochemical staining (B cell, T cell, CD8, HLA-DR) of tonsils. A phenotypic analysis (CD4, CD8, CD4DR, CD8DR) was also performed using flow cytometry. We estimated the effectiveness of steroid pulse therapy on tonsil immunity by above-mentioned data. Following characteristics seemed to be specific for IgA nephropathy compared with tonsils of chronic tonsillitis. Expansion of the interfollicular area and unclear border of follicle were observed. T cells were scattered in interfollicular area. The distribution of B cells in follicles and interfollicular area was irregular. The localization of CD8 T cells and CD4 T cells were irregular. HLA-DR T cells formed nodules (activated T cell nodules), and the number of these nodules was significantly increased. These findings suggest that there are some abnormal mechanisms in activation and maturity of tonsil immunity in IgA nephropathy. Tonsils removed after steroid pulse therapy had following findings. Intentionally reduction of the number of DR T cell nodules and the size of follicles were observed, whereas these changes were not observed in tonsils removed after oral corticosteroids therapy. There was a tendency for a follicle in tonsils to be large when CD4DR T cell counts were higher. It may suggest that activated CD4 T cells stimulate follicles to be large. The ratio of CD4DR/total CD4 cells decreased after steroid pulse therapy, and it persisted lower levels several months after steroid pulse therapy. CD8DR/total CD8 cells decreased in peripheral blood because of response to steroid pulse. These observations suggest that highly active T cell nodules cause the over production of B cells which may release aberrant IgA in tonsils of IgA nephropathy patients. The pulse dose of corticosteroid may be required for the apoptosis of memory T cells and suppression of B cells which produce aberrant antibody.

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C-P-05

A Repeat Biopsy Study on the Effect of Tonsillectomy Combined with Steroid Pulse Therapy on IgA Nephropathy Seiichiro Haraa, Shouichi Fujimotob, Hiroyuki Komatsub, Yuji Satob, Ryosuke Nishiurab, Shuji Iwatsubob, Mariko Tatsumotoa, Kazuhiro Yamadaa, Kazuo Kitamurab Division of Dialysis Unit and bDivision of Circulatory and Body Fluid Regulation, Faculty of Medicine, University of Miyazaki, Miyazaki, Japan

a

We previously reported that steroid pulse therapy combined with tonsillectomy is more effective in decreasing urinary abnormalities that steroid pulse monotherapy in patient with IgA nephropathy. The present study evaluates the effect of these therapies on histopathological lesions. Eighteen of 55 patients with IgA nephropathy who were enrolled in a previous prospective cohort study underwent a repeat renal biopsy 23.4  7.8 months after the initial treatment. Tonsillectomy plus steroid pulse therapy was administered to 11 patients (Group C) and 7 received steroid pulse monotherapy (Group M). The mean serum creatinine levels at the first and second renal biopsy remained unchanged between the two groups (Group C, 0.85–0.87 mg/dl; Group M, 0.87–0.99 mg/dl). The ratio (%) of patients with crescent formation decreased in both groups (Group C, 55–9%; Group M, 43–14%). The extent of mesangial proliferation after treatment was significantly improved as compared with that before treatment in Group C (mesangial proliferation score, 2.64  0.67 to 1.55  0.52; p  0.001), meanwhile, no improvement was evident in Group M (2.57  0.54 to 2.00  1.00; p  0.231). The distribution of IgA deposition in mesangial area was also significantly reduced in Group C (IgA deposition score, 3.09  0.70 to 2.45  0.69; p  0.011). Microscopic hematuria or proteinuria had persisted in 4 patients in Group C by the second biopsy and segmentally sclerotic glomeruli persisted in 3 of them. These findings suggest that steroid pulse therapy combined with tonsillectomy significantly reduces mesangial proliferation and IgA mesangial deposition compared with steroid pulse monotherapy. However, further treatment strategies should target the disappearance of urinary abnormalities when segmentally sclerotic glomeruli persist.

C-P-06

Expressions of TGF-Beta1 and CTGF mRNAs in the Tubulointerstitial Area Are Correlated with the Urinary Protein Level in IgA Nephropathy Sachiko Nonaka-Takahashia, Takayuki Fujitaa, Yuki Wadaa, Teruyuki Takahashib, Ken Itoa, Yoshinobu Fukea, Atsushi Satomuraa, Koichi Matsumotoa Division of Nephrology and Endocrinology and bDivision of Neurology, Department of Medicine, Nihon University School of Medicine, Tokyo, Japan

a

Background: IgA nephropathy (IgAN) is the most common form of primary glomerulonephritis. The degree of tubulointerstitial fibrosis is regarded as a poor prognostic indicator in IgAN. Recently, connective tissue growth factor (CTGF) was observed to be strongly upregulated in tubular epithelial cells (TECs) in human proliferative and fibrogenic diseases. In IgAN, CTGF may play an important role in the development and progression of tubulointerstitial fibrosis. In the present study, the authors examined that the expressions of TGF-beta1 and CTGF mRNAs in human renal tissues by using rapid in situ hybridization (rISH). Methods: Renal biopsy specimens were obtained from the 20 patients with IgAN. Based on the conventional patho-histological findings by PAS, Masson Trichrome and hematoxylin and eosin staining for light microscopy,

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the degree of tubulointerstitial damage was evaluated in all cases. In addition, all cases were categorized into a no or mild damage group (A) and moderate or severe damage group (B) based on the certain criteria which was previously reported by Shigematsu, et al. The expressions of transforming growth factor beta1 (TGF-beta1) and CTGF mRNAs in TECs in all cases of both groups (A and B) were examined by the technique of rISH. In rISH, each of the TGF-beta1 and CTGF mRNAs was detected by two commercially available oligonucleotide microprobes conjugated with Brigati-tail (FALMA, Tokyo Japan) 5-GTTCGCCAGGAATTGTTGC-Brigati tail for TGF-beta1 and ?-CTGTGAAAGTCCTTAGCCTTAGGACAGCT-Brigati tail for CTGF. The results of rISH were compared to conventional pathohistological findings and various clinical parameters such as creatinin clearance, urinary (u-) protein (24 hour accumulated) and serum creatinin, u-N-acetyl-beta-D-glucosaminidase activity and u-beta2 microglobrin (at the time of rising), etc., and statistically analyzed between groups A and B. Results: Significantly strong and diffuse expressions of TGF-beta1 and CTGF mRNAs were observed in proximal TECs in the cases of group B, whereas the expressions of these two mRNAs were weak in proximal TECs in group A and in glomerular resident cells in both groups. The degree and distribution of the expressions of these two mRNAs were almost consistent in both groups. The expressions of TGF-beta1 and CTGF mRNAs in proximal TECs were closely correlated with the degree of tubulointerstitial damage based on the conventional pathhistological findings. Moreover, these results also demonstrated most close correlation with the u-protein level statistically. Conclusion: Previously, the increasing u-protein level had been reported as one of the major causative agents leading to tubulointerstitial damage. Based on the present study results, the increase of the u-protein level is speculated one of the most important factors influencing the expression of TGF-beta1 and CTGF mRNAs in TECs. Both the u-protein level and the expressions of these two mRNAs in TECs are significantly correlated with the degree of tubulointerstitial damage in IgAN. These results suggest that proteinuria is a potent factor to accelerate tubulointerstitial fibrosis via the upregulation of TGF-beta1-induced CTGF.

C-P-07 Not submitted

C-P-08

Infiltration of Activated Macrophages and Their Disappearance Are Involved in the Effect of Tonsillectomy Combined with Steroid Pulse Therapy for Patients with IgA Nephropathy Kyoko Hara, Takahiro Nakayama, Yutsuki Yamamoto, Daisuke Komukai, Yoshihiko Inoue, Susumu Watanabe, Kiyoko Inui, Hiroyuki Morita, Ashio Yoshimura Division of Nephrology, Showa University Fujigaoka Hospital, Yokohama, Kanagawa, Japan Tonsillectomy combined with steroid pulse therapy (Tx-SPT) is now widely applied in patients with IgA nephropathy (IgAN) in Japan, and clinical remission has been achieved in many patients by Tx-SPT, so that it was reported that clinical remission halts subsequent progressive deterioration and improve renal prognosis of IgAN. However the mechanisms to mediate clinical modifications are unclear. Infiltration of macrophages and their activation are important in the progression of glomerular diseases. The significant increase in the number of

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macrophages in the urine is also induced in active proliferative glomerulonephritis patients. In the present study, we studied the change of macrophages in the blood and urine by flow cytometry during Tx-SPT from 21 patients with IgAN. The number of CD14 cells (macrophages, M), CD16 cells (activated macrophages) and Annexin V cells (apoptotic cells) in the blood and CD14 cells in the urine were studied by flow cytometry at four points: before (pre-Tx) and after (post-Tx) tonsillectomy, and before (pre-SPT) and after (post-SPT) steroid pulse therapy, respectively. In the blood, CD16 cells significantly decreased at post-SPT compared to pre-Tx, post-Tx, and preSPT respectively (p  0.002 vs. pre-Tx, p  0.00017 vs. post-Tx, p  0.0043 vs. pre-SPT, by non-parametric Friedman and 2 analysis). There was no siginificant change in the number of CD14 cells and Annexin V cells in the blood between the groups. In the urine, a significant decrease in CD14 cells was induced at post-SPT compared to pre-Tx (p  0.003) and post-Tx (p  0.0007). These findings indicate that the tonsillectomy combined with steroid pulse therapy suppresses the infiltration of activated macrophages that may be tightly associated with the therapeutic effect for IgA nephropathy. In the mechanism of decrease in the number of activated macrophages in the blood, apoptosis may not be importantly participated. Furthermore, the analysis of the number of macrophages in the urine may be important marker for the evaluation of the effect of this treatment.

C-P-09

Retention of TNFR I and II in IgA Nephropathy – A Potential Risk Factor for Cardiovascular Disease Sigrid Lundberga, Iva Gunnarssonb, Stefan H. Jacobsonc Department of Nephrology bDepartment of Rheumatology, Karolinska University Hospital, and Department of Nephrology, Danderyd University Hospital, Stockholm, Sweden

a

c

Background: IgA nephropathy is a common disease, associated with the risk of renal failure and cardiovascular disease. Soluble tumor necrosis factor receptors (sTNFR) are associated with the presence of cardiovascular disease. The aim of this study was to examine the concentration of sTNFR I and II in relation to renal function and progression of IgA nephropathy. Methods: Serum levels of sTNFR I and II were measured in 43 patients with IgA nephropathy within 6 months of biopsy-verified diagnosis. Patient characteristics: age 40  14 years, GFR 56  26 ml/min/1.73 m2, proteinuria 1.9  2.1 g/day, follow up 59.1  37.4 months (mean  SEM). sTNFR I and II were also determined in 25 healthy control subjects (age 41  13 years). In the patient group sTNFR I and II concentrations were related to s-creatinine. In patients with chronic kidney disease (CKD) stages 1–3 (GFR
30 ml/min) at diagnosis sTNFR I and II also were related to the progression in decline of renal function. For this purpose GFR was estimated with the MDRD Study equation. Results: sTNFR I and II (expressed in pg/ml) were significantly higher in patients compared to healthy controls [2,912  2,058 vs. 1,161  302 (p  0.001) and 5,287  3,039 vs. 2,080  562 (p  0.001)], a difference that was no longer significant after adjustment of sTNFR I and II for renal function (calculated as concentration of sTNFRs in pg/ml GFR/100). There was a linear correlation between sTNFR I and s-creatinine (r  0.94, r2  0.88) and sTNFR II and s-creatinine (r  0.89, r2  0.79). Almost the same correlation was obtained when 7 patients with Henoch-Schönlein nephritis were included (r  0.94, r2  0.88 and r  0.89, r2  0.80 for TNFR I and II respectively). Considering only the 33 patients with IgA nephropathy at CKD stages 1–3 at diagnosis (mean: GFR 67 ml/min/1.73 m2, creatinine 105 mol/l, follow up 66 months), the odds ratio (OR) for progression, defined as a rise in s-creatinine of more than 20% during follow up, was 5.19 when TNFR II was higher than 3,600 pg/ml (median for these 33 patients) compared to levels below 3,600 pg/ml. Similarly, OR for a decrease in GFR
3 ml/min/year was 4.5 for patients in CKD stages 1–3 with sTNFR II
3,600 pg/ml compared to patients with TNFR  3,600 pg/ml.

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Conclusion: In IgA nephropathyserum-sTNFR I and II concentrations are significantly increased but closely related to s-creatinine. This may be caused by an increased production of TNFR, but is more likely due to retention secondary to impaired renal function. Irrespective of origin, high TNFR I and II concentrations may be involved in the increased risk of cardiovascular disease in patients with IgA nephropathy and renal failure.

C-P-10

Association of Plasminogen Activator Inhibitor Type 1 Genotype 5G5G with Vascular Injury in IgA Nephropathy Shuwen Liu, Rui Ding, Xiangmei Chen Department of Nephrology, General Hospital of PLA, PLA Research Institute of Kidney disease, Beijing, P.R. China Objective: To look inside the relationship between plasminogen activator inhibitor type 1 (PAI-1) 4G/5G promoter polymorphism and the characteristics of immunoglobulin A (IgA) nephropathy. Subjects and Methods: Seven hundred and forty-nine biopsy-proven IgA nephropathy patients were enrolled. We collected the patients’ clinical data including hypertension, urinary protein excretion, serum creatinine, etc. The renal pathological changes were scored using Katafuchi Scoring. PAI-1 4G/5G polymorphism was determined by utilizing polymerase chain reaction and restriction-fragment length polymorphism. Results: (1) There was no significant difference in PAI-1 genotype frequency and allele frequency between IgA nephropathy patients and normal controls (p  0.272). (2) No significant difference of PAI-1 genotype frequency and allele frequency was found between groups with and without gross hematuria (p  0.660), hypertension (p  0.122), elevated serum creatinine (p  0.547), decreased creatinine clearance (Ccr) (p  0.717) and urinary protein excretion (UPE) 2.0 g/day (p  0.556). (3) In the group with hypertension accompanied by both UPE 2.0 g/day and Ccr 60 ml/min, or with severe renal injury, or with moderate to severe vascular injury, or with hypertension accompanied by moderate to severe vascular injury, there was a markedly higher 5G5G genotype frequency compared with its respective group (p  0.05). (4) In the 5G5G homozygote group, more severe renal vascular injury was found than that in the non-5G5G group (p  0.023). There was no significant difference among the three genotype groups in total Katafuchi scoring, glomerular scoring or tubulointerstitial scoring (p
0.05). (5) There was a significant higher 5G5G genotype frequency in the group with severe renal lesion than that in the group with mild to moderate renal lesion (p  0.025). Conclusion: Data present here suggest that the 4G/5G polymorphism of PAI-1 associates neither to the etiology nor to the progression of IgA nephropathy. 5G5G seems to be a risk factor for renal vascular injury in patients with IgA nephropathy.

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C-P-11

Urinary IL-6 and TGF-␤1 Levels in Steroid Treated Patients with IgA Nephropathy Dimitrios S. Goumenos, Sotirios Tsakas, Pantelitsa Kalliakmani, Jannis G. Vlachojannis Department of Internal Medicine – Nephrology, Patras University Hospital, Patras, Greece IgA nephropathy (IgAN) is the most frequently encountered primary glomerulonephritis and it is usually treated by corticosteroids. Various cytokines and growth factors are implicated in the development and progression of IgAN. Interleukin-6 (IL-6) and transforming growth factor-beta (TGF-1) have been identified within the glomeruli of patients with IgAN and implicated in the development of mesangial proliferation and renal scarring. In this study, IL-6 and TGF-1 urinary levels were determined in IgAN patients at diagnosis and after administration of corticosteroids in order to estimate the activity of the disease after treatment. Twenty-one patients (M/F 16/5), with well-preserved renal function (mean serum creatinine 1.16 mg/dl, Ccr 84 ml/min) and proteinuria 1.6 g/24 h were included. All patients were treated with methyl-prednisolone (0.7 mg/kg BW/d initially, followed by gradual tapering) for 18 months. In 5 patients, azathioprine or mycophenolate mofetil was given in addition to corticosteroids. The mean follow-up period was 5 years. Urinary IL-6 and TGF-1 levels were determined by ELISA in 24 h urine collection at diagnosis and at the end of the treatment period and compared to those of ten healthy subjects. Urinary IL-6 and TGF-1 levels at diagnosis were significantly higher in IgAN patients compared to the controls (14 vs. 21 ng/24 h and 310 vs. 473 ng/24 h, p  0.05 respectively). TGF-1 levels at diagnosis, were significantly higher in patients with proteinuria
1 g/24 h and/or severe mesangial proliferation, in comparison to those with less severe proteinuria and mesangial proliferation (524 vs. 405 ng/24 h, p  0.03 and 543 vs. 420 ng/24 h, p  0.05 respectively). Although a significant reduction of proteinuria (from 1.6 to 0.4 g/24 h, p  0.03) was observed with corticosteroid treatment, urinary IL-6 and TGF-1 levels were not significantly reduced after treatment (21 vs. 16 ng/24 h and 473 vs. 445, p  NS respectively). Deterioration of renal function (doubling of serum baseline creatinine) over the follow-up period was observed in 3 patients with IgAN (14%). All three patients had significantly higher urinary IL-6 levels at diagnosis in comparison to those who preserved their renal function (34 vs. 18 ng/24 h, p  0.03). In conclusion, increased urinary IL-6 and TGF-1 excretion is observed in patients with IgAN and related to the severity of the disease. The elevated IL-6 and TGF-1 urinary levels are maintained after administration of corticosteroids and this is probably due to an ongoing inflammatory process in the kidney. The serial measurement of IL-6 and TGF-1 in the urine of IgAN patients may provide significant information for the activity of the disease.

C-P-12

Urinary Complement Regulatory Proteins in Patients with IgA Nephropathy Kisara Onda, Hiroyuki Ohi, Mariko Tamano, Isao Ohsawa, Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan It is generally considered that complement activation may mediate the development of IgA nephropathy (IgAN). Glomerular deposition of C3, properdin (P) and factor H (H) was observed in patients with IgAN, but

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urinary excretion of complement components has not been examined. We conducted a study on urinary excretion of complement components in IgAN patients. Urine samples and clinical data were obtained from 75 patients with IgAN. As controls, 72 urine samples were collected from healthy volunteers. The IgAN patients were divided clinically into four prognosis groups, i.e. good prognosis group, relatively good prognosis group, relatively poor prognosis group and poor prognosis group at the time of renal biopsy by the findings of renal biopsy and clinical status. The levels of these urinary components were measured using a sandwich enzyme linked immunosorbent assay. Urinary P, H and membrane attack complex (MAC) in patients with IgAN were significantly higher than those in healthy controls. There was a significant correlation between urinary complement components (P, H and MAC) and other parameters such as urinary protein, N-acetyl-b-D-glucosaminidase and b2-microglobulin. There was also a significant correlation between H and renal function (SUN and s-Cr) in patients with IgAN. In the poor prognosis group, the levels of urinary P, H and MAC were significantly increased. It appears that the urinary complement regulatory proteins, especially H, might reflect pathological activity of IgAN.

C-P-13

Significance of Obesity-Related Factor in the Development of IgA Nephropathy Kentaro Koike, Yasunori Utsunomiya, Hidekazu Okamoto, Yoriko Ito, Satori Tokudome, Hideo Okonogi, Yoichi Miyazaki, Tesuya Kawamura, Tatuo Hosaya Division of Kidney and Hypertension, Jikei University School of Medicine, Tokyo, Japan Background: Recent large-scale clinical studies demonstrated that obesity, hyperlipidemia, hypertension, and hyperglycemia are risk factors for the onset for cardiovascular events. In addition, it has been also reported that metabolic disorder associated with obesity may have a potential of the development of chronic kidney disease. Although interest in the relationship between obesity and kidney disease is increasing, it is still unknown whether excess weight is a risk factor for the development of IgA nephropathy (IgAN). Therefore, to determine the association between obesity and renal damage, we examined obesity-related factors such as serum lipid profiles, and levels of several adipocytokines in patients with IgAN. Methods: Forty-six biopsy-proved patients with primary IgAN who visited our Hospital from January 2002 to December 2004 were included in this study. Patients who received repeated renal biopsy after steroid therapy and developed nephritic syndrome were excluded. The patients were divided into two groups by their body mass index (BMI), i.e. obesity group (BMI
25 kg/m2) and non-obesity group (BMI  25 kg/m2). Serum levels of adiponectin, plasminogen activator inhibitor-type 1 (PAI-1), resistin, and insulin were measured by enzyme-linked immunosorbent assay (ELISA) at the time of renal biopsy. In histological findings, the degree of mesangial proliferation and severity of wall thickening and hyaline change in small arteries or arterioles was semi-quantitatively determined. Glomerular diameter was measured by micrometer under light microscopy in all glomeruli cut at or near the hilus and calculated mean diameter in each patient. Results: As expected, serum adiponectin levels showed an inverse correlation with BMI. In addition, the concentrations of serum PAI-1 and resistin were significantly higher in the obesity group than in the non-obesity group. In clinical and laboratory findings, the levels of diastolic blood pressure, total cholesterol, and triglycerides increased in obesity group compared with non-obesity group. Conversely, HDL-C levels were low in obesity group. There was no difference in creatinine clearance and 24-hour urinary protein excretion between the groups. However, the degrees of urinalysis hematuria tended to be low in obesity group. In histological findings, glomerular diameter was enlarged and the degree of intrarenal arteriolar hyalinosis was increased in obesity group compared with non-obesity group. Of note, glomerular diameter was significantly correlated with BMI, estimated

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protein intake, serum levels of uric acid and PAI-1. Overweight and low levels of serum adiponectin were risk factors for the onset of intrarenal arteriolar hyalinosis. Conclusion: Our findings suggested that IgAN patients with obesity have the characteristic clinicohistogical features such as glomerulomegaly and intrarenal arteriolar hyalinosis in association with high BMI and the abnormality of adipocytokine secretion.

C-P-14

The Influence Factors on Hyperuricemia and Its Association with Intrarenal Arterial Lesions in IgA Nephropathy Qiang Qiu, Xiangmei Chen, Yuansheng Xie, Ribao Wei, Di Wu, Guangyan Cai, Shuwen Liu Institute of Nephrology, General Hospital of Chinese PLA, Beijing, China Objective: To analyze the independent influence factors on hyperuricemia and its association with intrarenal arterial lesions in IgA nephropathy. Methods: A retrospective study was carried out in 648 cases with IgA nephropathy diagnosed by renal biopsy. The definition of hyperuriceamia was serum uric acid 420 mol/l in males, and serum uric acid 360 mol/l, in females. The multivariate analysis was performed to identify the association between hyperuricemia and clinical and pathological factors, which including mesangial hypercellularity, tuft adhesion/small cellular crescent, crescent, segmental glomerular sclerosis, global glomerular sclerosis, tubular atrophy, interstitial fibrosis, interstitial cell infiltration and vascular lesions. Results: (1) The incidence of hyperuricemia in IgA nephropathy was 29.6%. The high level of serum creatinine (
110 mol/l), hypertriglyceride, hypertension, obesity and intrarenal arterial lesions were independently correlated to hyperuricemia (OR  7.43, 2.36, 4.48, 2.75, 2.76 and 2.89, respectively, all p  0.01). (2) The prevalence of hypertension and the level of serum triglyceride in the hyperuricemia group was significantly higher than that of the patients with normal serum uric acid (29.31 vs. 56.02%, p  0.001 and 1.76  1.31 vs. 2.70  2.46 mmol/l, p  0.001). (3) The level of serum uric acid was significantly associated with the prevalence of intrarenal arterial lesions in IgA nephropathy(R  0.5901, Spearman rank correlation). With the increase of serum uric acid level, the prevalence of intrarenal arterial lesions increased. Among patients (347 patients) whose serum uric acid levels were 360 mol/l (group A), the prevalence of intrarenal arterial lesion was 23.05%; the prevalence of intrarenal arterial lesion in group B (129 patients, serum uric acid 360–420 mol/l) was 64.34%; the prevalence of intrarenal arterial lesion in group C (85 patients, serum uric acid 420–480 mol/l) was 77.65%; in group D (85 patients) with serum uric acid
480 mol/l, the prevalence of intrarenal arterial lesions was 87.36%. Conclusion: To control hypertension and body weight, reduce blood lipid and uric acid level effectively would be helpful for alleviating the intrarenal lesions and delaying the progression of IgA nephropathy.

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C-P-15

Assessment of Serum High-Sensitivity C-Reactive Protein as a Marker of Inflammatory Pathogenesis in Patients with IgA Nephropathy Joo Eun Baek, Hyun Jung Seok, Tae Hee Kim, Won Seok Yang, Soon Bae Kim, Su-Kil Park, Sang Koo Lee, Jung Sik Park Division of Nephrology, Department of Internal Medicine, Asan Medical Center, Seoul, Korea Systemic inflammatory mediators such as cytokines and growth factors have been linked to the development and progression of renal injury in patients with IgA nephropathy (IgAN). C-reactive protein (CRP) is a representative acute phase protein that increases during systemic inflammation. At a low concentration, an immunoassay of high-sensitivity CRP (hs-CRP) can provide a more accurate value of CRP than a standard CRP assay. In this study, we investigated whether hs-CRP is a marker reflecting inflammatory pathogenesis of IgAN. We prospectively measured hs-CRP in patients diagnosed with IgAN through kidney biopsy at our hospital between January 2001 and March 2006. Patients with diabetes, evident cardiovascular disease, chronic inflammatory disease, malignancies and any acute illness or surgery in the previous 3 months were excluded from the study. There were 192 patients with IgAN (109 male, 83 female) and the mean age was 35.7  12.0 years. The patients with IgAN were classified according to Hass’ classification; Class I 13, Class II 18, Class III 99, Class IV 26 and Class V 8. We also measured hs-CRP in 998 healthy controls (543 male, 455 female, mean age 36.2  11.7 years) who visited Health Promotion Center of Asan Medical Center. Hs-CRP levels were correlated with serum albumin, cholesterol and complement levels, degree of hematuria, glomerular filtration rate, age, blood pressure, body mass index and other parameters. Hs-CRP was measured by a particle enhanced immunoturbidometric method using a COBAS INTEGRA 700 (Roche Diagnostic Systems). The median level of hs-CRP in the healthy population was 0.08 mg/dl (range: 0.03–2.76 mg/dl). Twenty five, seventy five and ninety five percentiles were 0.05, 0.12 and 0.33 mg/dl, respectively. The median level of hs-CRP in IgAN was 0.08 mg/dl (range: 0.03–3.13 mg/dl). Hs-CRP level in IgAN was not significantly different from that of the healthy population (p
0.05 by Mann-Whitney U test). Among the patients with IgAN, there were no significant differences in hs-CRP levels with different pathological classes (Class I 0.07 mg/dl (range: 0.03–0.27 mg/dl), Class II 0.06 mg/dl (range: 0.03–1.40 mg/dl), Class III 0.08 mg/dl (range: 0.03–3.13 mg/dl), Class IV 0.08 mg/dl (range: 0.03–0.83 mg/dl) and Class V 0.18 mg/dl (range: 0.03–0.62 mg/dl), p  0.334 by Kruskal-Wallis test). On multiple regression analysis, hs-CRP levels had an association with complement 3 (  0.307, p  0.001) and immunoglobulin A (  0.243, p  0.003) levels in IgAN. Our results suggest that hs-CRP does not closely reflect the inflammatory pathogenesis of IgAN and is not valuable as marker of disease activity.

C-P-16

Serum Cystatin C May Predict the Prognostic Stages of Patients with IgA Nephropathy Prior to Renal Biopsy Kensuke Saito, Yasuhiko Tomino Division of Nephrology, Department of Medicine, Juntendo University School of Medicine, Tokyo, Japan The relationship between the levels of serum cystatin C and the prognostic stages of IgA nephropathy was determined in Japan. The levels of serum cystatin C in patients with IgA nephropathy were measured by the Dade

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Behring N Latex Cystatin C assay. In 1995, the Joint Committee of the Special Study Group on Progressive Glomerular Disease, Ministry of Health and Welfare of Japan, and the Japanese Society of Nephropathy reported four prognostic stages. Prognostic criteria for patients with IgA nephropathy is divided into four groups by renal biopsy. These are: good prognosis group (Group I), relatively good prognosis group (Group II), relatively poor prognosis group (Group III) and poor prognosis group (Group IV) for this disease. Serum samples from 195 patients with IgA nephropathy and 111 patients with other glomerular disease were examined. There were no significant changes in the levels of serum creatinine or creatinine clearance between Group I and II. The mean value of serum cystatin C in Group II were significant higher than those in Group I (p  0.05). The mean value of serum cystatin C in Group III or IV were significantly higher than those in Group I (p  0.001, p  0.005 respectively). It is suggested that the measurement of serum cystatin C may predict the prognostic stages of patients with IgA nephropathy prior to renal biopsy.

C-P-17

The Efficacy of Tonsillectomy and Steroid-Pulse Combined Therapy (TSP) for Patients with IgA Nephropathy Naoto Miura, Wataru Kitagawa, Keisuke Suzuki, Masabumi Yoshino, Harutaka Yamada, Kazuhiro Nishikawa, Hirokazu Imai Division of Nephrology and Rheumatology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Japan Background: Tonsillectomy and steroid-pulse combined therapy (TSP) has been proposed as a curative method for IgA nephropathy by Hotta et al. There have been no randomized controlled trials, because tonsillectomy based on an informed consent has an ethical and practical issue for randomized controlled trial. Methods: We evaluated the efficacy of TSP in 21 consecutive IgA nephropathy patients who have received kidney biopsy. We monitored these patients in proteinuria, urinary occult blood reaction, and serum creatinine at 6 months before TSP, at 6 months and 12 months after TSP. Kruskal-Wallis test was used for statistical analysis in proteinuria and urinary occult blood reaction. Results: Urinary protein was significantly decreased by TSP at 6 months (p  0.00002) and 12 months (p  0.0004), and the remission rate in proteinuria was 38.1% and 53%, respectively. Urinary occult blood reaction was also significant improved (p  0.0008, p  0.0001). The remission rate at 6 months and 12 months was 33.3% and 64.7%, respectively. Serum creatinine levels were almost stable. We compared 9 responding patients (1 male and 8 female) and 8 non-responding patients (6 male and 2 female) at 12 months after TSP. Females was significantly higher responders (p  0.03). There is no significance in age, pathological activity, hypertension, and allergy between responders and non-responders. Conclusion: Approximately 50–60% of patients with IgA nephropathy showing proteinuria and hematuria reach complete remission at 12 months after TSP. The number needing treatment (NNT) with TSP for urinary abnormality in IgA nephropathy becomes almost 2. Female patients with IgA nephropathy respond better to TSP than male patients.

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C-P-18

Impact of Tonsillectomy Combined with Steroid Pulse Therapy (Tx-SPT) on IgA Nephropathy Kyoko Hara, Takahiro Nakayama, Eri Kawashima, Hironori Tayama, Fumihiko Koiwa, Hiroyuki Morita, Ashio Yoshimura Division of Nephrology, Showa University Fujigaoka Hospital, Yokohama, Kanagawa, Japan Tx-SPT is now widely applied to patients with IgA nephropathy (IgAN) in Japan, and clinical remission with disappearance of prolonged urinary abnormalities has been achieved in many patients, especially with mild form of IgAN. However, there is no consensus on whether Tx-SPT should be applied to progressive IgAN. We examined the effect of Tx-SPT retrospectively for 1 year in 21 patients with moderate to severe progressive form of IgAN as defined by kidney biopsy findings according to the criteria by the Japanese Society of Nephrology. After Tx-SPT, the patients (39.0  12.2 years old; 10 males and 11 females) were treated with prednisolone for 1 year (initially 30 mg every other day and thereafter reduced by 5 mg every 2 months). Urinary protein (U-p) was reduced by over 50% in 76 and 81.3% of the cases at 3 and 12 months (M) after Tx-SPT, respectively, with disappearance of proteinuria seen in 62% at 12 months. No patients showed any increase in U-p. The level of microhematuria was decreased in 76 and 81% of the patients at 3 and 12 months, respectively, with over 75% reduction in the sedimentary red blood cell number. Microhematuria disappeared in 92% at 12 months. Before TxSPT, 14 cases (67%) showed renal dysfunction with serum creatinine (SCr)
1.0 mg/dl, and 9 of these had advanced dysfunction with SCr
1.5 mg/dl and Cr clearance 70 ml/min. Renal function deteriorated only in 2 patients at 12 months who had over 3.0 g/day of U-p before the therapy. In the other 19 cases, U-p was less than 2.5 g/day, and renal function was either improved or maintained at 12 months. Thus Tx-SPT is one of the most powerful therapeutic strategies for patients with progressive IgAN. It can induce clinical remission with reduction/disappearance of proteinuria and microhematuria in many cases. Careful consideration is necessary on its use for patients with much severer proteinuria (
3.0 g/day).

C-P-19

Impacts of Tonsillectomy Plus One Course of Steroid Pulse Therapy on the Clinical Features of IgA Nephropathy Chihiro Yamazaki, Michiko Sasaki, Hideaki Nakajima, Kenichiro Kojima, Shunya Uchida Division of Nephrology, Department of Internal Medicine, Teikyo University School of Medicine, Tokyo, Japan Recently the efficacy of an adjunctive therapy of tonsillectomy and steroid pulse therapy in the treatment of IgA nephropathy has been advocated especially in Japan, but has yet to gain a world-wide agreement to the regimen. Thus, we have investigated whether tonsillectomy plus steroid pulse therapy has some impact on the clinical course of urinalysis and renal function in IgA nephropathy. Methylprednisolone pulse therapy was restricted to once with the aim of reducing its adverse effects. Dosing of oral prednisolone was between 15 and 30 mg daily. This is a prospective longitudinal study performed in a single institution to avoid clinical bias if any. Entry criteria for the present study included biopsy-proven primary IgA nephropathy that possessed either swollen tonsils or not-swollen tonsils but infected by alleged candidate pathogens such as Haemophilus parainfluenzae or Staphylococcus aureus. Patients enrolled in the present study were 14 (M:F  8:6, 18–64 years of age) and showed 0.3–3.0 g/day of proteinuria,  to 3 of hematuria and 0.5–2 mg/dl of serum creatinine (Cr). Creatinine clearance (Ccr)

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revealed 47–150 ml/min; the stages of chronic kidney disease were stage 1 (n  7), stage 2 (6) and stage 3 (1). Prognostic criteria based on the histopathologic findings consisted of relatively good (n  6), relatively poor (5) and poor prognoses (3) according to the classification by the Committee of IgA Nephropathy. Nephrotic level of proteinuria or serum Cr
2 mg/dl was excluded from the study. Doses of angiotensin converting enzyme inhibitor and angiotensin receptor blocker, if ever used, were unchanged during the study. At follow-up visits urine and blood samples were obtained and subjected to routine measurements. At 12 months proteinuria (1.9–0.5 g/day, ANOVA p  0.001) and hematuria (2.1–0.7, ANOVA p  0.005) significantly reduced, while Ccr remained constant (86–96 ml/min, ANOVA p  0.99). Serum IgA levels also decreased after the treatment (373–243 mg/dl, ANOVA p  0.04). Albeit in such a short course as 12 months, tonsillectomy plus one course of steroid pulse therapy followed by oral steroid therapy sufficiently alleviated the clinical features of IgA nephropathy. A long-term renal survival study is warranted in the future.

C-P-20

The Effects of Combined Therapy by Tonsillectomy and Corticosteroids for the Treatment of IgA Nephropathy Yasuhiro Abe, Yoshie Sasatomi, Satoru Ogahara, Toshiaki Murata, Hidetoshi Kaneoka, Takao Saito Division of Nephrology and Rheumatology, Fukuoka University School of Medicine, Fukuoka, Japan Background: As the treatment for IgA nephropathy, data have been accumulated about tonsillectomy and steroid administration. However the effects of the combination are still controversial. We compared efficacy between combined therapy and steroid alone administration and examine the short term prognosis. Method: Thirty six patients diagnosed as having biopsy-proven IgA nephropathy were enrolled in this study. Exclusion criteria were as followed: age younger than 18 years or older than 65 years, follow-up period less than 12 months, 24-hour urinary exertion under 0.5 g, number of glomeruli less than 10 in biopsy specimen. Twenty seven underwent tonsillectomy (Tx group). Afterwards nineteen of them were assigned for one or two courses of pulse intravenous therapy of methyl predonisolone at 500 mg daily for 3 consecutive days followed by oral steroid (OS); eight were OS only. As OS, 40 mg predonisolone (PSL) was given daily, gradually tapered and maintained alternative day therapy at 20 mg of PSL for 2 years. Nine patients were treated by oral administration of PSL alone (nonTx group). All the patients were given antiplatelet agents, anticoagulant agents and/or antihypertensive drugs (including angiotensin-converting inhibitors and/or angiotensin receptor blockers) as needed. The clinical findings in Tx group and Non-Tx groups were as follows; mean ages were 30.6  2.3 and 43.9  5.3 years old, proteinuria 1.4  0.1 and 1.3  0.4 g/day, serum creatinine 1.1  0.1 and 1.0  0.2 mg/dl, creatinine clearance 76.2  6.1 and 76.0  10.3 ml/min, respectively. Blood pressures were comparable in the two groups. Histologically, indexes of glomerular lesions (0–4) were 1.7  0.1 and 1.4  0.5, glomerular sclerosis rate 18.5  3.5 and 14.8  5.0%, and percentages of crescentic glomeruli were 8.3  1.5 and 16.5  4.8%, interstitial damage (0–3) 1.4  0.2 and 1.4  0.3, respectively. In each item, there were no significant differences between the groups. Results: Tx groups yielded positive effect on the reduction of proteinuria in both groups within 1 year. The number of RBC sediments was also significantly reduced in Tx group but not in Non-Tx group. The follow up observation over 2 years showed less than 0.3 g of urinary protein in Tx group whether they received following steroid pulse therapy or not, but urine protein increased from 0.3 to 0.6 g almost of Non-Tx group. Renal functions did not deteriorate in both groups. Clinical remission rate (defined as negative of proteinuria and hematuria) in Tx and Non-Tx group were 37.0% (10/27) and 22.2% (2/9) after 1 year, and 66.7% (10/15) and 22.2% (2/9) after 2 years, respectively. Severe adverse effects, such as gastro-intestinal bleeding or severe infection, were not observed during treatment.

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Conclusion: Combined therapy of tonsillectomy and steroids was effective for IgA nephropathy because both hematuria and proteinuria were improved for more than 2 years whether steroid pulse therapy were included or not.

C-P-21

Effectiveness of Corticosteroid Therapy According to Pozzi’s Protocol among Japanese IgA Nephropathy Patients: An Interim Report Sakamoto Izumi, Morita Yoshiki, Yasuda Yoshinari, Hayashi Hiroki, Mizuno Masashi, Maruyama Syouichi, Itoh Yasuhiko, Yuzawa Yukio, Matsuo Seiichi Departments of Nephrology, Nagoya University Graduate School of Medicine, Nagoya, Japan Background: IgA nephropathy is one of the most common causes of glomerulonephritis worldwide, which finally leads to the end-stage renal disease among 20–40% of patients within 20 years. Pozzi C et al. reported the effectiveness of methylpredonisolone pulse therapy combined with 0.5 mg/kg BW of oral corticosteroid administration on alternate days for 6 months (Pozzi’s protocol) by Italian multicenter randomized controlled trial (Lancet 1999;353:883–887 and J Am Soc Nephrol 2004;15:157–163); however, its effectiveness among the Japanese population has not yet been elucidated. Objective: To verify the effectiveness of corticosteroid therapy by Pozzi’s protocol, outcomes of IgA nephropathy patients treated by this regimen were retrospectively analyzed. Methods: All biopsy-proven IgA nephropathy patients were categorized using the clinical guideline reported by a joint committee of the Special Study Group on Progressive Glomerular Disease, Ministry of Health and Welfare of Japan including histological and clinical findings (Chairman, Hideto Sakai, MD, Clin Exp Nephrol 2003;7:93–97). Patients diagnosed as relatively poor or poor prognosis by the guideline, were treated according to Pozzi’s protocol after informed consent. Proteinuria and hematuria were evaluated by routine urinalysis. Primary and secondary end points were defined as 50% increase of serum creatinine level and as decrease of proteinuria less than 0.5 g/day, respectively. All treatments were conducted in Nagoya University Hospital. Results: Eighteen patients (6 males and 12 females) with serum creatinine level of 0.88  0.32 mg/dl (0.5–1.8 mg/dl) were introduced to the treatment according to Pozzi’s protocol. All 18 patients exhibited proteinuria over 0.5 g/day (1.46  0.88, 0.55–4.57 g/day) before the treatment. None of the patients were complicated with hypertension or diabetes mellitus. During the observation period of 33.7  16.5 months (12–59 months), none of the patients fall into primary end point of 50% increase of serum creatinine level. Percentages of patients achieving the secondary end point of proteinuria less than 0.5 g/day were 55.5% (10/18 cases) at 12 months, 72.8% (8/11) at 24 months and 57% (4/7) at 48 months, in good agreement with the previous report in Italy. Remission of hematuria (5 RBCs/HPF) constantly increased from 0% (0/18 cases) before treatment to 33.3% (8/18) at 12 months, 63.6% (7/11) at 24 months and 71.4% (5/7) at 48 months after corticosteroid therapy. No major complication by corticosteroid therapy was apparent during the observation period. Conclusion: Corticosteroid therapy according to Pozzi’s protocol reveals safety and effectiveness among Japanese IgA nephropathy patients as well, at least for the observation period of 30  6 months; however, further longer observation would be necessary to conclude its significance in Japan.

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C-P-22

Influence of Tonsillectomy on the Recurrence of Clinical Remission in Patients with IgA Nephropathy after Undergoing Steroid Pulse Therapy Yoshinobu Minamia, Haruhisa Otania, Masaki Ohyaa, Susumu Yukawaa, Yoshinori Toneb, Ryoichi Fujiib, Mastoshi Munec Wakayama Kidney Disease Clinic, Wakayama, bFujii Hospital, Anan, and cDepartment of Nutrition, Siebold University, Nagasaki, Japan

a

The combination of tonsillectomy and steroid pulse therapy has been known to probably result in clinical remission in IgA nephropathy patients. However, we have had patients who experienced clinical remission with steroid pulse therapy alone. We retrospectively investigated the differences in recurrence following clinical remission between a group of subjects who underwent tonsillectomy combined with steroid pulse therapy, and a group of subjects who underwent steroid pulse therapy alone. The clinical remission of IgA nephropathy was defined as the disappearance of urinalysis abnormality, that is, proteinuria and hematuria. The regimen for steroid pulse therapy was a high dose of methylprednisolone (0.5 g/day for three courses over a period of three days) followed by oral prednisolone at an initial dose of 15 mg/day, with the dosage being gradually decreased over a one-year period. Tonsillectomy for the combination treatment group was performed either before or within a year of the onset of steroid pulse therapy. The status of recurrence was investigated in 62 patients (20 males and 42 females) who were available for course observation during the year following after the completion of treatment. Recurrence was defined as the appearance of hematuria or proteinuria. At the onset of treatment, the mean age of patients was 34.6 years old, the mean serum Cr level was 0.9 mg/dl, and the mean protein level in urine was 876 mg/day. No differences were found in these parameters between the group with tonsillectomy combined with steroid pulse therapy (42 patients) and the group with steroid pulse therapy alone (20 patients). As a result, 15 patients (36%) experienced clinical remission in the group with tonsillectomy combined with steroid pulse therapy, compared with 7 patients (35%) in the group with steroid pulse therapy alone. The presence of recurrence was analyzed by using the Kaplan-Meier method. Recurrence was confirmed in 4 patients (26%) in the group with tonsillectomy combined with steroid pulse therapy, and in 5 patients (70%) in the group with steroid pulse therapy alone. Thus, the latter group had a significantly higher (p  0.01) incidence of recurrence. The period before recurrence was also significantly longer for the combination treatment group, as compared to the steroid pulse group (p  0.05, 26.5  10.0 months vs. 13.8  3.6 months). Multivariate Cox regression analysis also revealed that tonsillectomy has a significantly favorable effect on recurrence. In conclusion, tonsillectomy may extend the clinical remission in patients with IgA nephropathy after undergoing steroid pulse therapy.

C-P-23

Response of the Steroid Therapy and Histological Parameters of Childhood IgA Nephropathy Hiroshi Kitamuraa, Junko Udagawab, Toshiyuki Imasawaa, Kentaro Koikea, Hideaki Kurayamab, Kensuke Joha a Division of Immunopathology, Clinical Research Center, and bDepartment of Pediatrics, Chiba-East National Hospital, Japan

The purpose of this study is to clarify the effectiveness of the steroid therapy (ST; predonisolone 1 mg/kg/day) on the histological parameters of IgA nephropathy (IgAN) in children. The 100 cases (11.7  6 years old) who

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were diagnosed as IgAN by the 1st renal biopsy and underwent the 2nd renal biopsy more than 2 years after the 1st renal biopsy, were analyzed. Cases were divided into 4 groups according to either with () or without () ST, and either with continuance () or disappearance () of urine manifestation (UM; hematuria and/or proteinuria) at the time of 2nd renal biopsy, which consequently consisted of group A, 31 patients (ST, UM), group B, 16 patients (ST, UM), group C, 25 patients (ST, UM), and group D, 28 patients, (ST, UM). In each group, an extent of the active (A) and chronic (C) lesions (8 parameters) including glomerular mesangial cell proliferation (AGm), intracapillary hypercellularity (AGi), active extracapillary lesions (cellular and fibrocellular crescent) (AGe), interstitial inflammatory cell infiltration (Ai), global sclerosis (CGs), segmental sclerosis/hyalinosis or collapsing lesions (CGi), chronic extracapillary lesions (fibrous crescent or adhesion) (CGe) and tubulointerstitial fibrosis (Ci), were estimated. These 8 histological parameters were compared statistically between 1st and 2nd renal biopsy. In group A, AGm, AGi, AGe as well as CGe showed improvement, whereas CGs and Ci showed progression. In group D, progression of Ai, CGs, CGi, CGe, and Ci was shown. Between group A and B, the percentage of CGs was significantly higher in group B than those of group A before ST, whereas the percentage of AGi, AGe, and CGe was significantly higher in group B than those of group A after ST. In conclusion, ST was effective for improvement of active intracapillary and extracapillary lesions as well as chronic extracapillary lesions and was effective against progression of the other chronic lesions. However, the limited cases, which showed still active intracapillary as well as active and chronic extracapillary lesions even after ST, remains to be further analyzed on their process.

C-P-24

Tonsillectomy and Steroid Pulse Therapy Followed by Mizoribine Therapy for Pediatric Patients with Severe IgA Nephropathy Yuko Akiokaa, Kazuhiro Takahashib, Mikiya Fujiedac, Toshihiro Sawaid, Osamu Hottae, Motoshi Hattoria Department of Pediatric Nephrology, Tokyo Women’s Medical University, Tokyo, bDepartment of Pediatric Nephrology, Chiba Children’s Hospital, Chiba, cDepartment of Pediatrics, Kochi Medical School, Nankoku, dDepartment of Pediatrics, Shiga University of Medical Science, Otsu, and e Department of Nephrology, Sendai Shakaihoken Hospital, Sendai, Japan

a

Background: Short-term effect of steroid therapy for pediatric patients with IgA nephropathy has been established; however, there are considerable numbers of cases in whom their IgA nephropathy recurs after stopping the steroid therapy, and who result in carried over to adult. In other hand, long-term steroid administration will cause growth impairment. In this study, we examined the effects of a treatment regimen consisting of tonsillectomy and steroid pulse therapy followed by mizoribine to establish a curative therapy for achievement of a complete remission of pediatric IgA nephropathy without growth impairment. Method: For the study subjects, we included 13 patients (mean age: 17.4  5.4 years) who had childhoodonset IgA nephropathy. These subjects had glomerular hematuria, positive urine protein and acute segmental lesion in their renal biopsies. We excluded patients who showed rapidly progressive glomerulonephritis. We divided the subjects into two groups; one was an initial remedial group (Group A, n  5) and the other was an intractably relapsing group. The latter group was further subdivided into two subgroups; one was a normal renal function group (Group B, n  5) and the other was an impaired renal function group (Group C, n  3). Treatment regimen consisted of three courses of steroid pulse therapy after a tonsillectomy followed by an administration of mizoribine. The changes in hematuria, proteinuria, renal function, and adverse events were prospectively examined for at least more than 6 months.

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Result: The mean observation period was 7.4  1.5 months. In all cases, protein-creatinine ratio in urine decreased from a mean of 0.88  0.49 to 0.58  0.52 (p  0.01) and urinary sediment of red blood cells also improved (p  0.05). A significant decrease of serum IgA levels was observed early in postoperative period (p  0.01). A clinical remission defined as no urine abnormality was achieved in 20% of Group A patients, but was not observed in Group B and C patients. Remission of proteinuria was seen in 60% of Group A patients, but was not seen in Group B and C patients. Remission of hematuria was observed in 40% of Group A, 40% of Group B, and 33.3% of Group C patients. In addition, a further deterioration of renal function was not observed in Group C. There were no serious adverse events associated with this treatment regimen. Also, a growth impairment, moonshaped face or hypertension associated with a prolonged steroid therapy was not found in these cases. Conclusion: Although a continuous and careful monitoring of therapeutic effects and adverse reactions will be necessary, a treatment regimen consisting of tonsillectomy and steroid pulse therapy followed by mizoribine therapy can be a valuable addition to therapeutic options for treating patients with severe relapsing IgA nephropathy in children.

C-P-25

Renoprotection of Combined Low-Dose Predonisolone and Losartan in IgA Nephropathy Yoshio Horitaa, Ryu-ichi Ashidaa, Mayumi Hiua, Kouichi Taurab, Takashi Taguchic, Akira Furusud, Shigeru Kohnod a Department of Internal Medicine, National Nagasaki Medical Center, Omura, bDepartment of Internal Medicine, Nagasaki Municipal Medical Center, cDepartment of Pathology, and d Department of Internal Medicine, Nagasaki University Graduate School of Biomedical Sciences, Nagasaki, Japan

Background: This study investigates the ability of low-dose prednisolone, in combination with angiotensin II receptor blockers, to exert antiproteinuric effects in normotensive and proteinuric IgA nephropathy. Methods: A prospective randomized controlled clinical trial was performed to evaluate the effects of therapy with prednisolone (PSL, corticosteroid: 30 mg/dl, gradually tapered to 5 mg/dl during 2 years) plus 50 mg of losartan daily (LST, an angiotensin II receptor blocker) or PSL alone on normotensive and proteinuric IgAN. We divided 38 cases of IgAN (33  11 years old, serum creatinine 0.7  0.1 mg/dl, creatinine clearance 103  31 ml/min and proteinuria 1.6  0.5 g/day), glomerular score ranging 4–7 by Katafuchi’s grading (Clin Nephrol 1998), into two groups to be treated for 2 years with either PSL plus LST or PSL alone. There was no significant difference between the two groups in baseline clinical and histopathological data. The renal protective effects of each protocol were evaluated in terms of reduction in proteinuria, and preservation of renal function. After 2 years, proteinuria and creatinine clearance were compared. Results: Proteinuria was reduced in two groups (PSL plus LST: from 1.6  0.6 g/day to 0.3  0.1 g/day, PSL: from 1.6  0.3 g/day to 0.5  0.1 g/day, p  0.05, respectively). Creatinine clearance remained essentially unchanged for patients in PSL plus LST (from 104.3  36.4 ml/min to 100.4  38.9 ml/min). On the other hand, creatinine clearance decreased in PSL only (from 103.4  28.5 ml/min to 84.8  34.3 ml/min, p  0.05). Proteinuria significantly decreased at 2 years of treatment in both groups compared with baseline, and the therapeutic efficiency of PSL plus LST was better than that of PSL alone (p  0.05). Although creatinine clearance in both groups was almost the same at the start of study, there was a significant difference between them by the conclusion of the study (p  0.05). There were no significant differences between the groups with reference to side-effects or complications.

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Conclusion: Preliminary study had indicated that combined therapy with PSL plus LST was more effective than with PSL alone in reducing proteinuria and protecting renal function in patients with moderate proteinuric IgAN.

C-P-26

Mycophenolate Mofetil (MMF) in Patients with IgA Nephropathy, Grade I and III, Two-Year Follow-Up Ladislava Grcevska, Momir Polenakovic, Sonja Dzikova Department of Nephrology, Clinical Center, Skopje, R. Macedonia Up to 40% of the patients with IgA nephropathy progress to end-stage renal disease over 10–20 years. Currently, treatment is limited. MMF in glomerulonephritis combines two actions: as an immunosuppressive agent it reduces the inflammatory process early on and subsequently it interferes with the genesis of fibrosis. In order to document these two actions of MMF in patients with IgAN we used the drug in two groups of patients: grade I (minimal changes) and III (25–49% of glomeruli with crescents/segmental sclerosis) according to H.S. Lee’s glomerular grading system. First group consisted of 3 patients, 2 female and one male, aged 20.67  0.88, proteinuric 2.89  1.19 g/day with normal serum creatinine 58.33  7.31 and normal blood pressure. Second group consisted of 5 patients, all male, aged 31  2.5 proteinuric 3.37  0.73 g/day, with slight elevation of serum creatinine (155.4  25.89) and regulated hypertension in 4/5. The daily dosage of MMF for the first 6 months was 2 g, and 1.5 g for the further 18 months. Total follow-up was 2 years. Group I patients presented significant decrease of proteinuria to 0.51  0.2 g/day. One patient of grade III group presented slow worsening of the renal function, serum creatinine 160–286 mol/l, without significant change of proteinuria (5.48–4.46 g/day).The second one presented improvement of serum creatinine 255–187 mol/l, and decrease of proteinuria (2.42–0.79 g/day). The other three patients presented stable renal function and slight decrease of proteinuria. Besides the small number of treated patients we noted some benefit of MMF treatment in grade I and III IgAN patients.

C-P-27

Pharmacodynamic Monitoring of Mycophenolic Acid Treatment Investigating IMPDH-Activity, T-Cell Proliferation and Reactivity of Th1-Cells Lutz von Müllera, David Czockb, Alexandra Schillerb, Denise Dangelmeierb, Alexander Cariusb, Petra Glanderc, Steffen Bauerd, Thomas Mertensa, Frieder Kellerb, Franz M. Rascheb a

Department of Microbiology and Virology, and bDepartment of Internal Medicine, University of Ulm, Ulm, cDepartment of Internal Medicine-Nephrology and dInstitute of Clinical Pharmacology, Charite Campus Mitte, Humboldt University, Berlin, Germany

Mycophenolic acid (MPA) is established for immunosuppressive therapy of patients with transplantations and autoimmune diseases. In the present randomized cross-over study we investigated pharmacokinetics and pharmacodynamics of MPA in seven patients with progressive immunoglobulin A nephropathy (IgAN) treated with mycophenolate-mofetil (500 mg MMF, Cell Cept) or enteric coated MPA sodium (360 mg EC-MPS, Myfortic).

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Fourteen episodes were monitored, each at eight consecutive times (112 samples) and MPA and MPA-glucuronide (MPAG) in plasma, target enzyme activity (IMPDH) and also target cell function after stimulation with staphylococcal enterotoxin B (SEB) and CMV antigen were analyzed. Early and late target cell functions were investigated using Th1-cell reactivity (CD69 and IFN- expression) and the proliferation of CD4 and CD8 T-cells. CD4 and CD8 T-cell proliferation was inhibited dose dependently by MPA application in vitro and also using samples of patients with MPA therapy. Effector functions of Th1-cells detected by CD69 and IFN- expression were modulated neither by MPA application in vitro nor in patients after MPA treatment. MPA concentration was better correlated with target cell proliferation (CD4 and CD8 cells) compared with IMPDH. We conclude that T-cell proliferation but not effector functions of Th1-cells were inhibited by MPA. Investigation of T-cell proliferation is a promising tool for pharmacodynamic drug monitoring; however, the clinical value for individualized treatment regimes with MPA remains to be shown in future studies.

C-P-28

High-Dose Intravenous Immunoglobulin Pulse Therapy in Patients with Progressive IgA Nephropathy – A Long Term Follow-Up Franz M. Raschea, David Czocka, Lutz von Müllerb, Philipp Lepperc, Wolfram Kargesa, Frieder Kellera Department of Internal Medicine I and bDepartment of Microbiology and Virology, University Hospital of Ulm, Ulm, Germany; cDepartment of Intensive Care Medicine, University Hospital of Berne, Berne, Switzerland a

Introduction: In progressive IgA nephropathy (IgAN) intravenous immunoglobulin (IVIg) treatment has been used to delay disease progression, but the long term efficacy is largely unknown. Methods: We report the clinical outcomes after IVIg therapy in six male patients with progressive IgAN (median glomerular filtration rate (GFR) 31 ml/min per 1.73 m2) followed for a median observation period of 8 years. IVIg was given monthly at a dose of 2 g/kg body weight divided on two subsequent days over a period of 6 months. The course of renal function was assessed by linear regression analysis of GFR and proteinuria, and was compared to eight patients with IgAN (median GFR 29 ml/min per 1.73 m2) without IVIg as a contemporaneous control group. Results: IgAN disease progression was delayed after IVIg therapy on average for 3 years. The mean loss of renal function decreased from –1.05 ml/min per month to –0.15 ml/min per month (p  0.024) and proteinuria decreased from 2.4 g/l to 1.0 g/l (p  0.015). The primary end point (GFR 10 ml/min or relapse) occurred after 5.2 years (median; range 0.4–8.8) after the first IVIg pulse, and after 1.3 years (median; range 0.8–2.4) in the control group (p  0.043). In Kaplan-Meier analysis, the median renal survival time with IVIg was prolonged by 3.5 years (IVIg 4.7 years vs. control 1.2 years; p  0.006). Conclusion: IVIg pulse therapy may be considered as treatment option to reduce the loss of renal function and improve proteinuria in patients with progressive IgAN.

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Basic Advances (Update)

B-O-01

Down-Regulation of ␤1,3-Galactosyltransferase Gene in Tonsillar B Lymphocytes and Abberant Lectin Bindings to Tonsillar IgA as a Pathogenesis IgA Nephropathy Tatsuyuki Inouea, Hitoshi Sugiyamaa, Yoko Kikumotoa, Naomi Fukuoka-Todaa, Youhei Maeshimaa, Hisashi Hattoria, Kunihiro Fukushimab, Kazunori Nishizakib, Yoshiyuki Hikic, Hirofumi Makinoa a Department of Medicine and Clinical Science and bOtolaryngology, Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama, and c Department of Medicine, Fujita Health University School of Medicine, Tokoake, Japan

Human immunoglobulin A is a glycoprotein containing multiple O-linked glycans as well as N-linked carbohydrates. Under-O-glycosylation of the hinge in serum IgA and deposited IgA in glomeruli in patients with IgA nephropathy (IgAN) is reported. The clinical development and exacerbation of IgA nephropathy frequently are preceded by episodes of upper respiratory tract infections such as tonsillitis. Therefore, tonsils, which represent the predominant immunocompetent tissue of the upper respiratory tract, may be related to the pathogenesis of IgA nephropathy. Indeed, recent reports indicated that IgA1 molecules from tonsils are under-O-glycosylated in IgAN using the mass spectrometric analysis (Am J Kidney Dis 2003;42:486). However, the mechanism of production of underglycosylated IgA1 produced by tonsillar B lymphocytes has not been fully elucidated yet. Since O-glycans in IgA1 molecules are produced by enzymatic reaction by glycosyltransferases including galactosyltransferases and sialyltransferases, we hypothesized that dysregulation of the glycosyltransferases were associated with aberrant O-glycosylation in IgA1 molecules in tonsil extracts of patients with IgAN. Extracted tonsils were obtained from patients with IgAN, and chronic tonsillitis (CT) or sleep apnea syndrome (SAS) as controls after the approval by the Institutional Review Board of Okayama University. Gene expressions of various glycosyltransferases were examined by real-time RT-PCR in tonsillar CD19-positive B lymphocytes isolated by the magnetic-associated cell sorting. Glycosylation levels in IgA1 in tonsillar extracts were determined by enzyme-linked lectin binding assay (ELLA) using the following lectins: Jacaline, peanut agglutinin (PNA), Maackia amurensis (MAA), and Vicia villosa (VVL), which bound to alternative forms of O-glycan moieties. Gene expression of 1,3-galactosyltransferase (3GalT), the enzyme responsible for O-galactosylation, significantly decreased in tonsillar B cells in patients with IgAN (n  11) compared with those in SAS (n  5) (p  0.05). The levels of core 1 1,3galactosyltransferase-specific molecular chaperon (Cosmc), UDP-N-acetyl--D-galactosamine: polypeptide N-acetylgalactosaminyltransferase 2 (ppGalNAc-T 2), an essential enzyme for initiation of O-linked glycosylation of the IgA1 hinge region, sialyltransferases including 2,3-Gal-sialyltransferase (ST3Gal), and 2,6-GalNAc-sialyltransferase III (ST6GalNAc III) were not significantly different between the three groups. In ELLA, VVL and PNA bindings to IgA1 in tonsillar extracts from IgAN (n  16) significantly increased compared with those from SAS (n  9) or CT (n  15) (p  0.05). Since VVL binds to ungalactosylated terminal GalNAc residue, and PNA to the core 1 unsialylated Gal1,3GalNAc residue, the data indicate the loss of both galactose and sialic acids of IgA1 molecules. This study reports that in tonsillar B lymphocytes down-regulation of 3GalT may be involved in under-O-glycosylation of IgA1 from patients with IgAN and thus may be involved in the pathogenesis of the disease.

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B-O-02

Functional Evaluation of a Novel ST6GALNAC2 Promoter Haplotype Associated with Susceptibility to IgA Nephropathy Li Zhu, Hong Zhang, Gui-Sen Li, Hai-Yan Wang Renal Division of Peking University First Hospital, and Peking University Institute of Nephrology, Beijing, China Increasing evidence has implicated aberrant glycosylation of IgA1 molecules in the pathogenesis of IgA nephropathy (IgAN), including 2,6 sialic acid defects. Our previous case-control association study in 1,164 individuals revealed that a novel haplotype ADG (949G/A, 410-/CGGC(D/I), 96G/C, 1 corresponds to the transcription start site) in the promoter region of ST6GALNAC2 gene, encoding for Gal1,3GalNAc-specific GalNAc 2,6-sialyltransferase II (ST6GalNAc II), was associated with susceptibility to IgA nephropathy. For further functional evaluation of ADG haplotype, luciferase activity assay was performed. In the present study, a series of fragments containing truncated ST6GALNAC2 promoter regions (from 1,037, 923, 585, 274 to 12), and two DNA fragments containing the haplotype ADG and GDG, which is the most common haplotype in the population, were cloned into pGL3 promoterless vector to generate the constructs pGL3-1049, pGL3-935, pGL3597, pGL3-286 and pGL3-1049(ADG), and pGL3-1049(GDG). After these were transiently transfected into COS-7 cells, luciferase activities were measured. Among the four constructs containing truncated ST6GALNAC2 promoter regions, the most obvious increased luciferase activity was observed between the constructs pGL3-1049 and pGL3-935. Importantly, the fragment containing the ADG haplotype significantly reduced luciferase activity compared to that containing the GDG haplotype (196.43  21.55 versus 258.41  46.25, p  0.002). Progressive deletion analysis of ST6GALNAC2 promoter revealed that the region from 1,037 to 923 had the most remarkable positive effect on ST6GALNAC2 transcription. The ADG haplotype in the ST6GALNAC2 promoter region reduced promoter activity compared to the GDG haplotype. These findings suggest that the ADG haplotype in ST6GALNAC2 promoter region contributes to susceptibility to IgA nephropathy by reducing promoter activity and thus transcription efficiency of the ST6GALNAC2 gene.

B-O-03

Development of IgA Nephropathy-Like Disease with High Serum IgA Levels and Increased Proportion of Polymeric IgA in Beta-1,4Galactosyltransferase-Deficient Mice Toshikazu Nishiea, Osamu Miyaishib, Haruhito Azumac, Akihiko Kameyamad, Chie Narusea, Noriyoshi Hashimotoa, Hitoshi Yokoyamae, Hisashi Narimatsud, Takashi Wadae, Masahide Asanoa a

Division of Transgenic Animal Science, Advanced Science Research Center, Kanazawa University, Kanazawa, Ishikawa, bDepartment of Pathology, Aichi Medical University School of Medicine, Nagakute, cDepartment of Urology, Osaka Medical College, Osaka, dGlycogene Function Team, Research Center for Glycoscience, National Institute of Advanced Industrial Science and Technology (AIST), and eDepartment of Gastroenterology and Nephrology, Graduate School of Medical Science and Division of Blood Purification, Kanazawa University, Kanazawa, Japan

The glycosylation of glycoproteins is important for their biological activities, conformation, stability, and transport. Recent studies indicate that aberrant glycosylation causes various human disorders, such as metastasis of tumor cells, muscular dystrophy, and dyserythropoietic anemia.

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-1,4-galactosyltransferases (4GalTs) transfer galactose (Gal) from UDP-Gal to terminal GlcNAc of N- and O-glycans in a -1,4-linkage. Previously, we generated mice lacking 4GalT-I, a member of the 4GalT family, which is expressed strongly in almost all tissues except neural tissues. The 4GalT-I-deficient mice show semilethality before weaning due to growth retardation, and reduced inflammatory responses and delayed skin wound healing due to impaired leukocyte infiltration caused by the reduced biosynthesis of selectin ligands. In addition, the surviving 4GalT-I-deficient mice developed similarly to control mice after weaning, but they began to die sporadically from 10 weeks of age. Autopsy revealed that the kidney was small and pale. Here, we investigated the renal disorder of the 4GalT-I-deficient mice in more detail. Urinary albumin levels were significantly increased in 4GalT-I-deficient mice compared with control mice, while hematuria was detected only in a few 4GalT-I-deficient mice. Glomerular sclerosis was observed in 4GalT-I-deficient mice from 8 weeks of age and progressed with age. Furthermore, histological examination showed the IgA deposition of mesangial pattern and electron dense deposits in the paramesangial regions of 4GalT-I-deficient mice. These results demonstrate that 4GalT-I-deficient mice developed IgA nephropathy (IgAN)-like disease. Furthermore, high serum IgA levels with increased polymeric forms were detected in 4GalT-I-deficient mice. In humans, serum IgA derived from patients with IgAN is deficient in 3-galactosylation and sialylation on its O-linked glycans of the hinge region. However, mouse IgA doesn’t have O-glycans of the hinge region and has several N-glycans. As expected, 4-galactosylation and sialylation on the N-glycans of the serum IgA of the 4GalT-I-deficient mice was completely absent. This is the first report demonstrating that genetic remodeling of protein glycosylation causes IgAN. Our data suggest that serum IgA abnormalities (high serum IgA levels with increased polymeric forms and aberrant galactosylation of serum IgA N-glycans) cause IgAN. A hyper IgA (HIGA) mouse, a mouse model for human IgAN with high serum IgA levels, also shows reduced galactosylation of the N-glycans on serum IgA. These results support our hypothesis that aberrant N-glycosylation of IgA is involved in the pathogenesis of IgAN. It is reported that mutant human IgA1 molecule lacking the N-glycosylation sites tends to form higher polymeric forms. Therefore, we suppose that polymeric IgA in the 4GalT-I-deficient mice increased, because asialo-agalacto IgA tends to polymerize. Immune complexes including IgA, which are easily formed by the polymeric IgA due to its multivalent properties, may be trapped in the glomeruli. However, we cannot exclude the possibility that IgAN in 4GalT-I-deficient mice was caused by other mechanisms, because many glycoproteins were impaired in 4-galactosylation.

B-O-04

IgA Nephropathy (IGAN): Characterization of IgG Antibodies Specific for Galactose (Gal)-Deficient IgA1 Hitoshi Suzukia, Zina Moldoveanua, Stacy Halla, Rhubell Browna, Bruce A. Juliana, Robert J. Wyattc, Milan Tomanaa, Yasuhiko Tominob, Jan Novaka, Jiri Mesteckya a University of Alabama at Birmingham, Birmingham, Ala., USA; bDivision of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan; c University of Tennessee Health Sciences Center, Memphis, Tenn., USA

IgAN is characterized by IgA1-containing immune complexes in glomerular deposits and in the circulation. We have shown that the circulating immune complexes (CIC) are composed of Gal-deficient IgA1 bound to IgG or IgA1 antibodies specific for Gal-deficient IgA1. To analyze properties of the anti-glycan antibodies, we have determined binding of serum IgG from IgAN patients (n  15) and healthy controls (n  15) and IgG secreted by Epstein-Barr virus (EBV)-immortalized B cells from the same subjects to a panel of antigens coated on ELISA plates. These antigens were: enzymatically desialylated and degalactosylated IgA1 (dd-IgA1), Fab fragment of Galdeficient IgA1 containing part of the hinge region with O-glycans (Fab-IgA1), synthetic hinge-region peptide linked

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to bovine albumin (HR-BSA), and synthetic hinge-region glycopeptide with three GalNAc residues linked to BSA (HR-GalNAc-BSA). IgG-secreting EBV-immortalized cell lines were subcloned by limiting dilution. Total IgG concentrations and IgG subclasses were measured by ELISA. The levels of IgG in sera and supernatants directed against dd-IgA1 and Fab-IgA1 were significantly higher in IgAN patients than in controls (p  0.01). IgG from IgAN patients exhibited strong reactivity with HR-GalNAc-BSA, but not with HR-BSA. The IgG-secreting cell lines produced antibodies specific to dd-IgA1; the antigen-specific IgG was most frequently of the IgG2 subclass. In summary, sera and supernatants from IgG-secreting cell lines from patients with IgAN were characterized by high levels of IgG antibodies with specificity to the Gal-deficient O-linked glycans of IgA1. The immortalized cell lines provide stable and convenient source for IgG antibodies specific to the aberrant O-glycans in IgA1.

B-O-05

IgA Nephropathy (IgAN) and Henoch-Schoenlein Purpura Nephritis (HSPN): Aberrant Glycosylation of IgA1, Formation of IgA1-Containing Immune Complexes, and Activation of Mesangial Cells Jan Novaka, Zina Moldoveanua, Matthew B. Renfrowa, Takeshi Yanagiharaa, Hitoshi Suzukia, Alice Goepferta, Milan Tomanaa, Robert J. Wyattc, Bruce A. Juliana, Jiri Mesteckya a University of Alabama at Birmingham, Birmingham, Ala., USA; bAarhus University, Aarhus, Denmark; cUniversity of Tennessee Health Sciences Center, Memphis, Tenn., USA

IgA1 in the circulation and glomerular deposits of IgAN patients is aberrantly glycosylated; the hinge-region O-linked glycans are galactose-deficient. A similar defect is characteristic for HSPN patients. This aberrancy results in exposure of N-acetylgalactosamine-containing neoepitopes. These antigenic epitopes are recognized by naturally occurring IgG or IgA1 antibodies and, subsequently, immune complexes are formed. IgA1 contains 3–6 O-glycosylation sites per heavy chain; it is not known whether the glycosylation defect occurs randomly or preferentially at specific sites. Furthermore, it is not known whether the aberrant IgA1 molecules by themselves are pathogenic or whether the formed immune complexes are responsible for the effects. We sought to define the aberrant glycosylation of this IgA1, and analyze the formation of the immune complexes and their biological activities. We undertook the following approaches: in vitro formation of IgA1-containing immune complexes, culture of human mesangial cells to assess the capacity of size-fractionated complexes to induce cellular proliferation, and protocols to determine the glycosylation sites in the hinge region of IgA1. We have identified a galactose-deficient IgA1 myeloma protein that was able to replace IgA1 in the immune complexes from IgAN patients and used it in several studies as a model protein for aberrant IgA1. Supplementation of serum or cord blood serum with this IgA1 protein resulted in formation of new IgA1 complexes. These complexes stimulated proliferation of cultured human mesangial cells, as did the naturally occurring IgA1-containing complexes from sera of patients with IgAN and HSPN. Uncomplexed IgA1 did not affect cellular proliferation. Using specific proteases, lectin Western blots, and high-resolution mass spectrometry, we have determined the O-glycosylation sites in the hinge region of the IgA1 myeloma protein and IgA1 proteins from sera of IgAN patients. The IgA1 myeloma protein had galactosedeficient sites at residues 228 and/or 230 and 232. These 3 sites reacted with IgG specific to galactose-deficient IgA1. IgA1 proteins from the IgAN patients had galactose-deficient O-glycans at the same residues. However, the specific profiles differed among the patients. EBV-immortalized B cells from IgAN patients secreted polymeric IgA1 that was galactose-deficient. These cells thus offer a convenient source of aberrant IgA1 that will be further characterized. In summary, we have identified the neoepitopes on IgA1 in IgAN. These studies may lead to development of noninvasive diagnostic assays and future disease-specific therapy. Other contributing authors include Stacy Halla, Rhubell Browna, Mogens Kilianb, Knud Poulsenb, Wen-Qiang Huanga.

Basic Advances (Update)

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B-O-06

Patients with IgA Nephropathy Respond Strongly through Production of IgA with Low Avidity against Staphylococcus aureus Yoshio Shimizua, Kouichi Hirayamab, Masaki Kobayashib, Shuzo Kanekoa, Masahiro Hagiwaraa, Keigyo Yoha, Kunihiro Yamagataa, Akio Koyamac a

Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, Department of Nephrology, Tokyo Medical University Kasumigaura Hospital, Tokyo, and cIbaraki Prefectural University of Health Sciences, Ami, Japan b

Background: IgA nephropathy (IgAN) is thought to have a close relationship with bacterial infection, and post-methicillin resistant Staphylococcus aureus infection glomerulonephritis (post-MRSA infection GN) is seen after MRSA infection and is a similar disorder to IgAN. We have found that immune complexes composed of staphylococcal cell membrane antigen and IgA are deposited in the glomerular mesangial areas in patients with post-MRSA infection GN and in more than 50% of patients with IgAN (Kidney Int 2004;66:121–32). Aim: The aim of the study is to examine the characteristics of serum IgA obtained from patients with IgAN. Patients & Methods: Experiment 1. Serum samples were obtained from patients with IgAN (n  16) and post-MRSA infection GN (n  19), and from healthy donors (n  13). Serum IgA and IgG titers against S. aureus were measured using an ELISA. Experiment 2. Relative antibody avidities of serum IgA and IgG against S. aureus from patients and healthy donors were determined in a dissociation assay using an ELISA. Results: Experiment 1. IgA class titers of anti-S. aureus antibodies in patients with IgAN and post-MRSA infection GN were significantly higher than in healthy controls, and IgG class titers of anti-S. aureus antibodies in patients with post-MRSA infection GN were significantly higher than those in IgAN patients and healthy donors. A significant correlation between IgG and IgA titers was found across all cases, and this was particularly evident in patients with IgAN. Experiment 2. No significant correlation was observed between avidity and titer of IgA class antibodies. The avidity of anti-S. aureus IgG did not differ significantly among IgAN patients, postMRSA infection GN patients, and healthy donors, but the avidity of anti-S. aureus IgA from patients with IgAN was significantly lower than those from the other groups. There was a significant correlation between antibody titer and antibody avidity for the IgG class in patients with post-MRSA GN, but no significant correlation was observed for the IgA class. Conclusion: These results suggest that patients with IgAN have a strong response through production of IgA with low avidity against S. aureus.

B-O-07

The Switch of Proteasome to Immunoproteasome in Peripheral Lymphomonocytes of IgA Nephropathy (IgAN) Patients Is Corrected by Steroids and Not by ACE-Inhibitors A. Amorea, L. Peruzzia, A. Alfaranoa, A. Dal Cantonb, V. Sepeb, S. Balegnob, L. Bertolab, D. Mancusob, P. Tovoc, R. Coppoc a Nephrology, Dialysis and Transplant Unit, Regina Margherita Children’s Hospital, Turin, bChair of Nephrology, University of Pavia, Pavia, cChair of Pediatrics, University of Turin, Turin, Italy

The proteasome (PS) plays a key role in the activation of transcriptional factors, cytokines and presentation of HLA-I restricted peptides. In immune mature cells, as dendritic cells, the PS, under the action of interferon and

Abstracts

219

, becomes an immuno-proteasome (iPS), by substituting 3 catalytic units 1, 2, 5 with other low molecular weight proteins (LMP2 and LMP7) and an endopeptidase-like complex (MECL-1). This modification confers an optimal catalytic property for professional presentation of specific peptides to MHC Class I. Our group previously demonstrated that patients (pts) with IgA-Nephropathy (IgAN) have an increased expression of iPS catalytic subunit. In this study (that completely evaluates the PS and iPS subunit involved in the switch) we investigated the effects of common treatments for IgAN on this iPS switch. Peripheral Lymphomonocytes (PBMC) of 48 IgAN patients and 30 healthy control subjects (C), isolated by centrifugation gradient, were tested with real time PRC (Taqman methodology) to assess quantitatively the mRNA levels of  subunit (constitutive) and of the active subunit of PS (1, 2, 5) and iPS (LMP2, LMP7 and MECL-1). The normalized values and the ratio between mRNA of corresponding subunit of PS and iPS (p values vs. C calculated with Student’s t test, NS  not significant) are reported in the table.

Controls Not treated IgAN ACE-I IgAN Steroids IgAN

LMP2/1

p

LMP7/2

p

MECL-15

p

0.86  0.44 1.11  0.6 1.40  0.79 1.23  0.9

0.05 0.05 NS

1.01  0.25 1.94  0.8 2.26  0.9 1.23  0.48

0.01 0.001 NS

1.13  0.37 1.77  0.9 2.27  1.3 1.51  0.5

0.01 0.001 0.05

The patients with IgAN not treated and treated with ACE-I showed a switch of the catalytic units of the PS to iPS, which was blunted in the pts treated with steroids. The hyper-activation of PS system during IgAN would seem to be more sensitive to steroid therapy than to ACE-I.

B-O-08

Differential Binding and Biological Effect of in vitro Deglycosylated IgA1 with Different Molecular Weight on Human Mesangial Cells Yue-hua Gao, Lixia Xu, Junjun Zhang, Ying Zhang, Minghui Zhao, Haiyan Wang Renal Division and Institute of Nephrology of Peking University First Hospital, Beijing, P.R. China Background and Aims: Many studies suggested that abnormal physicochemical properties of circulating IgA1, such as size, charge and glycosylation, might play a pivotal role in the pathogenesis of IgA nephropathy. Recent studies had demonstrated that serum and mesangial IgA1 in IgA nephropathy were polymeric and deglycosylated. The aim of the current study is to investigate the binding capacity and biological effect of in vitro deglycosylated IgA1 with different size on human mesangial cells. Methods: Serum IgA1, purified by jacalin affinity chromatography, was desialylated (deSIgA1), desialylated and degalactosylated (deS/deGalIgA1) with neuraminidase, neuraminidase plus -galactosidase respectively. The monomeric IgA1 (mIgA1) and polymeric IgA1 (pIgA1) were separated by Sephacryl S-300 chromatography. IgA1 with molecular weight over 440 kDa was collected as polymeric IgA1, while IgA1 with molecular weight around 150 kDa was collected as monomeric IgA1. The polymeric native IgA1, polymeric deSIgA1 and polymeric deS/deGalIgA1 were defined as p1, p2 and p3 respectively. The monomeric native IgA1, monomeric deSIgA1 and monomeric deS/deGalIgA1 were defined as m1, m2 and m3 respectively. Binding capacities of the mIgA1 and pIgA1 to primary human mesangial cell (HMC) were evaluated by radioligand assay. Fibronectin (Fn) in culture supernatant was detected by enzyme-linked immunosorbent assay (ELISA).

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Results 1: The p1 and m1 could bind to HMC in a dose-dependent, saturable manner. The maximal binding capacity of p1 was significantly higher than that of m1 (p  0.05). Based on one IgA1 receptor binding one IgA1 molecule only, the binding sites/cell of p1 was significantly higher than that of m1 (p  0.05). However, the affinity of m1 was significantly higher than that of p1 as Scatchard analysis revealed that the dissociation constants (Kd) of m1 and p1 were 6.25  4.33 109 mol/l and 1.12  0.54 107 mol/l respectively (p  0.05). Production of supernatant fibronectin stimulated by p1 was significantly higher than m1 (p  0.05). 2: After desialytion and degalactosylation in vitro, the binding of the two deglycosylated mIgA1 to HMC was abolished. However, the maximal binding capacities of p2 and p3 to HMC were significantly increased compared with that of p1 (p  0.05, respectively), the binding sites/cell of p2 and p3 to HMC were also significantly increased compared with that of p1 (p  0.05, respectively). The maximal binding capacities and the binding sites/cell of p2 and p3 were similar. The affinity of p2 and p3 was similar to that of p1 as the Kd of p2 and p3 were 3.10  2.00 107 mol/l and 3.44  2.70 107 mol/l respectively (p
0.05). However, the production of supernatant fibronectin stimulated by p2 and p3 was significantly higher than that of p1 (p  0.05). Conclusion: The current study suggested that native polymeric and monomeric IgA1 might bind to different receptors on mesangial cells. Loss of sialic acid or/and galactose could enhance the binding capacity of polymeric IgA1 on HMC and could promote the secretion of fibronectin.

B-O-09

Cell Surface Beta 1,4-Galactosyltransferase Acts as a Novel IgA Receptor in Human Mesangial Cells Jonathan Barratt, Karen Molyneux, Alice Smith, John Feehally John Walls Renal Unit and Department of Infection, Immunity and Inflammation, University of Leicester, Leicester, UK The mechanism by which mesangial IgA deposition leads to glomerular injury in IgA nephropathy (IgAN) is poorly understood. Human mesangial cells (HMC) are believed to play a pivotal role in this process as they bind IgA in vitro and this binding stimulates pro-inflammatory cytokine production and superoxide generation. The nature of the IgA receptors expressed by human MCs has been an area of controversy for some time and currently it is believed that CD71, the Fc alpha/mu Receptor, an asialoglycoprotein receptor and an as yet uncharacterised Fc alpha Receptor are all involved. We have previously presented data demonstrating the expression of a novel Fc alpha Receptor by HMCs and now present data identifying this as beta 1,4-galactosyltransferase I (GALT I). A HMC cDNA library was expressed in HEK 293 cells and screened for IgA binding proteins by FACS. IgA binding affinity of positively screened clones was reconfirmed and those clones exhibiting greatest IgA binding were sequenced and GALT I identified. Simultaneous with this HMC membrane fractions were tested against IgAcoated IDM affinity beads and IgA binding membrane proteins identified using SELDI-TOFMS. A 51 kDa protein was identified consistent with IgA binding to GALT I. Western blotting of HMC membrane fractions confirmed expression of GALT I and expression of both the long (cell surface) and short (golgi apparatus) splice variants have been confirmed by RT-PCR and confocal microscopy. GALT I is most commonly found in the golgi apparatus where it is involved in the N-linked glycosylation of cellular proteins. GALT I can however also be expressed on the cell surface where it binds a number of proteins through their N-linked sugars. Furthermore, the long isoform of GALT I has been shown to have intracellular signalling capability. We propose that GALT I can act as a HMC IgA receptor binding the N-linked sugars in the Fc alpha region of the IgA molecule. The precise role of GALT I in the clearance of IgA from the mesangium and in MC activation in IgAN are areas of ongoing work.

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B-O-10

Transferrin Receptor Engagement by Polymeric IgA1 Induces Receptor Expression and Mesangial Cell Proliferation: Role in IgA Nephropathy Ivan C. Mouraa, Houda Tamouzaa, Charlotte Sadakaa, Michelle Arcos-Fajardoa, Yves Lepelletierb, François Vrtovsnikc, Marc Benhamoua, Renato C. Monteiroa Institut National de la Santé et de la Recherche Médicale (INSERM) U699, bCNRS FRE2444, Necker Hospital, and cNephrology, Bichat Hospital, Paris, France a

IgA nephropathy (IgAN) is characterized by IgA immune complex-mediated mesangial cell proliferation. We have previously identified the transferrin receptor (TfR) as an IgA1 receptor and found that, in kidney biopsies of patients with IgAN, TfR is overexpressed and co-localizes with IgA1 mesangial deposits. Interaction between IgA1 and TfR was confirmed with TfR-transfected chicken fibroblasts. Investigation of TfR/IgA interaction demonstrated that TfR bound only the IgA1 subclass. IgA1 binding was strikingly increased when IgA1 was hypogalactosylated and of high molecular weight polymeric IgA1 (pIgA1) as well as immunecomplexes containing IgA1, both features found in IgAN patients. Accordingly, binding of IgA1 from patients to TfR was higher than that of normal IgA1 and analysis of renal tissue from biopsies revealed an overexpression of TfR in the mesangium of patients that was colocalized with IgA deposition. Here, we show that purified polymeric IgA1 (pIgA1) is a major inducer of TfR expression (3 to 4-fold increase) in quiescent human mesangial cells (HMC) and Daudi cell line. In addition, sera from IgAN patients up-regulate TfR expression in cultured HMC in an IgA dependent manner. By contrast with known TfR function, IgA1-induced TfR upregulation is not associated with increased iron uptake. Rather, IgA1 induced HMC proliferation is dependent on TfR engagement and can be inhibited by both TfR1 and TfR2 ectodomains as well as by the anti-TfR mAb A24. Finally, activation of mesangial cells through pIgA1 binding to TfR induced secretion of IL-6 and TGF-beta from the cells, that could be involved, respectively, in the inflammatory and pro-fibrogenic developments observed in IgAN. We propose that deposited pIgA1 or IgA immune complexes could initiate an auto-amplification process involving hyper-expression of TfR allowing increased IgA1 mesangial deposition. Altogether, these data unveil a functional cooperation between pIgA1 and TfR for IgA1 deposition and HMC proliferation, features which are commonly implicated in the chronicity of mesangial injuries observed in IgAN. Thus, IgA triggers a TfR activation pathway that could be a new target in IgAN therapy.

B-O-11

Anti-Inflammatory Action of IgA Fc Receptor I and Its Therapeutic Potential in Renal Inflammation and Fibrosis Yutaka Kanamarua, François Vrtovsnika, Séverine Pfirscha, Michelle Arcos-Fajardoa, Yasuhiko Tominob, Ulrich Blanka, Renato C. Monteiroa a

Institut National de la Santé et de la Recherche Médicale (INSERM) U699, Paris, France; Division of Nephrology, Department of Internal Medecine, Juntendo University School of Medicine, Tokyo, Japan b

Inflammation is a hallmark of the initiation and progression of renal diseases leading to end-stage renal failure. No treatment exists besides dialysis and replacement therapy. We report here that monomeric targeting of the myeloid IgA Fc receptor (Fc alpha RI) reduces inflammation in two mouse models of renal disease of different aetiology. Kidneys from human Fc alpha RI transgenic mice subjected to unilateral ureteral obstruction showed decreased signs of fibrosis after treatment with anti-Fc alpha RI A77 Fab. Such treatment improved also renal functions in Fc

Basic Advances (Update)

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alpha RI transgenics subjected to anti-GBM-induced glomerulonephritis. In both models improvement was associated with decreased inflammatory cell infiltration. This involved a generalized inhibition of chemotactic responses and cytokine production as Fc alpha RI targeting inhibited MCP-1-, TNF alpha- and LPS-induced signaling in monocytic cell lines and blood monocytes. Inhibition was dependent on SHP-1 phosphatase as the siRNA-mediated decrease in SHP-1 levels reversed inhibition. IgA nephropathy patient serum-mediated responses of Fc alpha RIhumanized transfectants were also reduced demonstrating both Homologous and heterologous inhibitory capacity. Our findings indicate that Fc alpha RI monomeric targeting generally inhibits myeloid cell-mediated inflammation and may therefore represent a viable treatment option to limit progression of chronic renal diseases.

B-O-12

PDGF-D: A Mediator of Mesangioproliferative Glomerulonephritis and a Novel Therapeutic Target Jürgen Floegea, Frank Eitnera, Claudia Van Roeyena, Glennda Smithsonb, William Larochelleb Division Nephrology, University of Aachen RWTH, Aachen, Germany; bCuragen Corp., Branford, USA

a

We previously identified PDGF-B as a mediator of mesangial cell proliferation. PDGF-D like PDGF-B is mitogenic for mesangial cells and is not inhibited by a PDGF-B antagonist. Low levels of PDGF-D mRNA were detected in normal rat glomeruli. Following induction of mesangioproliferative nephritis in rats by anti-Thy 1.1 antibody, glomerular PDGF-D mRNA and protein expression increased significantly from days 4–9 in comparison to non-nephritic rats. To investigate the functional role of PDGF-D, fully human neutralizing monoclonal antibodies were generated utilizing the XenoMouse-Technology. Treatment of rats with these antibodies during the acute mesangioproliferative phase of the disease led to a dose dependent (up to 67%) reduction of glomerular cell proliferation at day 8. As judged by double immunostaining for BrdU and alpha-smooth muscle actin, glomerular mesangial cell proliferation was reduced by up to 57%. In contrast, injection of anti-PDGF-D antibody in normal rats was without effect. Next, the long-term consequences of PDGF-D inhibition in vivo were investigated. Rats with progressive mesangioproliferative glomerulonephritis (uninephrectomy plus anti-Thy 1.1 antibody) received the PDGF-D neutralizing antibody CR002 on days 3, 10 and 17 after disease induction, i.e. during the acute mesangioproliferative phase. Glomerular mesangioproliferative changes on day 10 were significantly reduced by anti-PDGF-D treatment as compared to control antibody. Eight weeks after disease induction anti-PDGF-D therapy significantly ameliorated focal segmental glomerulosclerosis, podocyte damage (i.e. de novo desmin expression), tubulointerstitial damage and fibrosis as well as the accumulation of renal interstitial matrix including type III collagen and fibronectin. Treatment with anti-PDGF-D also reduced the cortical infiltration of monocytes/macrophages on day 56, possibly related to lower renal cortical complement activation (i.e. C5b-9 deposition) and/or reduced epithelial-to-mesenchymal transition (i.e. preserved cortical expression of E-cadherin and reduced expression of vimentin and alpha-smooth-muscle actin). Finally, the effects of late anti-PDGF-D treatment in rats with progressive glomerulonephritis were assessed, i.e. antibody administration (days 17, 28 and 35 after disease induction) starting, when tubulointerstitial damage was already established. On day 100 following disease induction overall glomerulosclerosis was reduced by PDGF-D antagonism, but without a statistical significant difference compared to nephritic control animals that received irrelevant IgG. However, late PDGF-D antibody treatment significantly reduced proteinuria (days 50 and 78 after disease induction), total tubulointerstitial damage and matrix accumulation (day 100) as well as glomerular type III collagen and alpha-smooth muscle actin expression (day 100). In conclusion, these data provide evidence for a causal role of PDGF-D in the pathogenesis of renal scarring, both, in the acute mesangioproliferative phase as well as in later stages of glomerulonephritides. This opens the clinical perspective of treating patients with mesangioproliferative glomerulonephritis both in early active but also in late phases of their disease.

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B-O-13

Innate Response to Sendai Virus Disturbs Cytokine Polarity in BALB/c Mice and Alters Onset of Renal Failure in IgA Nephropathy Michifumi Yamashitaa, Subba R. Chintalacharuvub, Noriyoshi Kobayashic, John G. Nedruda, Yasuhiko Tominoc, Steven N. Emancipatora Department of Pathology, Case Western Reserve University, Cleveland, Ohio, bLilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, Ind., USA; cDivision of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Japan

a

The ability of committed T cells to function in altered cytokine environments is a central issue in autoimmune and hypersensitivity diseases. The tendency of CD4 T cells to differentiate to the Th1 or Th2 pathway is genetically regulated in each mouse strain under neutral conditions, i.e., without Th1 or Th2 priming, or in the absence of exogenous cytokines [1]. Most inbred mouse strains (e.g., B10D2, C57Bl/6, CBA, C3H, etc) spontaneously heal cutaneous lesions resulting from infection with Leishmania major, an obligate intracellular protozoan parasite of mononuclear phagocytes in mammalian hosts, but BALB/c mice are susceptible to chronic infection, developing severe progressing lesions [2]. Recently, we developed a murine model of IgA nephropathy (IgAN) induced by Sendai virus (SeV) [3]. In this experimental system without Th1- or Th2-polarizing immunization, BALB/c mice develop more severe nephritis with acute renal insufficiency than C3HeB mice, which rarely develop renal insufficiency [4]. To determine if Th1 or Th2 predominance influences the severity of experimental IgAN in different strains, we employed polarizing immunizations in a SeV-induced IgAN model in Th1-prone C57Bl/6 mice and Th2-prone BALB/c mice. C57Bl/6 mice, immunized with SeV  CFA or  IFA, showed: (1) equal increases in serum IgA and IgG antibody, and (2) clear cytokine polarity by splenocytes in recall assays. However, (3) total serum IgA and especially SeV-specific IgA from the IFA group showed a selective defect in galactosylation, not seen in the CFA group, and (4) serum creatinine (sCre) in the IFA group was higher than the CFA group or nonimmune controls. In contrast, neither IgA glycosylation, glomerular function nor the substantial IL-2, -4, -5, -13 and IFN- recall responses to SeV differ among BALB/c mice primed with CFA or IFA, despite the fact that each of these adjuvants promotes anticipated cytokine bias in response to a variety of other antigens in this strain. Moreover, spleen cells from naïve BALB/c mice produce IFN- (but not IL-2, -4, -5, or -13) upon stimulation with inactivated SeV in vitro. IFN- production by naïve splenocytes is blocked partially by anti-IL-12 blocking Abs, and completely by anti-IL-18R blocking Abs. By flow cytometry, IFN- producing cells are CD3(), CD19(), CD49b() cells, which are natural killer cells. In conclusion, in the SeV-induced IgAN model with Th1/Th2 priming, C57Bl/6 mice showed clear cytokine polarity, with underglycosylation of IgA and increased sCre with associated with Th2 bias. On the other hand, BALB/c mice did not show cytokine polarity or increased sCre, presumably due to the effects of IFN- response to SeV through the crosstalk between NK cells and dendritic cells via IL-18 signaling. Innate immunity may therefore influence risk for altered IgA glycosylation and/or decrements in GFR in IgAN.

References 1 2 3 4

Hsieh CS, Macatonia SE, O’Garra A, Murphy KM: J Exp Med 1995;181:713. Reiner SL, Locksley RM: Annu Rev Immunol 1995;13:151. Jessen RH, Emancipator SN, Jacobs GH, Nedrud JG: Lab Invest 1992;67:379. Chintalacharuvu SR: et al: Clin Exp Immunol 2001;126:326.

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B-O-14

Reconstitution of Glomerular IgA Deposition after Bone Marrow Cell Transplantation from IgA Nephropathy Model Mice into Alymphoplasia Mutant Mice Masashi Aizawa, Yusuke Suzuki, Hitoshi Suzuki, Masao Kihara, Kenji Yamaji, Takahiro Yamanaka, Toshinao Tsuge, Satoshi Horikoshi, Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan Previous studies indicated that IgA nephropathy (IgAN) may be a stem cell disease. Our recent study showed that the ‘Grouped ddY mouse’ is a useful model to approach the pathogenesis of IgAN (J Am Soc Nephrol 2005;16:1289). We also reported that bone marrow transplantation (BMT) from this mouse of this model reconstituted IgAN in healthy recipients with strong Th1-polarization. On the other hand, clinical reports demonstrated that tonsillectomy with steroid-pulse therapy improve proteinuria and hematuria with histological remission in IgAN patients, suggesting that mucosal immunity is involved in pathogenesis of this disease. However, in IgAN, responsible cell types, production sites of serum IgA and roles of the mucosal immune system and secondary lymphoid tissues remain unclear. The objective of the present study was to investigate the underlying mechanism in which BM cells induce glomerular IgA deposition, subsequent disease progression with alteration of Th polarity and roles of mucosa and secondary lymphoid tissues. We employed the onset ddY mice and alymphoplasia mutant mice (aly/aly) lacking all systemic lymph nodes, Payer’s patches (PP), isolated lymphoid follicles in the lamina propria (LP) and IgA producing cells because of NF-B-inducing kinase (NIK) mutation. A recent study showed that transfer of normal BM cells into aly/aly mice resulted in recovery of IgA producing cells in spleen and serum IgA, but not rescue gut IgA. We transplanted 1 107 BM cells from the onset ddY mice into aly/aly and original background mice (B6). At 12 weeks after BMT, elevation of serum IgA and IgAB220 plasma cells in the spleen, but not IgA producing cells in LP, were observed in aly/aly recipients. Although both transplanted aly/aly and B6 mice showed mesangial IgA deposition and co-localization of IgG, glomerular lesions with distinct IgG2a codeposition were detected only in B6 mice, suggesting that disease progression with Th1-polarization was absent in aly/aly mice. Adoptive transfer of 1 107 PP cells from the onset ddY mice to aly/aly mice reconstituted IgA cells in LP, but not glomerular IgA. Accordingly, the present results suggest that glomerular IgA deposition in this disease can be induced by BM-derived or -primed IgA producing cells independently of mucosal priming, at least in the gut. Furthermore, disease progression after IgA deposition may require immune responses in secondary lymphoid tissues.

B-P-01

A Rapid and Convenient Method for Detecting Underglycosylated IgA in Sera of Patients with IgA Nephropathy Daisuke Kondo, Ichiei Narita, Fuminori Sato, Minoru Sakatsume, Fumitake Gejyo Division of Clinical Nephrology and Rheumatology, Niigata University Graduate School of Medical, Japan and Dental Sciences, Niigata, Japan Background: IgA nephropathy (IgAN) is a disease characterized by an abnormal immune response with an increased synthesis of underglycosylated IgA1. Although the mechanisms of underglycosylation of IgA1 are still unclear, abnormally glycosylated IgA molecules have been shown to be apparently involved in the pathogenesis

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and progression of IgAN. However, the detection of such IgA molecules requires advanced techniques, which include isolation of highly pure IgA, glycosylation analysis utilizing gas chromatography and mass spectrometry. Mainly because of their complexity, it is difficult to apply them to clinical use for screening and/or follow-up of patients with IgAN. In the present study, we have attempted to develop a convenient and rapid method to detect abnormally glycosylated IgA. Methods: We used a streptococcal IgA binding peptide (SAP) to capture human IgA. The 50 amino acids synthetic peptide, SAP, which has recently been identified as having specific affinity to the Fc portion of human IgA, was purchased from Peptide Institute INC. (Osaka, Japan). Wells of microtiter plates for chemiluminescence detection were coated with the SAP peptide. After blocking, the wells were incubated with diluted sera from patients with IgAN or healthy individuals. The captured IgA was incubated with biotinylated lectins specific for GalNAc, which was exposed because of the deficiency of Gal in the O-linked side-chains of the hinge region of IgA1. The wells were incubated with horseradish peroxydase-conjugated streptoavidin and the signals of chemiluminescence, using high sensitivity substance, were measured with a multilabel plate reader (Mithras LB940, Berthold Japan). Results: By utilizing the SAP peptide, IgA molecules were successfully isolated with high specificity both in patients with IgAN and healthy individuals. IgA from sera of patients with IgAN was bound to biotinylated lectin Vicia villosa (VV), which is specific for GalNAc, more effectively than did IgA from healthy controls, suggesting obvious higher amount of IgA exposing GalNAc in sera of IgAN patients. In contrast, other lectins could not distinguish the serum IgA molecules of patients and controls. Discussion and Conclusion: The isolation of IgA from sera has some technical difficulties and, in most of the studies, Jacalin has been utilized for isolation of IgA. However, because Jacalin has its specificity for Gal beta1,3GalNAc, it accurately reflect binding to O-Glycans of glycated IgA1. For this reason, the use of Jacalin to purify IgA from sera of patients with IgAN may introduce a substantial bias, as a consequence of preferential isolation of Gal beta1, 3GalNAc bearing IgA1. This novel assay using SAP, GalNac specific lectin, and chemiluminescence detection is a simple and convenient method that can identify Gal deficient IgA in the serum. Further study with a large number of patients is needed to check the usefulness of this method to confirm the clinical significance of underglycosylation of IgA1 in IgAN. This method, if established, may be valuable for an accurate assessment of the disease activity of IgAN.

B-P-02

Identification of IgA1 Hinge Glycopeptides Using a SELDI-TOF-MS Lectin Assay Kazuo Takahashia, Yoshiyuki Hikia, Sachiko Shimozatoa, Hiroko Odania, Kazuko Inouea, Ikumi Matsumotoa, Hitoo Iwaseb, Nobuteru Usudaa, Satoshi Sugiyamaa a Department of Medicine and Department of Anatomy, Fujita Health University, School of Medicine, Toyoake, Aichi, bDepartment of Biochemistry, Kitasato University School of Medicine, Japan

Background: Aberrant O-glycosylation of IgA1 hinge peptides in IgAN has been found in several studies. We have previously developed an optimal experimental condition of the ProteinChip system for the analysis of the glycoform of the IgA1 hinge using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) and jacalin-immobilized ProteinChip arrays. In the present study, a SELDI-TOF-MS lectin assay was used to compare the peak distributions of IgA1 hinge glycopeptides trapped by various lectins that recognize alternative forms of O-glycan moieties. Methods: Preparation of IgA1 hinge: Serum IgA1 from healthy individuals was isolated by anti-IgA antibody column chromatography. IgA was dissolved in dithiothreitol and 4-vinyl pyridine for dissociation and

Basic Advances (Update)

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S-pyridylethylation of the disulfide linkage. Trypsin was then added for protein digestion. SELDI-TOF-MS analyses: The solution containing IgA fragments was added to the lectin-coupled PS10 ProteinChip array. The following lectins were used: jacalin, peanut agglutinin (PNA), and vilsa villosa lectin (VVL). The SELDI-TOF-MS peaks? which correspond to IgA1 fragments containing IgA1 hinge glycopeptides (33-mer amino acids and O-glycans) trapped by the coupled lectin? were then compared. Each peak was regarded as the corresponding glycoform of the hinge if the measured molecular weight of the peak was within 0.1% of the theoretical molecular weight of the hinge structure. To confirm the actual O-glycan structure in the IgA1 hinge region detected in this condition, the samples were purified by lectins coupled to affinity beads and then analyzed by electrospray ionization/liquid chromatography/MS or matrix-assisted laser desorption/ionization quadrupole ion trap TOF-MS. Result: There were 13, 4, and 3 peaks detected by jacalin-, PNA-, and VVL-immobilized ProteinChips, respectively, some of which were confirmed as glycopeptides by MS/MS analysis. Conclusion: A lectin-immobilized ProteinChip system can detect the O-glycan structure of an IgA1 hinge peptide directory. This method may be used to identify the aberrantly glycosylated hinge that is specific to IgAN.

B-P-03

Downregulation of Core 1 ␤1,3-Galactosyltransferase and Its Molecular Chaperone Cosmc by Th2 Cytokine Alters O-Glycosylation in the IgA1 Hinge Region Koshi Yamadaa, Noriyoshi Kobayashia, Tomomi Ikedab, Yusuke Suzukia, Toshinao Tsugea, Satoshi Horikoshia, Yasuhiko Tominoa Division of Nephrology, Department of Internal Medicine, and bDivision of Molecular and Biochemical Research, Juntendo University Graduate School of Medicine, Tokyo, Japan

a

O-linked glycan moieties in the hinge of serum and glomerular IgA1 are highly underglycosylated in IgA nephropathy (IgAN). Although increased production of Th2 cytokines by peripheral blood lymphocytes and a functional abnormality of core1 1,3-galactosyltransferase (C13Gal-T) have been proposed as mechanisms for the altered glycosylation of IgA1, the process is still obscure. To clarify the effect of T cell cytokines on IgA1 glycosylation, we analyzed mRNA expression of both C13Gal-T and its molecular chaperone Cosmc, C13Gal-T activity and glycosylation of IgA1 in human B cells under stimulation with these cytokines. The surface IgA1-positive human B lymphoma cell line, DAKIKI, was cultured with recombinant human IFN- , IL-2, IL-4 and IL-5. For analysis of cell proliferation, and production and glycosylation of IgA1, cells and supernatants were collected after 5 days. IgA1 content of supernatants and the glycosylation were determined by sandwich enzyme-linked immunosorbent assay (ELISA) and enzyme-linked lectin binding assay using Vicia villosa, respectively. The mRNA expression of C13Gal-T and Cosmc in cells stimulated with IFN- and IL-4 for 12, 24 and 48 hours was quantitatively measured by real-time PCR. Enzyme reaction products to measure C13Gal-T activity on GalNAc- pNp using homogenates of cells with IL-4 stimulation for 24 and 72 hours were subjected to high performance liquid chromatography (HPLC). The cells stimulated by IL-4 or IL-5 were significantly proliferated compared with those stimulated by either other cytokines or medium alone. The concentration of IgA1 in supernatant from IL-4 stimulated cells was significantly higher than in those stimulated by other cytokines except for IL-2 and medium alone. IL-2 stimulation significantly increased IgA1 production per cell. The terminal glycosylation of secreted IgA1 was altered in response to IL-4. IL-4 stimulation significantly decreased the mRNA expression of both C13Gal-T and Cosmc, and C13Gal-T activity. It appears that Th2 cytokines induced B cell proliferation and IL-4 may play a key role in both C13Gal-T and Cosmc mRNA expression, C13Gal-T activity, and subsequent altered glycosylation of the IgA1 hinge region. These results may have pathogenic significance for IgAN.

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B-P-04

ST6GALNAC2 Expression in Peripheral B Lymphocyte and Its Relation with Sialylation of Serum IgA1 in IgA Nephropathy Jia-xiang Ding, Li Zhu, Li-xia Xu, Ming-hui Zhao, Hai-yan Wang, Hong Zhang Renal Division of Peking University First Hospital, and Peking University Institute of Nephrology, Beijing, China Background: Altered IgA1 hinge O-glycosylation had been considered as an important part in the pathogenesis of primary IgA nephropathy (IgAN). Analysis of serum IgA1 from patients with IgAN revealed that 2,6sialic acid (SA) was reduced and associated with severer pathological phenotypes. It may be hypothesized that the abnormal sialyation of the hinge region glycans could result from a reduced quantity or enzymatic activity of 2,6sialyltransferase. The current study was carried out to elucidated the expression of a major coding gene for 2,6sialyltransferase, ST6GALNAC2, and its relation to sialylation of IgA1 in patients with IgAN. Subjects and Methods: 28 patients with IgAN and 8 healthy controls were included in this study. Peripheral B lymphocytes were isolated by Dynabeads M450 CD-19 magnetic beads. The expression level of ST6GALNAC2 was quantitatively measured by real-time reverse transcriptase polymerase chain reaction (RT-PCR). Serum IgA level and sialylation level were determined by ELISA and SNA lectin-binding assay. Results: The expression of ST6GALNAC2 gene in B peripheral lymphocyte was significantly lower in IgAN patients than that in normal controls (3.7  2.2 vs. 6.3  2.3, Mann-Whitney U  48.5, p  0.016), and, the level of ST6GALNAC2 gene expression showed a positive correlation with the level of 2,6-SA in serum IgA1 in patients (r  0.565, p  0.01). Moreover, the gene expression and 2,6-SA level of serum IgA1 were analyzed in various histological lesions in IgAN patients and controls. It was shown that expression of ST6GALNAC2 gene was 6.3  2.3 in normal controls (n  8), 3.7  2.6 in HAAS grading I (n  5) and 3.6  2.4 in HAAS III & IV (n  23). It was significant lower in the patients with severer pathological lesions than that in controls (Mann Whitney U  39.5, p  0.018). Furthermore, the level 2,6-SA of serum IgA1 was 0.84  0.06 in controls (n  8), 0.66  0.17 in patients with HAAS grading I (n  5) and 0.62  0.18 in HAAS III & IV (n  23). It indicated that the patient with more severe histological lesions were also with lower level of 2,6-SA in serum IgA1 (vs. normal control p  0.03). Whereas there was no correlation in expression of ST6GALNAC2 with proteinuria, serum creatinine and hypertension in investigated patients (p
0.05). Conclusion: These data suggested that the reduced sialylation of serum IgA1 was resulted from a decreased expression of ST6GALNAC2 gene in B lymphocyte in patient with IgAN. Decreased expression of ST6GALNAC2 was associated with severity of histological lesions in IgAN.

B-P-05

Epstein-Barr Virus (EBV)-Immortalized B Cells from Patients with IgA Nephropathy (IgAN) Secrete IgA1 with Galactose (Gal)-Deficient O-Linked Glycans Hitoshi Suzukia, Zina Moldoveanua, Huong L. Vua, Lea Novaka, Bruce A. Juliana, Robert J. Wyattc, Milan Tomanaa, Yasuhiko Tominob, Jiri Mesteckya, Jan Novaka University of Alabama at Birmingham, Birmingham, Ala., USA; bDivision of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan; c University of Tennessee Health Sciences Center, Memphis, Tenn., USA a

IgA1 contains multiple O-glycosylation sites in its hinge region. These O-glycans are built by stepwise addition of monosaccharides: N-acetylgalactosamine (GalNAc) is added to Ser/Thr residues of the IgA1 hinge region

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by enzyme GalNAc transferase 2 (GalNAcT2), Gal is attached to GalNAc by 1,3-galactosyltransferase (C1GalT1) in association with its chaperone Cosmc, and sialic acid may be linked to GalNAc by 2,6-GalNAc sialyltransferase 2 (ST6GalNAcII). Finally, 2,3-Gal sialyltransferases may add sialic acid to Gal. IgA1 in the circulation and glomerular deposits of IgAN patients is aberrantly glycosylated; the hinge-region O-linked glycans are Galdeficient. To characterize IgA1 glycosylation in IgAN and biosynthetic pathways of the O-linked glycans, we immortalized peripheral blood B cells using EBV and subcloned IgA1-producing cell lines from IgAN patients (n  10) and healthy controls (n  10). Gal-deficiency of IgA1 was determined in cell-culture supernatants and corresponding sera by ELISA using a GalNAc-specific lectin from Helix aspersa (HAA). IgA1 secreted by cell lines from IgAN patients was polymeric and had Gal-deficient O-glycans with terminal or sialylated GalNAc. IgA1 from healthy controls had predominantly normally galactosylated O-glycans. Serum levels of Gal-deficient IgA1 of the patients correlated with high HAA reactivity of IgA1 secreted by the corresponding cell lines. Western blotting confirmed Gal-deficiency of IgA1 in sera and cell culture supernatants from IgAN patients. Immunofluorescence staining showed that HAA co-localized with IgA1 in the cytoplasm of B cells in IgAN patients. For each cell line, gene expression levels of specific glycosyltransferases were quantitated by real-time RT-PCR. The results showed lower levels of gene expression in IgAN patients for GalNAcT2, C1GalT1, and Cosmc but higher levels for ST6GalNAc II. In summary, we have shown for the first time that immortalized cell lines from IgAN patients secrete polymeric Gal-deficient IgA1. This glycosylation aberrancy was related to complex changes in expression of specific glycosyltransferases. Thus, EBV-immortalized cell lines from IgAN patients offer a convenient model for studies of aberrant IgA1 glycosylation. Other contributing authors include Stacy HALLa and Rhubell BROWNa.

B-P-06

Primary IgA Nephropathy (IgAN) and IgAN in Patients with Hepatitis C Virus (HCV)-Induced Cirrhosis: Cellular Proliferation of Cultured Mesangial Cells after Stimulation with Circulating Immune Complexes (CIC) and Morphometric Analysis of Renal Biopsies Lea Novak, Jan Novak, Brendan M. McGuire, Britt Newsome, Milan Tomana, Jiri Mestecky, Devin E. Eckhoff, Steve Bynon Jr., Bruce A. Julian University of Alabama at Birmingham, Birmingham, Ala., USA IgAN is characterized by mesangial immunodeposits with IgA1 and elevated serum levels of IgA1-containing CIC. The mesangial immune deposits are likely derived from CIC that contain galactose-deficient IgA1 complexed with anti-glycan antibodies (IgG or IgA1). IgA1-CIC from patients with IgAN stimulate proliferation of cultured primary human mesangial cells. In a pilot study, we noticed a high frequency of immune-complex glomerulonephritis in patients undergoing liver transplantation for HCV-induced cirrhosis. IgAN was diagnosed in 7 of the 30 transplant patients. In the current study, we analyzed mesangial cellularity in the renal biopsy specimens and assessed the stimulatory properties of CIC from HCV-negative patients with primary IgAN (IgAN) and patients with IgAN secondary to HCV-induced cirrhosis (IgAN-HCV). Fractionated CIC were prepared from 7 IgAN patients, 4 IgAN-HCV patients, and 5 healthy controls using size-exclusion chromatography and then incubated with primary human mesangial cells. Cellular proliferation was measured by 3H-thymidine incorporation. Serum fractions of Mr 800–900 kDa from HCV-IgAN patients stimulated mesangial cell proliferation at least as much as did the fractions from IgAN patients. The stimulatory properties of these CIC correlated with the content of IgA1. Serum fractions from the controls containing proteins of similar sizes did not stimulate cellular proliferation. Glomerular cellularity was calculated from 10 glomeruli per renal biopsy as number of nuclei in a glomerulus divided by glomerular area. Cellularity of 10 IgAN patients and 7 IgAN-HCV patients was compared

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with that of 9 control patients with thin basement membrane or no pathology. IgAN-HCV patients had significantly more cells per square micrometer than did controls (70  9 104 vs. 55  6 104) but not the IgAN patients (70  10 104). The average glomerular area did not differ between the three groups. Compared to the IgAN patients, the IgAN-HCV patients were older (56  8 vs. 34  6 years), exhibited microscopic hematuria less frequently (17 vs. 100%), and had a lower urinary protein/creatinine ratio (0.17  0.20 vs. 1.33  0.78). Serum creatinine did not differ between the two groups. In summary, IgA-CIC in patients with primary IgAN and secondary IgAN associated HCV-induced cirrhosis stimulated cellular proliferation of cultured human mesangial cells and renal biopsies of both groups showed increased mesangial cellular proliferation. We conclude that IgA1CIC in patients with IgA-HCV activate mesangial cells in a fashion similar to that in IgAN patients. Other contributing authors include Rhubell BROWN and Stacy HALL.

B-P-07

Analysis of Urinary Polypeptides from Patients with IgA Nephropathy (IgAN) and Henoch-Schoenlein Purpura Nephritis (HSPN) Jan Novaka, Stefan Wittkeb, Zina Moldoveanua, Matthew B. Renfrowa, Claretha R. Nicholsa, Candace Kirkseya, Robert J. Wyattd, Brendan McGuirea, Harald Mischakb,c, Bruce A. Juliana University of Alabama at Birmingham, Birmingham, Ala., USA; bMosaiques-diagnostics GmbH and cMedical School Hanover, Hanover, Germany; dUniversity of Tennessee Health Sciences Center, Memphis, Tenn., USA a

Polypeptides in urine may include disease-specific markers. Using ELISA, SDS-PAGE, Western blotting, and capillary electrophoresis-mass spectrometry (CE-MS), we have analyzed urine samples from patients with biopsyproven IgAN (n  14), Henoch-Schoenlein purpura (HSP) with (n  2) or without (n  1) nephritis, primary non-IgAN glomerulonephritis (n  3), and cirrhosis induced by hepatitis C virus (HCV) (n  10, all with glomerulonephritis; 3 had IgAN) or of non-viral origin without renal disease (n  1), and healthy controls (n  12). Furthermore, we used SDS-PAGE and Western blotting to analyze samples from additional HSP patients without nephritis (n  6) and with nephritis (n  15; 8 had biopsy-proven HSPN). Amounts of urinary IgA and IgG, and IgG- and IgA-containing immune complexes were significantly higher in patients with IgAN than in patients with primary non-IgAN glomerulonephritis or healthy controls. The samples were also analyzed for disease-specific polypeptide patterns using CE-MS. Urine aliquots were subjected to ultrafiltration on a 20-kDa cut-off membrane to remove high-molecular-weight proteins, such as albumin. Subsequently, the ultrafiltrate was desalted on a PD10 column. The eluate was lyophilized and resuspended in water before CE-MS analysis. Mass spectral ion peaks representing identical molecules at different charge states were deconvoluted to a single mass using MosaiquesVisu® software. CE migration time and ion signal intensity (amplitude) were normalized using internal polypeptide standards. We found markers of glomerulonephritis in IgAN patients and patients with nonIgAN glomerulonephritis, including HCV-positive patients with minimal abnormalities by renal biopsy. CE-MS data correctly identified 39 of 43 samples. Of the 4 misclassified samples, two samples from convalescent IgAN patients and one IgAN sample with low levels of IgA and IgG were classified as healthy, and one primary nonIgAN glomerulonephritis sample with very high levels of IgA and IgG was identified as IgAN. Notably, two HSP nephritis samples were identified as IgAN-related while the HSP (no nephritis) sample was classified as normal. The additional samples from HSP patients were analyzed by SDS-PAGE and Western blotting with IgA- and IgGspecific antibodies. Sample loading was normalized to urinary creatinine concentration. The protein profiles of IgAN and HSPN samples were similar, with elevated concentration of IgA heavy chain and its proteolytic fragments. In contrast, HSP samples from patients without nephritis had similar profiles as healthy controls showing only small amounts of IgA and no proteolytic fragments. Overall, the data from the ELISA, SDS-PAGE, and

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CE-MS closely correlated. Identification of the biomarker polypeptides by tandem mass spectrometry will clarify pathological processes in the injured kidney. These novel analytical approaches may lead to development of noninvasive procedures for diagnosis of IgAN and HSPN and monitoring the renal disease in HCV-positive patients with glomerulonephritis. Other contributing authors include Petra Zurbigb, Marion Haubitzc, Jeannette Leea, Karel Matousovica, Catherine V. Barkera, Rhubell Browna, Stacy Halla, Jiri Mesteckya.

B-P-08

Disordered Production of Immunoglobulin A in the Tonsil of Some IgA Nephropathy Patients Akihiko Itoha, Hitoo Iwaseb, Naomi Iwanamic, Toru Takatanid, Yoshiyuki Hikie Department of Otolaryngology, bDepartment of Biochemistry, cDepartment of Pediatrics, Kitasato University, Sagamihara, Kanagawa, dRenal Division, Akebono Hospital, Machida, and e Division of Nephrology, Department of Internal Medicine, Fujita Health University School of Medicine, Toyoake, Japan

a

Introduction: There are some reports on the relationship of tonsillectomy and IgA nephropathy (IgAN). According to some papers, tonsillectomy can improve the urinary findings, keep stable renal functions and have a favorable effect on long term renal survival in IgAN patients. But the mechanism that tonsillectomy have effectiveness for IgAN patients is unknown. Recently, we reported that over-production of aberrant IgA1 in tonsillar tissue from IgAN patients. On the other hand, the disordered balance in IgG- and IgA-producing cell in IgA nephropathy patients was reported previously by means of histochemical method. There is a report for the different subclass distribution (IgA1/IgA2) of IgA in secreted fluids such as serum (89/11), colostrum (65/35), saliva (63/37), jejunal fluid (63/37), colonic fluid (35/65), hepatic bile (74/26), nasal fluid (95/5) and bronchial fluid (67/33). The aim of this experiment is to determine the IgA1/IgA2 ratio in tonsil tissue and to learn whether the IgA subclass distribution in the tonsil is the same as in the controls and IgAN patients. Experimental procedure: Right-side tonsils were obtained from 32 patients with IgAN (15 males and 17 females, age range 17–60 years, mean age 34.1  12.8), who had been identified by IF examination of biopsy specimens by Kitasato University. Tonsils from 10 patients with chronic tonsillitis (5 males and 5 females, age range 18–36 years, mean age 27.2  6.6) without renal disease were used as the control group. IgA in tonsillar extract and serum was separated into passed fraction (IgA2) and bound fraction (IgA1) by a jacalin-agarose column. Isoelectric focusing (IEF) analysis was carried out using an IPGphor instrument. The content of the IgA in each sample was analyzed by enzyme-linked immunosorbent assay (ELISA). Results: The IgA1/IgA2 ratio of the tonsillar extract of controls and IgAN patients was compared. The distribution of the ratio indicated statistically significant differences from each other. The mean ratio for the control tonsil was 61/39. However, the ratio from one-fourth of 32 IgAN patients exhibited a higher value than mean  2SD (standard deviation) of the controls. Among them, three patients exhibited 92/8. Meanwhile, the ratios for serum by this method were close to the previously reported 89/11. Conclusions: This was the first report about IgA subclass distribution in tonsillar tissue. The ratio 61/39 for tonsillar IgA differed from the value (over 90% of IgA1) in the previous histochemical report. The value was rather similar to the previous report for colostrum, whole saliva, jejunal fluid and bronchial fluid. The presence of highIgA1-producing group in IgAN patients meant the excess production of hypoglycosylated IgA1 in their tonsil.

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References 1

2

Itoh A, Iwase H, Takatani T, Nakamura I, Hayashi M, Oba K, Hiki Y, Kobayashi Y, Okamoto M: Tonsillar IgA1 as a possible source of hypoglycosylated IgA1 in the serum of an IgA nephropathy patient. Nephrol Dial Transplant 2003;18: 1108–1114. Horie A, Hiki Y, Odani H, Yasuda Y, Takahashi M, Kato M, Iwase H, Kobayashi Y, Nakashima I, Maeda K: IgA1 molecules produced by tonsilar lymphocytes are under-O-glycosylated in IgA nephropathy. Am J Kidney Dis 2003;42: 486–496.

B-P-09

Efficacy of Tonsillectomy with Steroid Pulse Therapy in Patients with IgA Nephropathy Who Showed Strong Expression of Tonsillar TLR9 Tatsuya Kano, Yusuke Suzuki, Hitoshi Suzuki, Toshinao Tsuge, Satoshi Horikoshi, Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan IgA nephropathy (IgAN) patients show macroscopic hematuria and/or proteinuria after upper respiratory tract infections such as tonsillitis. Antigens related to tonsillar focal infections are considered to be involved in the pathogenesis of IgAN. However, the underlying mechanism (of) the therapeutic effects remains unclear. Toll-like receptors (TLR) are key molecules (that) trigger innate immune responses and thus (defend the) host against infections through production of inflammatory mediators by immune-competent cells including macrophage or dendritic cells (DC). Recent papers reported that TLR2, 4, and 9 might be involved in the pathogenesis of various glomerulonephritides. In the present study, we investigated TLR expression in tonsils from IgAN and determined their cell types. In addition, correlations of magnitude of TLR expression and (therapeutic) outcome were examined. Sixteen patients who were diagnosed (in the) poor or (relatively) poor prognosis group in accordance with the prognostic criteria for IgAN in Japan and treated (by) tonsillectomy were (used in) this study. At least 2 weeks after the tonsillectomy, 3 courses of steroid pulse therapy (methylprednisolone (0.5 g/day) 3 days) were performed every 2 months. During steroid pulse therapy, prednisolone (at) 0.5 mg/kg was given once every two days. Clinical data was collected before tonsillectomy or each steroid pulse therapy. Real-time PCR for TLR2, 4, and 9 and cytokines (IFN-alpha/gamma) using RNA isolated from tonsils was performed. Immunohistochemical (analysis) of tonsils was performed to identify localization of TLR expression and cell types. mRNA (expression) of TLR2 and 4 in tonsils (did) not (differ) in each patient, although (marked) expression of TLR9 was observed in three patients. These three patients also showed (marked) expression of IFN-alpha and -gamma, (with) a significant correlation among (them). We defined these three patients as ‘(the high) group’ and other patients as ‘(the low) group’. Immunohistochemical analysis confirmed (marked) TLR9 expression in tonsils of (the high) group. TLR9 expression (was) mainly localized in BDCA-2 and CD123 positive plasmacytoid dendritic cells (pDC). Numbers of pDC in tonsils of (the high) group were higher than those of (the low) group. However, there was no significant difference in serum level of IFN-alpha and -gamma between (the two) groups. Seven patients (High; n  3, Low; n  4) finished steroid pulse therapy. (The high) group showed a significant improvement in proteinuria (p  0.05) and hematuria (p  0.01) just after therapy. Moreover, serum IgA level and IgA/C3 ratio were also significantly decreased in (the high) group (serum IgA: p  0.05, IgA/C3 ratio: p  0.01).

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It is suggested that tonsillectomy with steroid pulse therapy may (provide rapid) and (good) (therapeutic) outcomes in IgAN patients who show high expression of TLR9 in tonsillar pDC. These (findings) indicated that TLR9 activation in the mucosa may be involved in the (pathogenesis) of IgAN.

B-P-10 Not submitted

B-P-11

Analysis of IgA1-BP (Binding Protein) in IgA Nephropathy (IgAN) Using Liquid Chromatography Electron Spray Ionization Mass Spectrometry (LC-ESI TOF MS) Keiko Obayashi, Yusuke Suzuki, Reiko Mineki, Tsutomu Fujimura, Hikari Taka, Takashi Ueno, Satoshi Horikoshi, Kimie Murayama, Yasuhiko Tomino Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan Background: The major pathogenic abnormality of IgAN is considered to be present in the systemic immune system rather than in the kidney. However, the pathogenesis of this disease still remains unclear. Abnormalities of IgA including underglycosylation and polymeric IgA immune complex (IC) formation are discussed in the pathogenesis. Since upper respiratory infection exacerbates IgAN and tonsillectomy has therapeutic effects on this disease, previous studies have examined the contribution of antigens to the pathogenesis. However, obvious antigens that form IgAIC have not been identified. Some groups reported that altered IgA itself may act as an antigen. Aim: In the present study, we analyzed IgA binding protein from serum of IgAN patients by LC-ESI TOF MS to examine the possibility that exogenous antigens are involved in IgAIC formation. Materials and Methods: Six patients with IgAN (n  1 good prognosis, n  4 relatively poor prognosis, n  1 poor prognosis) and five healthy volunteers were used in this study. To determine IgA1 and IgA1-BP, 400 l of serum was applied to jacalin. Then, IgA1/IgA1-BP was eluted by 0.8 M galactose. The eluted protein showed 4 peak fractions. (170 kDa, 340 kDa, 680 kDa, and
680 kDa) on a Superdex 200 10/300 GL column. When 1.5 g protein from each fraction was electrophoresed on acrylamide gel, 30–40 bands confirmed by silver staining of each sample were analyzed by LC-ESI-TOF MS. To quantitatively evaluate the identified protein, 1 g of protein from each fraction was applied to Western blotting. Moreover, in three patients (n  2 poor prognosis, n  1 relatively poor prognosis) who had a tonsillectomy and 3 courses of steroid pulse therapy, changes in IgA1-BP by this therapy were also analyzed. The protocol of the steroid therapy was as follows: intravenous pulse administration of methylprednisolone of 0.5 g/day 3 days in every two months and oral administration of prednisolone of 0.5 mg/kg every other day after each pulse therapy. Results: In each fraction of IgA1/IgA1-BP, MS identified apolipoprotein, immunoglobulin (Ig) heavy chain (, , ) and light chain, Ig J chain, fibronectin, C1INH, C4bp and 1MG. C4bp was frequently observed in the patient group (patients; 3/6, volunteers; 1/6). This system failed to find obvious exogenous antigen in the IgA1-BP. Interestingly, tonsillectomy with steroid pulse therapy increased concentration of the 170 and higher than 680 kDa peaks and decreased that of the 340 kDa peak. After this therapy, IgA1-binding IgG decreased in the 340 kDa and 680 kDa fractions, while IgA1-binding C4bp decreased in the 680 kDa and higher fractions.

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Conclusion: Although we could not confirm exogenous antigen in IgA1-BP, the present findings suggested that IgG and C4bp might play an important role in the pathogenesis of IgAIC.

B-P-12

Staphylococcal Cell Membrane Antigen, a Possible Antigen in Post-Methicillin-Resistant Staphylococcus aureus (MRSA) Infection Glomerulonephritis (GN) and IgA Nephropathy, Interacts Directly with Cultured Mesangial Cells Yoshio Shimizua, Masanori Sekia, Hideko Sakuraia, Yoh Arakawaa, Fumiyo Watanabea, Keigyo Yoha, Kunihiro Yamagataa, Akio Koyamab a Graduate School of Comprehensive Human Sciences, University of Tsukuba, Tsukuba, Ibaraki, and bIbaraki Prefectural University of Health Sciences, Ami, Japan

Background: Post-MRSA infection glomerulonephritis (GN) is a mesangial proliferative GN that occurs following Staphylococcus aureus infection and leads to a severe type of IgA nephropathy, due to glomerular IgA and C3 deposition in its clinical course. It has been assumed that staphylococcal superantigens and other cellular antigens are necessary for development of post-MRSA infection GN and we have identified a staphylococcal cell membrane antigen (GenBank accession number: BAB41819.1) as a possible antigen in post-MRSA infection GN. We have also shown that this antigen is deposited in glomerular mesangial areas in patients with post-MRSA infection GN and in more than 50% of patients with IgA nephropathy. Aim: The aim of the study is to determine whether the staphylococcal cell membrane antigen interacts directly with mesangial cells. Methods: Experiment 1. FLAG-tagged staphylococcal cell membrane antigen was used in the study. Cells from a mouse mesangial cell line (ATCC: CRL-1927) were stripped from a dish, washed twice, and then mixed with purified FLAG-tagged antigen under various conditions. After washing twice more, the cells were stained with fluorescein isothiocyanate (FITC)-conjugated anti-FLAG antibody and cellular attachment of the FLAGtagged staphylococcal cell membrane antigen was analyzed by flow cytometry. Experiment 2. Culture dishes were coated with recombinant staphylococcal cell membrane antigen dissolved in phosphate-buffered saline (PBS). Mouse mesangial cells were cultured with or without the antigen, and the growth stimulation effect of the antigen on these cells was determined using an ELISA with bromodeoxyuridine (BrdU) incorporation. Results: Experiment 1. FLAG-tagged staphylococcal cell membrane antigen attached to cultured mesangial cells in the exponential growth phase, whereas mature cells did not interact with the antigen. Incubation of the mesangial cells with the antigen for 15 minutes at 37C resulted in higher fluorescence intensity in flow cytometry, compared to incubation at 4C. An incubation time of more than 30 minutes caused the mesangial cells to aggregate. Experiment 2. Treatment with recombinant staphylococcal cell membrane antigen attenuated incorporation of BrdU by mouse mesangial cells, and this effect was dependent on the antigen dose. Conclusion: These results suggest that staphylococcal cell membrane antigen, which is a possible antigen in post-MRSA infection GN and IgA nephropathy, interacts directly with mesangial cells and transmits growth inhibitory signals in mesangial cells through this interaction.

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B-P-13

Differential Expression of Receptors for BMP-7 by Human Mesangial Cells Cultured with Polymeric IgA from Patients with IgA Nephropathy Joseph C.K. Leung, Sydney C.W. Tang, Loretta Y.Y. Chan, Y.M. Yuen, Kar Neng Lai Department of Medicine, University of Hong Kong, Hong Kong Bone morphogenetic protein-7 (BMP-7) is required for normal renal development and the expression of BMP-7 remains high in adult kidneys. Recent studies suggest that BMP-7 may reduce glomerular and tubulointerstitial fibrosis in various experimental models of acute and chronic renal injury. Preliminary reports showed that BMP-7 represses the basal and increased expression of pro-inflammatory cytokines including interleukin-6 (IL-6) and interleukin-1 (IL-1), and chemokines in human proximal tubule epithelial cells induced by tumor necrosis factor- (TNF-). We have demonstrated that polymeric IgA (pIgA) from patients with IgA nephropathy (IgAN) is capable of inducing the synthesis of TNF- in cultured human mesangial cell (HMC). Base on the counteracting effect of BMP-7 on biological actions of TNF-, we hypothesized that IgA from IgAN patients modulates the receptors for BMP-7 in the kidney. We also tested whether exogenous BMP-7 could prevent the pro-inflammatory response of pIgA on cultured human mesangial cells. BMPs signal through serine/threonine kinase receptors, composed of type I and II subtypes. Three type I (ALK2, ALK-3 and ALK-6) and type II receptors (BMPR-II, ActR-II and ActR-IIB) have been shown to bind BMP ligands. These receptors are expressed differentially in various tissues and both receptors are essential for signal transduction. Using RT-PCR, constitutive expression of five BMP receptors including ALK-2, ALK-3, ALK-6, ActR-II and BMPR-II was demonstrated in cultured HMC. The expression of these receptors was further confirmed by immunofluorescence staining and flow cytometry analysis. We then examined whether these receptors are modulated by pIgA in IgAN. Polymeric IgA prepared from IgA isolated by jacalin chromatography was further separated using size exclusion chromatography. Compared with pIgA isolated from healthy subjects, pIgA from patients with IgAN significantly decreased the receptor expression of ALK-2, ALK-6 and BMPR-II (p  0.01) when cultured with HMC in a dose- and time-dependent manner. Polymeric IgA from patients significantly up-regulated TNF-, IL-1 and IL-6 in cultured human mesangial cells. Interestingly, the up-regulation of these proinflammatory cytokines was blunted by pre-incubating cultured mesangial cells with recombinant BMP-7. Our result suggests that BMP-7 and its receptors could play an important role in the pathogenesis of IgAN.

B-P-14

Polymeric IgA1 Up-Regulated Fibronectin Secretion by Human Mesangial Cells and Was Associated with the Pathological Phenotypes of IgA Nephropathy Lixia Xu, Yuehua Gao, Ying Zhang, Minghui Zhao, Haiyan Wang Renal Division and Institute of Nephrology, Peking University First Hospital, Beijing, China Background: Many studies had found that IgA1 with different molecular weights had differential effects on mesangial cells. However, whether the IgA1 in patients with different pathological phenotypes have different effects on human mesangial cells (HMC) is not investigated yet. The current study was conducted to investigate the effects of IgA1 isolated from patients with different pathological phenotypes of IgAN on secretion of fibronectin (Fn) and IL-6 by HMC.

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Methods: Sera from nine patients with minimal mesangial proliferative IgAN (mIgAN), nine patients with proliferative and sclerosing IgAN (sIgAN) and ten healthy age-matched normal controls was collected and pooled respectively. After jacalin affinity chromatograph and sephacryl 300 size separation, the IgA1 was divided into polymeric IgA1 (
440 kDa) and monomeric IgA1(150 kDa). The primary cultured HMCs was stimulated with IgA1 at a final concentration of 100 ng/ml for 3 h, 6 h, 12 h, 24 h and 48 h. The supernatants were collected and Fn and IL-6 concentration were detected by ELISA. Result: Both polymeric and monomeric IgA1 preparations could up-regulate Fn secretion by HMC. All the three polymeric IgA1 could induce more Fn secretion than their corresponding monomeric IgA1 (p  0.05). Furthermore, polymeric IgA1 from patients with sIgAN could induce more Fn secretion than that of patients with mIgAN and normal controls (p  0.05 respectively). However, there was no significant difference in polymeric IgA1 induced Fn secretion between the patients with mIgAN and normal controls. Compared with blank control, polymeric IgA1 from normal control could significant up-regulate IL-6 production from 6 h to 24 h (p  0.05 respectively); and polymeric IgA1 from patients with mIgAN could only significant up-regulate IL-6 production at 24 h (p  0.05), however, polymeric IgA1 from patients with sIgAN could not up-regulate IL-6 production. There was no significant difference of IL-6 production between monomeric and polymeric IgA1 from the same group. Conclusion: Polymeric IgA1 could up-regulate Fn production of HMC than monomeric IgA1, and polymeric IgA1 from patients with proliferative and sclerosing IgAN had stronger effect. However, polymeric IgA1 from patients with IgA nephropathy could not promote IL-6 production.

B-P-15

IgA Fc Receptor I Signals Apoptosis through the FcR Gamma ITAM Yutaka Kanamarua, Houda Tamouzaa, Séverine Pfirscha, Delphine Elmehdia, Claudine GuerinMarchanda, Marina Pretolania, Yasuhiko Tominob, Ulrich Blanka, Renato C. Monteiroa a

Institut National de la Santé et de la Recherche Médicale (INSERM) U699, Paris, France; Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, Japan b

The IgA Fc receptor (Fc alpha RI) has dual pro- and anti-inflammatory functions that are transmitted through the immunoreceptor tyrosine-based activation motifs (ITAM) of the associated FcR gamma subunit. While the involvement of Fc alpha RI in inflammation is well documented, little is known of its anti-inflammatory mechanisms. Here we show that monomeric targeting of Fc alpha RI triggers apoptosis in human monocytes and human Fc alpha RI RBL mast cell transfectants. The physiological ligand IgA induced apoptosis only when cells were cultured in low serum conditions, indicating differences with the induction of anti-inflammatory signaling. Apoptosis signaling required the FcR gamma ITAM, as cells transfected with wild-type Fc alpha RI or with a chimeric Fc alpha RI-FcR gamma receptor responded to death-activating signals, while cells expressing a mutated Fc alpha RIR209L unable to associate with FcR gamma, or an ITAM-mutated chimeric Fc alpha RI-FcR gamma, did not respond. Fc alpha RImediated apoptosis signals were blocked by treatment with the pan-caspase inhibitor zVADfmk, involved proteolysis of procaspase-3, and correlated negatively with SHP-1 concentration. Anti-Fc alpha RI Fab treatment of nude mice injected subcutaneously with wild-type Fc alpha RI RBL transfectants prevented tumor development and halted the growth of established tumors. These findings demonstrate that, upon monomeric targeting, Fc alpha RI functions as an FcR gamma ITAM-dependent apoptotic module that may be fundamental for controlling inflammation and tumor growth.

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B-P-16

Fc␣RI-FcR␥ Crosslinking Induces Leukocyte Chemotaxis and Promotes Aggravation of Glomerulonephritis Michelle Arcos Fajardo, Yutaka Kanamaru, Ivan C. Moura, Hélène Cohen, Marie Essig, Francois Vrtovsnik, Pierre Launay, Marc Benhamou, Ulrich Blank, Renato C. Monteiro INSERM U699, Bichat Medical School, Paris, France Myeloid cells bear FcR at their cell surfaces that mediate inflammatory signaling through ITAM containing FcR signaling adaptors. In severe immune-mediated glomerulonephritis (GN) activation of Fc R mediates kidney damage associated with significant leukocyte infiltration. Myeloid cells also express FcRI (CD89), which are expressed with or without FcR ; however, the role of membrane FcRI, and the mechanisms by which FcRs mediate cell infiltration remain poorly understood. IgA-immune complexes (IC) that could activate FcRI may play a major role in the pathogenesis of IgA nephropathy (IgAN), one of major causes of end-stage renal disease. To analyze the involvement of FcRI activation in GN, we used transgenic mice expressing the mutated, signaling incompetent, human FcRIR209L that cannot associate with FcR , and compared them to mice expressing wild-type (wt) FcRIwt. Activation of FcRI in FcRIwt, but not in FcRIR209L, transgenic mice resulted in marked inflammation with severe proteinuria and leukocyte infiltration in both models. The molecular mechanism involved recruitment of FcRI cells. Crosslinking of FcRIwt on macrophages activates MAP kinases and production of TNF and MCP-1. Thus, FcRI activation also represents a crucial mechanism in the progression of kidney disease. The molecular mechanism involves the activation of a cytokine/chemokine cascade that promotes leukocyte recruitment and kidney damage. The molecular mechanism involves the activation of a cytokine/chemokine cascade that promotes leukocyte recruitment and kidney damage. The potential relevance in human pathology is highlighted by the capacity of IgA-IC from patients with IgAN to activate FcRI and induce TNF production. These results demonstrate that activation of FcaRI/FcRg2 inflammatory signaling by infiltrating leukocytes represents an integral component of the pathophysiological changes underlying the progression of glomerular injury in GN.

B-P-17

Relationship between TGF-␤-Overproduction in Payer’s Patches and Glomerular IgA Deposition – Analysis in GATA3 Transgenic Mouse Takahiro Yamanakaa, Hidekazu Tamauchib, Yusuke Suzukia, Hitoshi Suzukia, Toshinao Tsugea, Satoshi Horikoshia, Masazumi Terashimac, Sonoko Habud, Yasuhiko Tominoa a

Division of Nephrology, Department of Internal Medicine, Juntendo University School of Medicine, Tokyo, bDepartment of Microbiology, Kitasato University School of Medicine, Sagamihara, Kanagawa, cPharmacology Research Laboratories, Dainippon Sumitomo Pharma Co. Ltd., Osaka, and dDivision of Host Defense Mechanism, Department of Immunology, Tokai University School of Medicine, Isehara, Kanagawa, Japan It is discussed that IgA nephropathy (IgAN) has Th2 bias. In addition, mucosal immunity is considered to contribute to pathogenesis of IgAN. We recently generated GATA-3/TCR-Tg and non GATA-3 (wild littermates: WT)/TCR-Tg mice by mating between GATA-3 transgenic mice (GATA-3 Tg) and ovalbumin (OVA)-specific T cell receptor transgenic mice (TCR-Tg). Although GATA-3 Tg mice showed Th2-dominant immune responses including IgA overproduction only after antigen challenges, there was no significant difference in immune

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response in the absence of the antigen between GATA-3/TCR-Tg and WT/TCR-Tg mice. In the present study, to examine contribution of mucosal immunity and Th2 polarity to glomerular IgA deposition, we sensitized these strains of mice orally or intraperitoneally with OVA. Cholera enterotoxin (CT) was used as a mucosal adjuvant for Th2-dominant immune responses to disrupt mucosal tolerance in the oral immunization while alum was used as an adjuvant for general immunopotentitation in the intraperitoneal immunization. GATA-3/TCR-Tg and WT/TCR-Tg mice were orally pre-immunized with OVA/CT or CT, OVA alone. Only GATA-3/TCR-Tg orally immunized with OVA/CT showed mesangial cell proliferation, matrix expansion and glomerular IgA/C3 deposition with serum elevation of OVA-specific IgA. To examine the underlying mechanisms in this model, we evaluated histopathological findings and cytokine profiles in payer’s patches (PPs), mesenteric lymph nodes (MLNs) and spleens (SPs) after in vivo and in vitro antigen stimulation. Interestingly, only GATA-3/TCR-Tg mice orally immunized with OVA/CT showed that CD4CD25Foxp3 T cells, known as professional regulatory T cells (Treg), were diffusely detected in not only dome and interfollicular areas but also follicle areas of PPs. Moreover, IgA positive cells also increased mainly in follicle areas of PPs. FACS analysis indeed confirmed the increase of CD4CD25 T cells. Cytokine assays with MLNs cells from these mice demonstrated that in vitro OVA restimulation increased IL-5 and IFN- in GATA-3/TCR-Tg mice orally immunized with OVA alone, while IL-4 and IL-5 were decreased in GATA-3/TCR-Tg mice orally immunized with OVA/CT. On the other hand, cytokine assays with PPs cells by OVA re-stimulation showed that not only IL-5 but also TGF-, known as critical mediators for IgA switching of B cells, were strongly increased only in GATA-3/TCR-Tg mice orally immunized with OVA/CT. Accordingly, our present data suggest that oral immunization with OVA/CT in animals with Th2-dominant immune responses may induce IL-5 and TGF- overproduction in PPs, presumably by Treg, and increase of antigen (OVA)-specific IgA and subsequent glomerular IgA deposition.

B-P-18

Acute Aggravation of Mesangioproliferative Glomerulonephritis through Activation of Coagulation System in a High IgA (HIGA) Strain of ddY Mice Triggered by Lipopolysaccharide Takahiko Onoa, Makiko Shimosawaa, Ning Liua, Kazuhide Uemuraa, Fumiaki Nogakib, Noriko Moric, Eri Musod, Haruyoshi Yoshidae a Department of Molecular Medicine, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka, bDepartment of Nephrology, Shimada Municipal Hospital, Shimada, cDepartment of Nephrology, Shizuoka General Hospital, Shizuoka, dDepartment of Nephrology, Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka and eDepartment of Clinical Laboratory Medicine and Nephrology, Faculty of Medical Sciences, Fukui University, Fukui, Japan

Tissue factor activates factor X to Xa; and factor V is a membrane-bound potent cofactor for the conversion of prothrombin to thrombin by factor Xa. Previously, we showed that factor V was colocalized with fibrin in the mesangial area in an active type of IgA nephropathy with mesangial cell proliferation (Liu et al. Kidney Int 58: 2000), and that coagulation process proceeded on cultured human mesangial cells via expression of factor V (Ono et al. Kidney Int 60: 2001). Recently we have shown that factor Xa induces cellular proliferation through the activation of ERK1/2 via protease-activated receptor 2 (PAR2) in cultured mesangial cells (Tanaka et al. Kidney Int 67: 2005). The episodes of macroscopic hematuria in IgA nephropathy are occasionally found in upper respiratory infection, and urinary tract infections or gastroenteritis are much less commonly observed. In a recent study, measuring high sensitivity C-reactive protein, Nelson et al. have concluded that inflammation may be a common process in various renal diseases including IgA nephropathy, and may accelerate renal dysfunction (Nephrol Dial Transplant 20: 2005).

Basic Advances (Update)

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The high IgA (HIGA) strain of ddY mice is an inbred model of IgA nephropathy, and show mesangioproliferative glomerulonephritis (MsPGN) with mesangial IgA deposition (Muso et al. Kidney Int 50: 1996). Recently, Nogaki et al. have identified the genetic loci of hyper-IgA and mesangial IgA deposition by QTL analysis (Kidney Int 68: 2005). In the present study, aggravating mechanisms in HIGA mice through activation of coagulation system triggered by lipopolysaccharide (LPS) were investigated. HIGA mice, and BALB/c mice as normal control, were intraperitoneally injected with LPS twice at an interval of 3 days, and kidney specimens were collected 7 days after the second LPS injection. In an intervention study to clarify the role of factor Xa in MsPGN, a factor Xa inhibitor, danaparoid was injected intraperitoneally after the first LPS injection for 7 days. Although LPS induced total number of glomerular cells, macrophage infiltration, and fibrin deposition both in HIGA and Balb/c mice, the increase was greater in HIGA than Balb/c mice. In this context, other coagulation factors including tissue factor, factor V, factor X, PAR1, and PAR2 depositions were negative in saline-injected Balb/c mice, weakly positive in saline-injected HIGA mice, and apparently positive in LPS-injected HIGA mice. Interestingly, treatment of danaparoid abolished PCNA-positive cells from LPS-injected HIGA mice, and brought to the basal levels in saline-injected Balb/c mice together with the reduction of proteinuria and fibrin deposition. The reason of these phenomena is suspected that HIGA mice strain has basically the feature of MsPGN, which shows mesangial cell proliferation in contrast to Balb/c mice strain. The hyper-reaction to LPS in HIGA mice may be through upregulated expression of PAR1 and PAR2. In conclusion, the results of the present study suggest that tissue factor and factor V induction by LPS may aggravate MsPGN through activation and accumulation of factor X, at least in part, which further conduct proinflammatory and procoagulant mechanisms. LPS-injected HIGA mice strain would be an acute aggravation model of IgA nephropathy, triggered by inflammatory infection on the standby state with prompt response.

B-P-19

Angiotensin Receptor Blocker (ARB) Prevents the Induction of Active Crescentic Glomerular Lesion by IL-12 Possibly through Suppression of Oxidative Stress with IFN-␥ and TNF-␣ Expression in High IgA Strain (HIGA) of ddY Mice Toshiyuki Komiya, Yukako Iwasaki, Hidemoto Asada, Sachiko Tahara, Takeshi Sugishita, Tomomi Tsujii, Fumiko Fukuuchi, Tatsuo Tsukamoto, Eri Muso Division of Nephrology and Dialysis, Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka, Japan In IgA nephropathy, active lesion with crescent formation has been suggested to be induced by immunomodulation with Th1 shift. In HIGA mice, potent murine model of IgA nephropathy, we have reported that Th1 shift by IL-12 induced active crescentic lesion (Nogaki NDT, 2002), which was attenuated by angiotensin converting enzyme inhibiter (Komiya ASN, 2004 abstract). In this study, the effect of ARB in this system was investigated. 46-week-old female HIGA mice underwent daily intraperitoneal IL-12 (300 ng/mouse) treatment for five days with or without ARB (Olmesartan). We analyzed histopathological findings and renal cortex mRNA expression of ACE, INF- , TNF- and thioredoxin (TRX; the oxidative stress marker which is highly increased under local ischemia) by realtime RT-PCR. Histopathological analysis revealed that the crescent formation and induced by IL-12 treatment were significantly prevented in ARB administered mice. ACE mRNA expression was significantly increased by IL-12 administration. Furthermore, increased IFN- , TNF- and TRX mRNA expression induced by IL-12 administration were significantly attenuated by ARB. These results suggested that ARB might prevent the progression of acute renal lesion induced by Th1 shift, not only through its hemodynamic but antioxidant in IgA nephropathy.

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B-P-20

The Role of Podocytes in the Activation of Tubular Epithelial Cells in IgA Nephropathy Kar Neng Laia, Loretta Y. Chana, Sydney C. Tanga, Moin A. Saleemb, Peter W. Mathiesonb, Joseph C. Leunga Department of Medicine, University of Hong Kong, Hong Kong, China; bRenal Unit, University of Bristol, Bristol, UK

a

The severity of tubulointerstitial injury in IgA nephropathy (GN) correlates closely with the declining renal function and the long-term clinical outcome. We had recently demonstrated the tubular epithelial cells (TEC) are activated by mediator released from human mesangial cells (HMC) following mesangial IgA deposition (Kidney Int 2005;67:602–612). There is no binding of IgA to TEC that express no known IgA receptors. We documented that TNF-alpha is the mediators involved in the glomerulo-tubular communication in the development of interstitial damage in IgAN. Since podocytes are positioned in the strategic path in the glomerulotubular trafficking, in this study, we examine the role of podocytes in activation of TEC in IgAN. IgA were isolated from 30 IgAN patients, 30 healthy subjects and other glomerulonephritic controls. Using flow cytometry assay, there was no binding of IgA from patients with IgAN to human podocytes. When podocytes were cultured with purified IgA from patients with IgAN, there was no increase in the release of growth factors or cytokines including TNF-alpha by RT-PCR, ELISA or Western blot. Furthermore, podocytes did not express mRNA of known IgA receptors including ASGPR, Fc-alpha-R, pIgR and Fc-alpha/MuR, except TfR. We next examined the effect of IgA-conditioned medium (IgA-HMC medium), prepared by culturing HMC with IgA for 48 hours, on the synthesis of TNF-alpha by podocytes. IgA-HMC medium prepared from IgAN patients significantly increased the mRNA expression and protein release of TNF-alpha by podocyte (p  0.05) with a five-fold concentration above that of HMC cultured with IgA. This up-regulation of TNF-alpha expression by podocyte was only abolished by a neutralizing antibody against TNF-alpha but not by antibodies against IL-1 beta, IL-6, VEGF, TGF-alpha, HGF, PDGF or basic FGF. Moreover, exogenous TNF-alpha also up-regulated the synthesis of TNF-alpha by podocytes in an autocrine fashion. Our study suggests podocytes also play a contributory role in the development of interstitial damage in IgAN by amplifying the TEC activation with enhanced TNF-alpha synthesis following inflammatory changes of HMC.

B-P-21

Functional Variations of L-Selectin in Immunoglobulin A Nephropathy Takashi Takeia, Masayuki Yoshidab, Shigeru Ohtsuboa, Keiko Uchidaa, Ken Tsuchiyaa, Wako Yumuraa, Yusuke Nakamurac, Kosaku Nittaa Department of Medicine, Kidney Center, Tokyo Women’s Medical University, bVascular Medicine and Medical Biochemistry, Graduate School of Medicine, Tokyo Medical and Dental University and cHuman Genome Center, The Institute of Medical Science, University of Tokyo, Tokyo, Japan

a

Although genetic factors are assumed to be involved in the development and progression of Immunoglobulin A nephropathy (IgAN), the pathogenesis remains largely unknown. To identify genetic variations that might confer susceptibility to IgAN, we previously performed a case-control association study using gene-based approximately 80,000 single-nucleotide polymorphisms (SNPs) markers. As a result, we discovered candidate genes,

Basic Advances (Update)

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PIGR (polymeric immuoglobulin receptor) gene, HLA-DRA gene and selectin gene. These genes have been known as the molecular mechanisms of Immunoglobulin A. In the selectin gene cluster, several polymorphisms have been described in association with the pathogenesis of atherosclerosis and myocardial infarction. SNPs in selectin genes, we discovered, were 712C
T (P238S) in L-selectin, -642A
G in the promoter region of Lselectin, and 1402C
T (H468Y) in E-selectin. Interestingly, these SNPs were in nearly complete linkage disequilibrium, thus 2 haplotypes, disease-associated TGT and wild type (Wt) CAC, were constructed. To investigate the functional significance of the disease-associated TGT haplotype, a stable CHO transfectant expressing a P238S-L-selectin variant (CHOa-varL) and a recombinant adenovirus vector containing an H468Y-E-selectin variant (Ad-varE) were established and compared to their Wt counterparts. Under flow, CHO-vrL exhibited significantly less adhesion over IL-1b-activated human umbilical vein endothelial cells (HUVEC) monolayers compared to CHO-wtL (p  0.004). In contrast, there was no significant difference between the adhesion of HL60 cells to Ad-varE-transduced HUVEC and those transduced with Ad-wtE. Further, a luciferase reporter construct containing a 1-kb promoter region of the L-selectin variant (luc-varL) exhibited significantly less transcription activity compared to Wt luc-wtL (p  0.0001) when transfected into HEK293 cells. These results suggest that the adhesive interactions and expression level of L-selectin in disease-associated haplotypes are significantly compromised, indicating a potential role of these SNPs in the pathogenesis of IgAN.

B-P-22

Markers of Tubulo-Interstitial Injury in IgA Nephropathy Dimitrios S. Goumenos, Evangelos Papachristou, Anastasios Drakopoulos, Jannis G. Vlachojannis Department of Internal Medicine – Nephrology, Patras University Hospital, Patras, Greece IgA nephropathy, the most common type of idiopathic glomerulonephritis, is often followed by histological changes in the tubulointerstitial area. Various factors such as Transforming Growth Factor (TGF-1), myofibroblasts (-SMA() cells) and apoptosis have been implicated in the development of renal fibrosis. The purpose of this study is to identify markers of tubulointerstitial injury in the renal tissue of patients with IgA nephropathy and to estimate their relation to the clinical outcome. Twenty-two patients (M/F 15/7) with a mean baseline serum creatinine 1.2 mg/dl and proteinuria 1.7 g/24 h at presentation were included in the study. Sections of renal tissue obtained from the diagnostic biopsies of all patients and normal renal tissue from 10 patients who underwent nephrectomy for hypernephroma were examined immunohistochemically. Using specific antibodies, the expression of TGF-1, -SMA() cells, bax and bcl-2 apoptosis related proteins as well as the presence of peritubular capillaries (anti-CD34() cells) was investigated. The extent of immunostaining was estimated by morphometric analysis and correlated to classic histology and clinical outcome (doubling of serum baseline creatinine over a follow-up period of 5 years). The extent of immunostaining for all the above markers, apart from CD34() cells in the peritubular area, was significantly higher in patients with IgA nephropathy compared to controls. TGF-1, bax and bcl-2 proteins were mainly identified within tubular epithelial cells and in the interstitium whereas -SMA() cells in the renal interstitium and CD34() cells within the peritubular capillaries (endothelial cells). A significant positive correlation of the expression of -SMA() cells with the degree of glomerular sclerosis and interstitial fibrosis (r  0.694, p  0.01) was observed. A negative correlation of CD34() cells (peritubular capillaries) with glomerulosclerosis (r  0.416, p  0.05) and interstitial myofibroblasts (r  0.496, p  0.05) was also identified. Deterioration of renal function during the follow-up period was observed in 4 of 22 patients (18%). A more intense interstitial myofibroblast expression was found in the renal tissue of patients who

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had progressive renal disease in comparison to those who preserved renal function over the follow-up period (stained points in morphometric analysis: 53 vs. 30%, p  0.01). Tubulointerstitial injury is related to an unfavorable outcome of patients with IgA nephropathy. The identification of certain markers of tubulointerstitial injury such as -SMA() and CD34() cells might be valuable for a better estimation of the severity of renal damage and prognosis.

Basic Advances (Update)

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Closing Remarks Richard J. Glassock The David Geffen School of Medicine at UCLA, Los Angeles, Calif., USA Over almost four decades IgA nephropathy has morphed from an interesting and seminal observation derived from application of a new technique to renal biopsy specimens to one of the commonest kidney diseases in the world. Indeed, the prevalence of undiagnosed IgA nephropathy likely greatly exceeds the diagnosed disorder. This International Symposium devoted to the topic of IgA nephropathy, like others that have preceded it, has again illustrated the incremental advances in the understanding of fundamental biology and its clinical applications in this still puzzling disorder. The phenotype of IgA nephropathy is still undergoing genetic dissection and we now have at least 480 genes at three independent loci on separate chromosomes as possible candidates for the familial forms of the disease (and perhaps the sporadic forms as well). Sorting out this oligo/polygenic background will be a challenging task requiring broad-based collaboration and fraught with numerous false paths and technical hurdles. Pathogenetic mechanisms are now focused on auto-immune reactions to an abnormally glycosylated IgA1 molecule with resultant formation of phlogistic immune complexes. The fundamental cause of the glycosylation abnormality is still elusive, but it appears to be a property of only a sub-set of B-cells. Both environmental and genetic factors appear to play important roles, and the deposition of immune complexes in affected tissues is receptormediated. These important findings offer tantalizing clues to new therapeutic approaches, not dependent on nonspecific suppression of immunity or inflammation. The phenotype of IgA nephropathy also includes a wide variety of clinical and pathological forms, and classification schema and clinico-pathological correlations have been inconsistent. A greatly needed and long-awaited international agreement on pathological classification appears close to realization. Much can be gained from standardization of the approach to classification of IgA nephropathy, but substantial work still needs to be done to validate the clinical utility of the new schema. Surprisingly, even after more than three decades of effort, many uncertainties still exist regarding the specific contribution of renal pathology to estimating prognosis in individual patients, over and above simple clinical assessment. Progress in identifying disease-specific biomarkers (in renal tissue, blood or urine) has been slow, but the pace is quickening with the advent of urinary proteomics, advances in immuno-histochemistry, and sensitive assays for abnormally glycosylated IgA1. Prognostic tools have sharpened and the focus has shifted from baseline to follow-up observations, but much still needs to be done to improve prognostication for individual patients as opposed to groups of patients with IgA nephropathy. Improvements in treatment continue at a reasonable pace, but most therapeutic trials remain small in size with limited follow-up. Inhibition of angiotensin II has emerged as the first evidence-based step in therapy, but we still do not know when to start treatment or what combination of drugs is best. Steroids or combinations of steroids and cytotoxic or antiproliferative agents appear to be indicated in patients with severe and progressive disease. A specific role for inhibition of platelet aggregation, anti-coagulation or tonsillectomy remains to be proven by suitably designed randomized and controlled trials. Recurrence of IgA nephropathy in the renal allograft is common and not preventable, at least by currently available approaches.

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Author Index

(A) ⫽ Abstracts Abe, Y. 208(A) Adachi, M. 197(A) Afiani, A. 185(A) Aizawa, M. 164, 225(A) Akioka, Y. 211(A) Alfarano, A. 219(A) Amore, A. 27, 175(A), 219(A) Amoroso, A. 80, 183(A) Arakawa, Y. 234(A) Arcos-Fajardo, M. 144, 222(A), 237(A) Asada, H. 239(A) Asano, M. 125, 216(A) Ashida, R.-i. 212(A) Azuma, H. 125, 216(A) Baek, J.E. 205(A) Balegnob, S. 219(A) Barratt, J. 13, 221(A) Bauer, S. 213(A) Benhamou, M. 144, 222(A), 237(A) Berthoux, F.C. 185(A), 187(A) Bertola, L. 219(A) Bisceglia, L. 80, 183(A) Blank, U. 148, 222(A), 236(A), 237(A) Boor, P. 153

Brown, R. 129, 134, 217(A) Buren, M. 50, 180(A) Bynon, S., Jr. 229(A) Cai, G. 204(A) Camilla, R. 27 Canton, A.D. 219(A) Carius, A. 213(A) Cerullo, G. 80, 183(A) Chan, L.Y. 235(A), 240(A) Chen, P. 189(A) Chen, W. 186(A) Chen, X. 189(A), 201(A), 204(A) Chintalacharuvu, S.R. 159, 224(A) Cohen, H. 237(A) Cook, T. 13, 44, 179(A) Coppo, R. 13, 27, 175(A), 219(A) Cox, S. 80, 183(A) Czock, D. 109, 193(A), 213(A), 214(A) Dangelmeier, D. 213(A) Deprele, C. 187(A) Ding, J.-X. 187(A), 228(A) Ding, R. 189(A), 201(A) Drakopoulos, A. 241(A)

Dzikova, S. 185(A), 213(A) Eckhoff, D.E. 229(A) Eitner, F. 223(A) Elmehdi, D. 236(A) Emancipator, S.N. 50, 159, 180(A), 224(A) Endoh, M. 195(A) Essig, M. 237(A) European IgA Nephropathy Consortium 80, 183(A) Feehally, J. 13, 174(A), 221(A) Floege, J. 153, 223(A) Frascà, G. 80 Fujieda, M. 211(A) Fujii, R. 210(A) Fujimoto, S. 198(A) Fujimura, T. 233(A) Fujita, T. 198(A) Fuke, Y. 198(A) Fukui, S. 90, 188(A) Fukuoka-Toda, N. 120, 215(A) Fukushima, K. 120, 215(A) Fukuuchi, F. 239(A) Fumi, T. 192(A) Furusu, A. 212(A)

244

Gao, Y. 220(A), 235(A) Gejyo, F. 225(A) Ghiggeri, G.M. 80 Glander, P. 213(A) Glassock, R.J. 169, 243(A) Goepfert, A. 134, 218(A) Goumenos, D.S. 202(A), 241(A) Grcevska, L. 185(A), 213(A) Guerin-Marchand, C. 236(A) Gunnarsson, I. 200(A) Habu, S. 237(A) Hagiwara, M. 139, 219(A) Hall, S. 129, 134, 217(A) Hara, K. 199(A), 207(A) Hara, S. 99, 198(A) Hashimoto, N. 125, 216(A) Hattori, H. 120, 215(A) Hattori, M. 211(A) Hideyuki, K. 192(A) Higa, Y. 99 Hiki, Y. 120, 215(A), 226(A), 231(A) Hirakata, H. 190(A) Hirayama, K. 219(A) Hiroki, H. 209(A) Hiu, M. 212(A) Horii, A. 191(A) Horikoshi, S. 164, 225(A), 227(A), 232(A), 233(A), 237(A) Horita, Y. 212(A) Hosaya, T. 203(A) Hoshino, J. 99 Hosoya, T. 114, 194(A) Hotta, O. 94, 104, 190(A), 193(A), 197(A), 211(A) Houzawa, K. 197(A) Huang, W.-Q. 134 Ieiri, N. 104, 193(A) Ikeda, K. 190(A) Ikeda, T. 227(A) Imai, E. 191(A)

Author Index

Imai, H. 190(A), 206(A) Imasawa, T. 195(A), 196(A), 210(A) Inoue, K. 226(A) Inoue, T. 120, 215(A) Inoue, Y. 199(A) International IgA Nephropathy Network 13 Inui, K. 199(A) Ishii, T. 114, 194(A) Itoh, A. 231(A) Ito, K. 198(A) Ito, T. 191(A) Ito, Y. 203(A) Iwanami, N. 231(A) Iwasaki, Y. 90, 188(A), 239(A) Iwase, H. 226(A), 231(A) Iwatani, H. 191(A) Iwatsubo, S. 198(A) Izumi, S. 209(A) Jacobson, S.H. 200(A) Japanese Multicenter Study Group on the Treatment of IgA Nephropathy (JST-IgAN) 94, 190(A) Jiang, L. 186(A) Joh, K. 195(A), 196(A), 210(A) Julian, B.A. 19, 56, 129, 134, 174(A), 181, 217(A), 218(A), 228(A), 229(A), 230(A) Jyunichi, H. 192(A) Kalliakmani, P. 202(A) Kameyama, A. 125, 216(A) Kanamaru, Y. 148, 222(A), 236(A), 237(A) Kaneko, S. 139, 219(A) Kaneoka, H. 208(A) Kano, T. 232(A) Karges, W. 193(A), 214(A) Katafuchi, R. 190(A) Katori, H. 99

Kawamura, T. 37, 114, 176(A), 194(A), 195(A), 203(A) Kawashima, E. 207(A) Kazufumi, N. 192(A) Keller, F. 109, 193(A), 213(A), 214(A) Kenmei, T. 192(A) Kihara, M. 164, 225(A) Kikumoto, Y. 120, 215(A) Kilian, M. 134 Kim, S.B. 205(A) Kim, T.H. 205(A) Kirksey, C. 230(A) Kitagawa, W. 206(A) Kitamura, H. 195(A), 196(A), 210(A) Kitamura, K. 198(A) Kobayashi, M. 219(A) Kobayashi, N. 159, 224(A), 227(A) Kohno, S. 212(A) Koike, K. 195(A), 196(A), 203(A), 210(A) Koiwa, F. 207(A) Kojima, K. 207(A) Komatsuda, A. 94, 190(A) Komatsu, H. 198(A) Komiya, T. 90, 188(A), 239(A) Komukai, D. 199(A) Kondo, D. 225(A) Koyama, A. 139, 219(A), 234(A) Kubo, T. 191(A) Kumagai, H. 190(A) Kurayama, H. 210(A) Lai, K.N. 235(A), 240(A) Lamm, M.E. 159 Larochelle, W. 223(A) Launay, P. 237(A) Laurent, B. 185(A), 187(A) Lee, S.K. 205(A) Lepelletier, Y. 222(A)

245

Lepper, P.M. 109, 214(A) Leung, J.C.K. 235(A), 240(A) Li, G.-S. 216(A) Liu, G. 186(A) Liu, N. 238(A) Liu, S. 189(A), 201(A), 204(A) Lundberg, S. 200(A) Lu, Y. 189(A) Lv, J.-C. 186(A), 187(A) McGuire, B.M. 229(A), 230(A) Maeshima, Y. 120, 215(A) Makino, H. 120, 215(A) Mancuso, D. 27, 219(A) Mariat, C. 185(A), 187(A) Masashi, M. 209(A) Mathieson, P.W. 240(A) Matousovic, K. 19, 56, 181(A) Matsumoto, I. 226(A) Matsumoto, K. 198(A) Mertens, T. 213(A) Mestecky, J. 19, 56, 129, 134, 181(A), 217(A), 218(A), 228(A), 229(A) Minami, Y. 210(A) Mineki, R. 233(A) Mischak, H. 230(A) Miura, N. 206(A) Miyaishi, O. 125, 216(A) Miyazaki, M. 94, 190(A) Miyazaki, Y. 203(A) Mizumasa, T. 190(A) Mohey, H. 185(A) Moldoveanu, Z. 19, 56, 129, 134, 181(A), 217(A), 218(A), 228(A), 230(A) Molyneux, K. 221(A) Monteiro, R.C. 64, 144, 148, 181(A), 222(A), 236(A), 237(A) Mori, N. 238(A) Morita, H. 199(A), 207(A)

Author Index

Moura, I.C. 144, 222(A), 237(A) Mune, M. 210(A) Murata, T. 208(A) Murayama, K. 233(A) Muso, E. 90, 188(A), 238(A), 239(A) Nagasawa, Y. 191(A) Nagura, H. 197(A) Nakai, S. 94, 190(A) Nakajima, H. 207(A) Nakamura, Y. 240(A) Nakanishi, S. 99 Nakayama, T. 199(A), 207(A) Nakazato, T. 195(A), 196(A) Naoki, S. 192(A) Narimatsu, H. 125, 216(A) Narita, I. 225(A) Naruse, C. 125, 216(A) Nedrud, J.G. 159, 224(A) Newsome, B. 229(A) Nichols, C.R. 230(A) Ninomiya, T. 190(A) Nishie, T. 125, 216(A) Nishikawa, K. 206(A) Nishiura, R. 198(A) Nishizaki, K. 120, 215(A) Nitta, K. 240(A) Nogaki, F. 238(A) Nomura, K. 99 Nonaka-Takahashi, S. 198(A) Novak, J. 19, 56, 129, 134, 174(A), 181(A), 217(A), 218(A), 228(A), 229(A), 230(A) Novak, L. 228(A), 229(A) Obayashi, K. 233(A) Odani, H. 226(A) Ogahara, S. 208(A) Ogura, M. 114, 194(A) Ohashi, K. 99 Ohi, H. 202(A) Ohsawa, I. 202(A)

Ohtsubo, S. 240(A) Ohya, M. 210(A) Okamoto, H. 203(A) Okonogi, H. 203(A) Onda, K. 202(A) Ono, T. 238(A) Ostendorf, T. 153 Otani, H. 210(A) Pang, H. 164 Papachristou, E. 241(A) Park, J.S. 205(A) Park, S.-K. 205(A) Peruzzi, L. 27, 175(A), 219(A) Pfirsch, S. 222(A), 236(A) Polenakovic, M. 185(A), 213(A) Popovska, M.M. 185(A) Poulsen, K. 134 Pretolani, M. 236(A) Qiu, Q. 204(A) Rasche, F.M. 109, 193(A), 213(A), 214(A) Raska, M. 134 Renfrow, M.B. 134, 218(A), 230(A) Ristovska, V. 185(A) Roberts, I. 13 Sadaka, C. 222(A) Saito, K. 205(A) Saito, T. 208(A) Sakatsume, M. 225(A) Sakurai, H. 234(A) Saleem, M.A. 240(A) Sasaki, M. 207(A) Sasatomi, Y. 208(A) Sato, F. 225(A) Sato, Y. 198(A) Satomura, A. 198(A) Sawa, N. 99 Sawai, T. 211(A) Schena, F.P. 80, 183(A) Schiller, A. 213(A)

246

Scolari, F. 80, 183(A) Seiichi, M. 209(A) Seki, M. 139, 234(A) Seok, H.J. 205(A) Sepe, V. 219(A) Shen, L. 189(A) Shigeko, H. 192(A) Shimizu, Y. 139, 219(A), 234(A) Shimosawa, M. 238(A) Shimozato, S. 226(A) Shohei, N. 192(A) Shoji, T. 94, 190(A) Smith, A. 221(A) Smithson, G. 223(A) Sogawa, Y. 99 Sugishita, T. 90, 188(A), 239(A) Sugiyama, H. 120, 215(A) Sugiyama, S. 226(A) Suwabe, T. 99 Suzuki, H. 56, 129, 134, 164, 181(A), 217(A), 218(A), 225(A), 228(A), 232(A), 237(A) Suzuki, K. 206(A) Suzuki, Y. 50, 70, 164, 182(A), 225(A), 227(A), 232(A), 233(A), 237(A) Syouichi, M. 209(A) Taguchi, T. 212(A) Taguma, Y. 94, 104, 190(A), 193(A) Tahara, S. 239(A) Taka, H. 233(A) Takahashi, K. 211(A), 226(A) Takahashi, T. 198(A) Takaichi, K. 99 Takasu, A. 90, 188(A) Takatani, T. 231(A) Takechi, H. 197(A) Takei, T. 240(A) Takemoto, F. 99

Author Index

Tamano, M. 202(A) Tamauchi, H. 237(A) Tamouza, H. 144, 222(A), 236(A) Tamura, M. 191(A) Tanaka, M. 90, 188(A) Tang, S.C.W. 235(A), 240(A) Tatsumoto, M. 198(A) Tatsuya, S. 192(A) Taura, K. 212(A) Tayama, H. 207(A) Terashima, M. 237(A) Tiwari, M. 144 Tokudome, S. 203(A) Tomana, M. 56, 129, 134, 181(A), 217(A), 218(A), 228(A), 229(A) Tomino, Y. IX, 1, 8, 50, 70, 129, 159, 164, 182(A), 195(A), 202(A), 205(A), 217(A), 222(A), 224(A), 225(A), 227(A), 228(A), 232(A), 233(A), 236(A), 237(A) Tone, Y. 210(A) Torres, D.D. 80, 183(A) Tovo, P. 219(A) Tsakas, S. 202(A) Tsuboi, N. 114 Tsuchiya, K. 240(A) Tsuge, T. 225(A), 227(A), 232(A), 237(A) Tsujii, T. 90, 188(A), 239(A) Tsukamoto, T. 90, 188(A), 239(A) Ubara, Y. 99 Uchida, K. 240(A) Uchida, S. 207(A) Udagawa, J. 210(A) Uemura, K. 238(A) Ueno, T. 233(A) Usuda, N. 226(A) Utsunomiya, Y. 114, 194(A), 203(A)

van Roeyen, C. 153, 223(A) Vende, F. 144 Vlachojannis, J.G. 202(A), 241(A) von Müller, L. 109, 193(A), 213(A), 214(A) Vrtovsnik, F. 144, 222(A), 237(A) Vu, H.L. 228(A) Wada, T. 125, 216(A) Wada, Y. 198(A) Wakai, K. 195(A) Wang, H. 186(A), 187(A), 216(A), 220(A), 228(A), 235(A) Wang, S. 186(A) Watanabe, F. 234(A) Watanabe, S. 199(A) Wei, R. 204(A) Wittke, S. 230(A) Wu, D. 204(A) Wu, J. 189(A) Wyatt, R.J. 19, 129, 134, 217(A), 218(A), 228(A), 230(A) Xie, Y. 204(A) Xu, L. 187(A), 220(A), 228(A), 235(A) Yamada, H. 206(A) Yamada, K. 198(A), 227(A) Yamagata, K. 139, 219(A), 234(A) Yamaji, K. 164, 225(A) Yamamoto, R. 191(A) Yamamoto, Y. 199(A) Yamanaka, T. 225(A), 237(A) Yamashita, M. 50, 159, 180(A), 224(A) Yamazaki, C. 207(A) Yanagihara, T. 56, 134, 181(A), 218(A) Yang, W.S. 205(A)

247

Yasuhiko, I. 209(A) Yasunaga, C. 94, 190(A) Yasushi, H. 192(A) Yoh, K. 139, 219(A), 234(A) Yoko, S. 192(A) Yokoyama, H. 125, 216(A) Yonemoto, S. 90, 188(A) Yoshida, H. 238(A) Yoshida, M. 240(A) Yoshifumi, U. 192(A)

Author Index

Yoshikawa, N. 178(A) Yoshiki, M. 209(A) Yoshimura, A. 199(A), 207(A) Yoshinari, Y. 209(A) Yoshino, M. 206(A) Yuen, Y.M. 235(A) Yukawa, S. 210(A) Yukio, Y. 209(A) Yumura, W. 240(A)

Zaza, G. 80, 183(A) Zhang, H. 186(A), 187(A), 216(A), 228(A) Zhang, J. 220(A) Zhang, Y. 220(A), 235(A) Zhao, M. 187(A), 220(A), 228(A), 235(A) Zhu, L. 216(A), 228(A) Zou, W. 186(A)

248

Subject Index

Absolute renal risk (ARR), end-stage renal failure 198, 199 Adiponectin, IgA nephropathy levels 216, 217 aly/aly mouse, see Alymphoplasia mouse Alymphoplasia (aly/aly) mouse, bone marrow cell induction of IgA nephropathy bone marrow transplantation 166, 167 overview 165, 238 Peyer’s patch cell adoptive transfer 166 study design 165 Angiotensin blockade, IgA nephropathy management angiotensin II actions 27, 28 angiotensin-converting enzyme inhibitors comparison with receptor blockers 32, 33 overview 9 blood pressure targets 34 clinical trials 31, 32, 188, 189 combination heparin/warfarin in progressive IgA nephropathy 115–118, 207 combination therapy 33 complications 33 ddY mouse studies 253 guidelines by prognosis 11 losartan-corticosteroid combination therapy 225, 226 proteinuria reduction 29–31 rationale 29

Angiotensin-converting enzyme (ACE) deletion allele and IgA nephropathy prognosis 202 inhibitors, see Angiotensin blockade Azathioprine, sequential immunosuppressive therapy in progressive IgA nephropathy 109, 112 Biomarkers, IgA nephropathy 20–23, 255 Biopsy, see Renal biopsy Blood pressure, targets in IgA nephropathy management 34 Bone marrow alymphoplasia mouse and bone marrow cell induction of IgA nephropathy bone marrow transplantation 166, 167 overview 165 Peyer’s patch cell adoptive transfer 166 study design 165 dysregulation in IgA nephropathy cellular immunity 75, 76 innate immunity 76, 77 mucosa-bone marrow axis abnormalities in IgA nephropathy 71, 72, 78 nephritogenic memory cell reservoir 73, 74 transplantation and IgA nephropathy remission 164, 165 Bone morphogenetic protein-7 (BMP-7), mesangial cell expression 248 Candidate genes, IgA nephropathy association studies 85, 86

249

Candidate genes, IgA nephropathy (continued) DNA microarray studies 83, 84 European IgA Nephropathy Consortium Biobank search 81, 82, 196, 197 high-throughput technologies 87 linkage analysis 83 prospects for study 86, 87 serial analysis of gene expression studies 84 CD89, see Fc␣RI Chemokines, altered expression in IgA nephropathy 50–54 Children, IgA nephropathy management 191, 223–225 Classification, see International IgA Nephropathy Network classification Clinical trials, design in IgA nephropathy 172, 173 Complement, urinary regulatory proteins in IgA nephropathy 216 Connective tissue growth factor (CTGF), proteinuria correlation in IgA nephropathy 211, 212 Corticosteroids, IgA nephropathy management children 191, 223–225 clinical trials 38, 39 cytokine responses 215 disease severity and efficacy 39 losartan combination therapy 225, 226 Pozzi’s protocol analysis 222 prednisolone dosing 38 proteasome-to-immunoproteasome switch inhibition 232, 233 retrospective analysis 189, 190 tonsillectomy combination therapy biopsy outcomes 211 corticosteroid pulse therapy efficacy analysis 219–222, 203–205 methylprednisone 99–103, 205 tonsillectomy effects after pulse therapy 223 tonsil Toll-like receptor expression response 245, 246 JST-IgAN 95–98, 203 macrophage changes 213

Subject Index

prospects for study 182, 183 urine health checkup influence on outcomes 104–107, 206 C-reactive protein (CRP), inflammation marker analysis in IgA nephropathy 218 Cyclophosphamide, sequential immunosuppressive therapy in progressive IgA nephropathy 109–112, 206, 207 Cystatin C, serum levels and IgA nephropathy prognosis 218, 219 ddY mouse angiotensin receptor blocker studies 253 genome-wide scanning 3 IgA nephropathy onset 165 pathogenesis studies 1–3, 73, 74 lipopolysaccharide effects 252 Diet therapy, IgA nephropathy management guidelines by prognosis 10–12 DNA microarray, candidate gene searching 83, 84 Docosahexaenoic acid, see Fish oil Eicosapentaenoic acid, see Fish oil European IgA Nephropathy Consortium Biobank association studies 85, 86 candidate gene search 81, 82, 196, 197 DNA microarray studies 83, 84 high-throughput technologies 87 linkage analysis 83 serial analysis of gene expression studies 84 Family-based association test (FBAT), candidate gene searching 85 Fc␣/␮RI, immunoglobulin A receptor in IgA nephropathy 67 Fc␣RI activation studies 149–151 anti-inflammatory functions 149–151, 235, 236 apoptosis induction 249, 250 FcR␥ crosslinking effects 250

250

immunoglobulin A receptor in IgA nephropathy 65–68 properties 194, 195 signaling 149–151, 249, 250 Fibronectin, upregulation in mesangial cells by polymeric immunoglobulin A1 249 Fish oil anti-inflammatory actions 33 IgA nephropathy management 33, 34, 178 side effects 34 ␤-1,3-Galactosyltransferase (C1GALT1) expression in IgA nephropathy tonsils 120–123, 228, 240 immunoglobulin A1 glycosylation detection of underglycosylated antibody 238, 239 role 86, 121 sites 135–137, 239, 240 immunoglobulin A receptor activity 234 knockout mice and IgA nephropathy-like disease development 126–128, 229, 230 GalNAc, immune complexes in IgA nephropathy 59, 60 Gas6, warfarin inhibition 117 GATA3, transgenic mouse studies 85, 251 Glomerular basement membrane (GBM), thickness as end-stage renal failure risk in IgA nephropathy 200 Glucocorticoids, see Corticosteroids gp70, deposition in ddY mouse 2 Granulin, candidate gene studies 83 Henoch-Schonlein purpura nephritis immunoglobulin A aberrant glycosylation 135–137, 231 urine polypeptide analysis 232, 244 Heparin, combination angiotensin blockade therapy in progressive IgA nephropathy 115–118, 207 Hepatitis C virus (HCV), cirrhosis and IgA nephropathy 242, 243 Histone deacetylase 5, candidate gene studies 83 Hypertension, IgA nephropathy progression 29

Subject Index

IGAN1 familial IgA nephropathy role 20, 21, 81 function 20 locus 20 mouse homolog 3, 74 Immune complex (IC) GalNAc-specific antibodies 59, 60 generation 60, 61, 194 IgA as antigen 57–59 mesangial cell activation 136, 137, 145, 146, 231, 235 Immunoglobulin A anti-inflammatory functions 149 detection of underglycosylated antibody 238, 239 glycosylation, see ␤-1,3Galactosyltransferase; HenochSchonlein purpura nephritis; Immunoglobulin G; Sendai-virusinduced IgA nephropathy immune complex, see Transferrin receptor immunoglobulin A receptor in IgA nephropathy 66–68 receptors IgA nephropathy pathogenesis mechanisms 64–68 therapeutic targeting 68 types 145 sialylation IgA nephropathy prognosis 200, 201 ST6GALNAC2 peripheral B lymphocyte expression 241 promoter haplotype and IgA nephropathy susceptibility 229 Staphylococcus aureus avidity in IgA nephropathy 140–142, 232 Immunoglobulin-A1-binding protein, serum analysis in IgA nephropathy 246, 247 Immunoglobulin G B cell line subcloning from IgA nephropathy patients 130 galactose-deficient immunoglobulin A reactivity in IgA nephropathy antibody characterization 230, 231 hinge region epitope 130, 132 overview 130

251

Immunoglobulin G (continued) methicillin-resistant Staphylococcus aureus titers 141 Immunosuppressive therapy, see Azathioprine; Cyclophosphamide; Mycophenolate mofetil Innate immunity dysregulation in IgA nephropathy 76, 77 modulation of immunoglobulin A immunity 193 Insulin, IgA nephropathy levels 216, 217 Interferon-␥ (IFN-␥) interleukin-12 and interleukin-18 in production 162 Sendai-virus-induced IgA nephropathy expression 161, 162 Interleukin-2 (IL-2), candidate gene studies 83 Interleukin-6 (IL-6), corticosteroid response in IgA nephropathy 215 Interleukin-12 (IL-12), receptor blockade studies in Sendai-virus-induced IgA nephropathy 162, 163 Interleukin-18 (IL-18), receptor blockade studies in Sendai-virus-induced IgA nephropathy 162, 163 Interleukin-21 (IL-21), candidate gene studies 83 International IgA Nephropathy Network classification clinical dataset 16 data analysis 16, 17 goals and strategy 14, 15, 187 origins 13, 14 pathological definitions and scoring 16 patient cohort testing 16 Interstitial fibrosis, see Renal interstitial fibrosis Intravenous immunoglobulin (IVIg), IgA nephropathy management 227 Japanese Multicenter Study Group on the Treatment of IgA Nephropathy (JST-IgAN), tonsillectomy-corticosteroid combination therapy trial 95–98, 203 Joint Committee of the Japanese Ministry of Health, Labor, and Welfare, IgA

Subject Index

nephropathy management guidelines by prognosis 10–12 Kidney transplantation, IgA nephropathy recurrence 256 Lee’s glomerular grading system, renal survival 198 Linkage analysis, candidate gene searching 83 Lipopolysaccharide (LPS), ddY mouse effects 252 Losartan, corticosteroid combination therapy 225, 226 Lymphocyte homing, chemokine-altered expression in IgA nephropathy 50–54 Macrophage, tonsillectomy-corticosteroid therapy effects 213 MAdCAM, lymphocyte localization role 51 Mast cell, interstitial infiltration in IgA nephropathy pathogenesis 5, 6 Memory cell, see Nephritogenic memory cell Mesangial cell activation by immunoglobulin A immune complexes 136, 137, 145, 146, 231, 235 bone morphogenetic protein-7 expression 248 deglycosylated immunoglobulin A-binding studies 233, 234 fibronectin upregulation by polymeric immunoglobulin A1 249 Methicillin-resistant Staphylococcus aureus, see Staphylococcus aureus Methylprednisone pulse therapy, see Corticosteroids Mizoribine, IgA nephropathy management in children 224, 225 Mucosa-bone marrow axis abnormalities in IgA nephropathy 71, 72, 78, 195, 196 bone marrow as reservoir of nephritogenic memory cells 73, 74 dysregulation in IgA nephropathy cellular immunity 75, 76

252

innate immunity 76, 77 Mycophenolate mofetil (MMF), IgA nephropathy management grade I and grade III patients 226 overview 41, 42 pharmacodynamic monitoring 226, 227 retrospective analysis 189, 190 sequential immunosuppressive therapy in progressive IgA nephropathy 109–112, 206, 207 Nephritogenic memory cell, bone marrow as reservoir 73, 74 Obesity, IgA nephropathy influences adipocytokine levels 216, 217 biopsy findings 91, 92, 201 study design 91 transforming growth factor-␤ upregulation 93 treatment response 92 Peyer’s patch adoptive transfer in alymphoplasia mouse 166 transforming growth factor-␤ overproduction and IgA nephropathy 251 Plasminogen activator inhibitor-1 (PAI-1) levels in IgA nephropathy 216, 217 polymorphism and vascular injury in IgA nephropathy 214, 215 Platelet-derived growth factor (PDGF) isoforms 154 PDGF-D mesangioproliferative glomerulonephritis mediation 155, 156, 236 renal interstitial fibrosis role 156, 157 receptors 153–155 Podocyte loss in IgA nephropathy pathogenesis 3–5, 181 tubular epithelial cell activation in IgA nephropathy 253, 254 Prednisolone, see Corticosteroids Prognosis, IgA nephropathy management guidelines by prognosis 10–12

Subject Index

Proteasome, immunoproteasome switch 232, 233 Proteinuria angiotensin blockade and reduction 29–31 IgA nephropathy monitoring 24 progression 29 transforming growth factor-␤ and connective tissue growth factor expression correlation in IgA nephropathy 211, 212 obese patients 93 warfarin effects 117, 118 Real-time polymerase chain reaction, ␤-1,3galactosyltransferase expression in IgA nephropathy tonsils 120–123 Receiver operating characteristic (ROC) curve, renal functional impairment analysis in IgA nephropathy 209, 210 Renal biopsy diagnostic value 170, 179 histological scoring system 199, 208–210 immunohistochemistry 45 indications 176 interpretation 192 limitations in IgA nephropathy 46–48 obese patient findings 91, 92 prognostic value 175 purpose in IgA nephropathy 45, 46, 48 tonsillectomy with steroid pulse therapy outcomes 211 Renal interstitial fibrosis, platelet-derived growth factor D role 156, 157 Resistin, IgA nephropathy levels 217 L-Selectin,

functional variations in IgA nephropathy 254 Sendai-virus-induced IgA nephropathy cytokine profiles and receptor-blocking studies 161–163, 237 glomerular immune deposits and renal function 160 immunoglobulin A glycosylation 160, 161 model overview 160

253

Serial analysis of gene expression (SAGE), candidate gene searching 84 SHP-1, Fc␣RI recruitment 151 Single-nucleotide polymorphisms (SNPs), IgA nephropathy development and progression 24 ␣-Smooth muscle actin (␣-SMA), tubulointerstitial injury marker in IgA nephropathy 255 Staphylococcus aureus immunoglobulin A avidity in IgA nephropathy 140–142, 232 methicillin-resistant Staphylococcus aureus glomerulonephritis association 140 immunoglobulin titers 141 staphylococcal cell membrane antigen role in kidney disease 247, 248 ST6GALNAC2 peripheral B lymphocyte expression 241 promoter haplotype and IgA nephropathy susceptibility 229 T helper cell polarization in IgA nephropathy 74–76, 85, 86, 160, 164, 167, 181, 237 Th2 cytokines and ␤-1,3galactosyltransferase downregulation 240 Therapeutic window, IgA nephropathy 182 Toll-like receptors (TLRs), tonsil expression in IgA nephropathy 10, 76, 77, 245, 246 Tonsil immunoglobulin A production in IgA nephropathy 244 immunohistochemistry study 210, 211 mucosa-bone marrow axis abnormalities in IgA nephropathy 71, 72 Toll-like receptor expression in IgA nephropathy 10, 76, 77, 245, 246 Tonsillectomy, IgA nephropathy management clinical trials 40, 41 corticosteroid combination therapy biopsy outcomes 211 corticosteroid pulse therapy

Subject Index

efficacy analysis 219–222, 203–205 methylprednisone 99–103, 205 tonsillectomy effects after pulse therapy 223 tonsil Toll-like receptor expression response 245, 246 JST-IgAN 95–98, 203 macrophage changes 213 prospects for study 182, 183 urine health checkup influence on outcomes 104–107, 206 indications 177 overview 9, 10 rationale 39, 40 retrospective analysis 189, 190 Transferrin receptor expression induction by immunoglobulin A 145, 146, 235 immunoglobulin A receptor in IgA nephropathy 66–68, 235 mesangial cell proliferation induction by immune complex 145, 146, 235 Transforming growth factor-␤ (TGF-␤) corticosteroid response in IgA nephropathy 215 Peyer’s patch overproduction and IgA nephropathy 251 proteinuria correlation in IgA nephropathy 211, 212 tubulointerstitial injury marker in IgA nephropathy 255 upregulation in obese patients 93 TRPC3, candidate gene studies 83 Tumor necrosis factor receptor (TNFR), retention in IgA nephropathy 213, 214 Uric acid hyperuricemia incidence in IgA nephropathy 217 intrarenal arterial lesion role 217 serum monitoring in IgA nephropathy 24, 177 Urine health checkup, influence of annual checks on IgA nephropathy remission 104–107, 206

254

IgA nephropathy biomarkers 20–23 proteomics polypeptide analysis in kidney disease 232, 244 prospects 176

Subject Index

Warfarin, IgA nephropathy management combination angiotensin blockade in progressive IgA nephropathy 115–118, 207 Gas6 warfarin inhibition 117 guidelines by prognosis 11

255