Progress in Kidney Transplantation

PROGRESS IN KIDNEY TRANSPLANTATION

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PROGRESS IN KIDNEY TRANSPLANTATION

DOMINICK W. MANCUSO EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2006 by Nova Science Publishers, Inc.

All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers’ use of, or reliance upon, this material.

This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Library of Congress Cataloging-in-Publication Data Progress in kidney transplantation / Dominick W. Mancuso (editor). p. ; cm. Includes bibliographical references and index. ISBN 978-1-60876-514-0 (E-Book) 1. Kidneys--Transplantation. 2. Chronic renal failure. I. Mancuso, Dominick W. [DNLM: 1. Kidney Transplantation. WJ 368 P964 2006] RD575.P76 2006 617.4'610592--dc22 2006019706

Published by Nova Science Publishers, Inc. New York

Contents Preface

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Chapter I

Renal Transplantation in Patients with Fabry’s Disease Renzo Mignani and Leonardo Cagnoli

Chapter II

Can Estimated Renal Graft Function Serve as a Valid Endpoint in Clinical Trials? Christophe Mariat, Eric Alamartine and François Berthoux

1

23

Chapter III

The Role of ‘Statins’ in Renal Transplantation Martin Lee and Lavjay Butani

37

Chapter IV

Pharmacogenetics of Immunosuppressive Drugs Eric Thervet and Dany Anglicheau

55

Chapter V

Brain-Death-Induced Gene Regulatory Networks in Donor Kidneys 89 Frans Gerbens, Rutger J. Ploeg and Theo A. Schuurs

Chapter VI

Kidney Transplant: The Search for Better Quality of Life Claire Terezinha Lazzaretti and José Miguel Rasia

Chapter VII

The Cost and Cost Effectiveness of Kidney Transplantation: A Systematic Review and Economic Evaluation Cyril F. Chang and Stephanie C. Steinberg

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Chapter VIII

Ethical Aspects of Kidney Transplantation Paolo Bruzzone

137

Chapter IX

State of the Art in Kidney Xenotransplantation Cristina Costa and Rafael Manez

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Index

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Preface Kidney transplantation has revolutionized the treatment of end-stage renal failure. Not only does it offer the best hope for complete rehabilitation, but it has also proved to be the most cost-effective of all treatment options, including dialysis. The surgical techniques involved have been mastered for half a century and are now considered routine. Nevertheless, this should not prevent us from appreciating the range and complexity of the issues surrounding kidney transplantation. This new book examines the latest research in this field including rejection. Fabry disease is a rare X-linked recessive disorder resulting from deficient activity of the lysosomal enzyme, α-galactosidase A (α−Gal A) that leads to progressive accumulation of the glycosphingolipid globotriaosylceramide (Gb3) in fluids and tissues including vascular endothelium, connective tissues, kidney, heart, brain and peripheral nerves. Kidney involvement, more often heralded by the onset of proteinuria, usually begins by the fourth decade and typically progresses by the fifth decade to the end stage renal disease (ESRD) requiring renal replacement therapy (RRT) with haemodialysis and/or kidney transplantation. In patients on dialysis treatment high morbidity and mortality occur due to late cardiological and neurological complications that represent the main cause of mortality in these patients. Kidney transplantation successfully corrects renal failure and quite often improves clinical symptoms, resulting in a good graft and patient survival. Nevertheless, some aspects of Fabry’s disease like myocardial involvement sometimes persist and progress also after transplantation despite a normal renal function. Historically, the treatment of Fabry disease has been non-specific and palliative. Recently, a targeted treatment for Fabry disease, enzyme replacement therapy (ERT) with recombinant human α−Gal A Agalsidase, was approved in Europe and USA. Randomized clinical trials and clinical application of Agalsidase have demonstrated to be safe and effective in the improvement of clinical symptoms of the disease. Moreover, in the patients with normal renal function or mild chronic renal failure Agalsidase leads to the disappearing of extra renal clinical symptoms and to the stabilization or the improvement of renal function and cardiomyopathy. In chapter one, the authors’ previous studies have demonstrated the safety of ERT in kidney transplant patients with Fabry disease. In these patients ERT could be beneficial in long term stabilization of allograft function and preventing or attenuating further damages such as cardiac and nervous involvements.

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Chronic allograft dysfunction is at present, one of the main challenges in kidney transplantation, and many therapeutic strategies are emerging in order to improve long-term graft survival. In this respect, short-term endpoints commonly used in clinical trials progressively become meaningless and new surrogate markers of long-term outcome are warranted. Among them, the evaluation of graft function has recently received great attention. Renal function is at best described by the glomerular filtration rate (GFR) and numerous mathematic equations are routinely employed to estimate the GFR. However, several recent reports have strongly challenged the performance of these equations when applied to transplant patients, and thereby have raised some major concerns regarding their widespread use as a suitable tool to determine graft function in clinical trials. In chapter two, the authors review the recent studies that have lead to question the validity of these equations. Then they emphasize the potential dangers of predicting renal graft function by using these equations. Finally, they discuss what should currently be the standards for GFR evaluation in kidney transplantation trials and, what could be the possible future alternatives. Chronic kidney disease is well recognized to be a state of aberrant lipid metabolism and inflammation, both of which contribute to the cardiovascular morbidity and mortality encountered in this population. Even after successful renal transplantation, both dyslipidemia and the inflammatory milieu persist, and lead not only to premature atherosclerosis, but also to the development of chronic allograft nephropathy and eventual graft loss. The independent adverse effects of, and also the complex interplay between, dyslipidemia and inflammation on vascular health will be discussed in the first part of chapter three. The statins are a group of agents that have potent lipid lowering effects and are being used commonly in the posttransplant period. More recently, the statins have been demonstrated to have immunomodulatory effects independent of their lipid-lowering action. These immunologic effects of the statins may turn out to be of much greater significance for patients with CKD and those with transplants. The latter part of this chapter will address the clinical data demonstrating the beneficial effects of statins in transplant recipients and also discuss the mechanisms by which the statins might mediate these benefits. Organ transplantation is the treatment of choice, if not the only treatment, for patients with end-stage organ failure. Recent data have shown a constant improvement in graft survival during the past decade. This improvement essentially results from the discovery of new immunosuppressive drugs which prevent the occurrence of acute rejection episodes. However, there are still some major concerns in this field as reported in chapter 4. The first one is the need for organ donors, because of the growing discrepancy between the number of transplantations performed and the number of patients awaiting transplantation. The second concern is the absence of a major improvement in long-term graft survival, partly because of the chronic nephrotoxicity associated with the use of some immunosuppressive treatments. Lastly, one of the major causes of graft failure in the long-term is now the premature death of patients with a functioning graft, mainly due to cardiovascular disease. Other major causes include stroke, infections and malignancies. Brain death has been shown to affect hormone regulation, hemodynamic stability and inflammatory reactivity. In transplant models, kidneys, livers and lungs retrieved from braindead (BD) rats do suffer from increased primary non-function and deteriorated graft survival. However, the mechanism(s) by which brain death leads to these processes have remained unclear, yet. To further unravel these mechanisms the authors of chapter five performed DNA

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microarray studies with RNA isolated from kidneys from BD rats. Kidneys from sham operated animals were used as controls. Oligonucleotide arrays were manufactured using the Sigma/Genosys Rat Oligonucleotide Library harbouring 4854 unique, gene-specific rat sequences. In kidneys from normotensive donors 63 genes were identified that were either up (55) or down (8) regulated. By using PubMed searches and GeneOntology, genes were assigned to different functional clusters: Metabolism and Transport (including water channel Aqp-2), Inflammation and Coagulation (containing the largest number (16) of up regulated genes), Growth/Regeneration and Fibrosis (including genes as KIM-1 involved in tubular regeneration) and Defense and Repair (with the cytoprotective genes HO-1, Hsp70 and MnSOD2). Also genes encoding transcription factors and proteins involved in signal transduction (such as Pik3r1) were identified. In addition, Pathway AssistTM software was used for further interpretation of their microarray data in the context of pathways, gene regulatory networks and protein interactions. These type of analyses will allow them to create a better understanding of the brain-death-related biochemical pathways which are either induced or repressed. Ultimately, these approaches will help us to design specific interventions in the brain dead donor to better maintain or even repair organ viability and protect against ischemia /reperfusion injury. The aim of kidney transplants, besides preserving life, permits patients to live their life as normally as possible, offering a better life expectancy, if not equal, at least close to nontransplanted patients of the same age. Patients put all their hope in an organ transplant to stay alive. However, their perspectives on their health problems are generally part of an individual conceptual model, with roots and meanings coming from their cultural context. With that point of view, the illness becomes part of the psychological, moral and social dimension of a particular culture, and this should be considered to understand the way patients interpret and answer their health problems. Starting from here, it is possible to think that the individuals who submitted themselves to a kidney transplant can adapt themselves to the adverse life circumstances, since they feel satisfied with the life they live, even though it is necessary to transform the concept of normality. Chapter six attempts to measure the dimension of how chronicle renal diseases affect the life of people and investigates on how the transplant, a highly innovative modality of medical science, can contribute to the rescue or reconstruction of the identity, giving back their “freedom” of life. A hundred kidney transplanted patients participated in a life quality study using the WHO questionnaire. In this sample, twenty-two patients were randomly selected to be interviewed following a non-directive methodology to analyze the subjective conditions on their perception of life quality. The results of the selfanswered questionnaire as well as the results of the qualitative results were analyzed. Although the majority of the individuals show that the transplant increased their quality of life, the authors can conclude that the disease and the post-transplant situation may lead to a particular meaning to the subject, frequently sheltering a subjective position of unhealthy. This situation shows that the concept of health escapes from the statistic calculations which define normality, that neither health can be reduced to mere balance or capacity to adapt, but that it should be thought of as the capacity to establish new principles in adverse situations. The purpose of chapter seven is to summarize the current knowledge on the economics of kidney transplantation. It systematically reviews and evaluates articles in the peer-reviewed literature on topics relating to the cost and cost effectiveness aspects of kidney transplantation. The literature review is conducted through the identification and classification of articles by topic and by the economic evaluation methodology applied by the

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authors. It includes only articles with publication dates from January 1, 2000 to June 2005 to meet the criteria for "new" research findings in transplantation. Studies in non-English language journals, abstracts, conference papers, and posters are excluded. The main data source of research literature is PubMed, a service of the National Library of Medicine which includes over 15 million citations for biomedical and health services research articles back to the 1950's. Kidney transplantation from living donors is widely performed all over the world. As discussed in chapter eight, living nephrectomy for transplantation has no direct advantages for the donor other than an increased self-esteem, but at least remains an extremely safe procedure, with a worldwide overall mortality of 0.03%. This theoretical risk for the donor seems to be justified by the socioeconomic advantages and increased quality of life of the recipient, especially in selected cases, such as pediatric patients, when living donor kidney transplantation can be performed in a pre-uremic phase, avoiding the psychological and physical stress of dialysis, which in children is not well tolerated and cannot prevent the retarded growth. According to the Ethical Council of the Transplantation Society, commercialism must be effectively prevented, not only for ethical but also medical reasons. The risks are too high not only for the donors, but also for the recipients, as a consequence of poor donor screening and evaluation with consequent transmission of HIV or other infective agents, as well also of inappropriate medical and surgical management of donors and also of recipients, who are often discharged too early. Most public or private insurance companies are considering kidney donation a safe procedure without long-term impairment and therefore do not increase the premium, while recipients’ insurances of course should cover hospital fees for the donors. "Rewarded gifting" or other financial incentives to compensate for the inconvenience and loss of income related to the donation are not advisable, at least in the authors’ opinions. The authors’ Centre does not perform anonymous living organ donation or “cross-over” transplantation. In the last few years kidney, liver, pancreas, heart and lungs transplantation have been increasingly performed with declining morbidity and mortality. However the inadequate availability of cadaver human donors significantly reduces the number of patients who can undergo this kind of treatment. An alternative procedure is xenotransplantation, introduced in Europe in 1966 by the authors’ Institution using primates as donors. More recently other animal species, such as pigs, who are not under risk of extinction, have been preferred; this "discordant" model of xenotransplantation is associated with unreversible hyperacute rejection on a humoral basis, involving complement activation. This phenomenon could be prevented by creating transgenic pigs expressing on their cell membranes complement inhibitors such as Decay Accelerating Factor (DAF), Membrane Cofactor Protein(MCP) and CD59. Research in kidney xenotransplantation aims to solve the great shortage of kidneys for transplantation. In fact, more than half of the patients in the waiting list remain without transplant. The potential benefits of xenotransplantation raised great expectations in the early 90s, but the combination of several hurdles has precluded its clinical application. The main impediment is the strength of the immune response triggered by the xenograft. Porcine kidneys were chosen for its availability and human-like physiology as the best candidates for

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clinical xenotransplantation. However, transplantation of such kidneys in nonhuman primate models results in xenograft rejection in a period of weeks to months in spite of all available immunosuppression. This process is called acute humoral xenograft rejection (AHXR) because it comprises a very strong humoral immune response and thrombosis. The presence of an innate cellular component (NK cells and macrophages) has also been described in chapter nine. The approaches developed to date, including genetic engineering of the donor pig to express human complement regulatory proteins or removal of the Gal α1,3-Gal antigen, have averted xenograft hyperacute rejection (HAR). Moreover, the prolonged survival of kidneys from pigs deficient in the Gal α1,3-Gal antigen (up to 83 days for the thymokidney) suggests that this carbohydrate also contributes to AHXR. Nevertheless, further modifications of the donor pig are needed that target other key pathways of AHXR, such as coagulation incompatibilities, to allow progress toward the clinic. Finally, potential xenozoonotic infections and ethical factors are not to be left aside. In this regard, all studies to date suggest these issues can be addressed with careful monitoring and regulation. In summary, great advances have been made in the xenotransplantation field that encourage to actively continue this research. Further genetic engineering of the donor organ may lead to successful kidney xenotransplantation.

In: Progress in Kidney Transplantation Editor: Dominick W. Mancuso, pp. 1-22

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter I

Renal Transplantation in Patients with Fabry’s Disease Renzo Mignani* and Leonardo Cagnoli Department of Nephrology and Dialysis Infermi Hospital – Rimini – ITALY

Abstract Fabry disease is a rare X-linked recessive disorder resulting from deficient activity of the lysosomal enzyme, α-galactosidase A (α−Gal A) that leads to progressive accumulation of the glycosphingolipid globotriaosylceramide (Gb3) in fluids and tissues including vascular endothelium, connective tissues, kidney, heart, brain and peripheral nerves. Kidney involvement, more often heralded by the onset of proteinuria, usually begins by the fourth decade and typically progresses by the fifth decade to the end stage renal disease (ESRD) requiring renal replacement therapy (RRT) with haemodialysis and/or kidney transplantation. In patients on dialysis treatment high morbidity and mortality occur due to late cardiological and neurological complications that represent the main cause of mortality in these patients. Kidney transplantation successfully corrects renal failure and quite often improves clinical symptoms, resulting in a good graft and patient survival. Nevertheless, some aspects of Fabry’s disease like myocardial involvement sometimes persist and progress also after transplantation despite a normal renal function. Historically, the treatment of Fabry disease has been non-specific and palliative. Recently, a targeted treatment for Fabry disease, enzyme replacement therapy (ERT) with recombinant human α−Gal A Agalsidase, was approved in Europe and USA. Randomized clinical trials and clinical application of Agalsidase have demonstrated to be safe and effective in the improvement of clinical symptoms of the disease. Moreover, in the patients with normal renal function or mild chronic renal failure Agalsidase leads to the disappearing of extra renal clinical symptoms and to the stabilization or the improvement of renal function and cardiomyopathy. Our previous studies have *

Corresponding author: Renzo Mignani, MD; Department of Nephrology and Dialysis, Infermi Hospital; via Settembrini 2; 47900 Rimini, Italy; +39-0541-705288; +39-0541-705544; [email protected]

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Renzo Mignani and Leonardo Cagnoli demonstrated the safety of ERT in kidney transplant patients with Fabry disease. In these patients ERT could be beneficial in long term stabilization of allograft function and preventing or attenuating further damages such as cardiac and nervous involvements.

Introduction Fabry disease is a rare X-linked recessive disorder resulting from deficient activity of the lysosomal enzyme α-galactosidase A (α−Gal A). It is the second most common Lysosomal Storage Disease (LSD) with an estimated frequency ranging from 40,000 to 117,000 live births [1]. The α-Gal A gene is localized in the X-chromosomal region q22.1 [2] and several mutations have been discovered at the moment but a genotype-phenotype correlation has not been yet documented [3]. The enzymatic defect leads to progressive accumulation of the glycosphingolipid globotriaosylceramide (Gb3) in fluids and tissues including vascular endothelium, kidney, heart, connective tissue, and peripheral nerves [4]. In hemizygous males suffering from the classic form of Fabry disease, the most common presenting symptoms are painful paresthesias in the extremities, fever, hypohidrosis, angiokeratoma and corneal opacities (cornea verticillata). Progressed disease manifestations involving the kidneys, heart and central nervous system are the main causes of the high morbidity and mortality of the disease, causing a cumulative survival rate significantly lower in patients with Fabry disease in comparison with the general population [5]. In male patients the residual serum and leukocytes α−Gal A enzyme activity is nearly 0-5%. In some cases residual α−Gal A activity is associated with milder clinical expression but still now everyone consider it a not reliable predictor of phenotype [5]. In heterozygote females the clinical expression of the disease is extremely variable with some female suffering of the same manifestations affecting hemizygotes males and others women asymptomatic. By analogy, the residual α-Gal A activity is variable as well ranging from 0 to 100 % [6] with the possibility that affected woman may also have the α-GalA activity in a normal range [7]. This variability is consequent to the Lyon hypothesis that attributes the phenotype diversity to the random X-inactivation of chromosome alleles during ontogeny[8]. Moreover, early studies on clinical manifestations in heterozygotes have recognized a low incidence of signs and symptoms of the disease while most serious manifestations of the disease were estimated to occur in only 1% of female [9]. However, further cohort studies have documented a higher incidence of Fabry’ s manifestations in heterozygotes females with a frequency of severe clinical symptoms over 30% of the females population. Probably, in heterozygotes the disease appears later and progresses slowly respect hemizygous males. Moreover, due to the high frequency of mild and severe manifestation of the disease, also in female patients the cumulative survival rate is significant worst with respect the general population [6]. In addition to the Classic Fabry disease phenotype two atypical “cardiac and renal variants” have been identified. Cardiac Variant has been documented in some adult or old hemizygous male patients with a residual α−Gal A activity corresponding to 10 % of normal serum concentration where the solely clinical manifestation was a typical hypertrophic cardiomyopathy not associated with other signs or symptoms of Fabry disease [10,11]. By analogy, some males patients on chronic haemodialysis for ESRD have been found to have a reduced but not completely depressed serum/leukocytes α−Gal A activity (less than 5%) and

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only symptoms of kidney involvement supposing the existence of an atypical Renal Variant of Fabry disease [12]. Historically, the treatment of Fabry disease has been non-specific, symptomatic and palliative based on the use of antihypertensive drugs, or gabapentin and carbamazepin for the treatment of chronic neuropathic pain [13]. Recently, a enzyme replacement therapy (ERT) with the recombinant human α−Gal A enzyme Agalsidase has been proposed as a specific treatment of Fabry disease. Early trials demonstrated the feasibility, the efficacy and the safety of enzyme replacement therapy either to reduce and clear Gb3 storage in several organs and tissues or to improve signs and symptoms of the disease such as neuropathic pain, sweating and quality of life [14,15]. Kidney involvement in Fabry disease is one of the most early and severe manifestation and also one of the main responsible of morbidity and mortality of the disease. Fabry nephropathy most often heralds by the onset of proteinuria in teenagers and usually progresses rapidly by the fourth decade to end stage renal disease (ESRD) requiring renal replacement therapy (RRT) with haemodialysis and kidney transplantation [16,17]. In patients on haemodialysis extra renal symptoms such as neurophatic pain, hypohidrosis and gastrointestinal manifestations persist and both neurological and cardiac manifestations occur frequently [18,19]. By contrast, kidney transplant corrects some extra renal manifestations such as acroparesthesias, seems to reduce neuro and cardiac events and, above all, permits a longer patient and graft survival rate [18,20,21]. For all these reasons, renal transplant is now considered the treatment of choice that should be prompted and supported in patients with Fabry disease that underwent to ESRD. However, also in allograft patients the cardiomyopathy progress and a recurrence of symptoms as pain is possible several years after the transplant. The recent, even if limited, application of the ERT in transplanted patients seems to restore extra renal clinical symptoms of the disease and, above all, seems to slow down the progression of the cardiac involvement without any significant interference with the allograft function [22].

Clinical Manifestations of Fabry Disease Clinical manifestations of Fabry disease result from the progressive accumulation of globotriaosylceramide in almost all cells type and in particular in the vascular endothelium. In the classic form of Fabry disease that occurs prevalently in hemizygotes males, clinical onset occurs during childhood and adolescence. In these cases, the early manifestations include pain in the extremities (acroparesthesias), hypohidrosis, angiokeratomas, corneal opacities, hearing loss, gastrointestinal disturbances. With the progression of the disease the involvement of heart, kidney and central nervous system also appear. The neuropathic pain begins in the childhood and frequently manifests itself as a sudden attack of paresthesias at the extremities of hands and feet, often associated or induced by fever, exercise, fatigue [4,23]. Together with hypoydrosis, the neuropathic pain is probably the expression of an autonomic dysfunction secondary to the deposition of the substrate into the nervous cells of the peripheral nervous system [24]. Acroparesthesias are very debilitating and compromise severely the quality of life of these patients [25] that may obtain only partial benefit by the treatment with Gabapentin or conventional analgesic therapy [26]. With

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availability of the ERT, both symptoms relief very clear and rapid only few months after from the start of Agalsidase treatment [27]. Angiokeratomas are typical skin lesions that appear as small, red-black vascular lesion occurring as a small number of single lesions or as a large number of diffused lesions covering areas of the genital areas, buttocks and thighs (1). At present, there are no data to support angiokeratomas extension as a surrogate marker of progression of Fabry disease or improving of the disease after the introduction of ERT [28]. Ocular involvement occurs mainly in the cornea with typical corneal opacities that are observed only with the slit-lamp microscopy. In the advanced cases, these opacities appear as whorled streaks extending from a central vortex to the periphery (Cornea Verticillata). A second and possible ocular manifestation of Fabry disease is a posterior cataract observed by retroillumination. Both lesions do not interfere with visual activity [4]. Hearing loss and sudden deafness are frequently documented in Fabry disease patients. It is reasonable that hearing loss is due to the accumulation of glycosphingolipid within the ganglion cells of the inner ear and so as an expression of the deterioration of the peripheral nervous system as well as pain and hypoydrosis [29]. A prolonged treatment with agalsidase therapy appears to improve gradually hearing deterioration [30]. Gastrointestinal symptoms manifest early and frequently in Fabry disease. They include abdominal pain, diarrhoea, bloating, nausea, vomiting and contribute to the poor quality of life of these patients [4]. Before the ERT the palliative treatment for these disturbances were oral pancreatin supplements [31]. Limited and cohort studies with both Agalsidase formulations have documented an encouraging efficacy of the ERT in the relief of gastrointestinal symptoms [32]. Fabry disease is associated with an increased incidence of thrombotic events including retinal thrombosis, deep vein thrombosis, stroke, myocardial infarction [33]. Hypercoagulability and thrombophilia depends mainly on the antithrombin III and protein C deficiencies. A resistance to Activation of Protein C (APC) is frequently recognised in patients with Fabry disease and consequently it is considered the most common risk factor for hypercoagulability and thrombosis in these patients [34]. Therefore, a prophylactic therapy with anticoagulant drugs has been proposed [33,34]. Central nervous involvement is very frequent in both hemi and heterozygotes. The high rate and the severity of this involvement are considered to be some of the main causes of the morbidity and mortality of patients with Fabry disease [1]. It is a cerebrovascular disease that include thrombosis, transient ischemic attacks, aneurysm, seizures, hemiplegia or frank cerebral haemorrhage mainly localized in the inner side of white-matter. Neurological complications are related to the progressive accumulation of storage material in the endothelium of cerebral vessels and are associated with an impaired cerebral blood flow. The diagnosis of cerebral manifestations is based on the examination of the magnetic resonance imaging (MRI). While ERT seems to improve regional blood flow [35] , up to date, it seems not to be able to prevent or stabilize the rate of cerebrovascular events [36]. Supporting this data is the recognition of sub clinical white-matter lesions in a cohort of female with a serum and leukocytes α−Gal A enzyme activity in the normal range and consequently with a supposed low deposition of substrate in cerebrovascular endothelium [37]. Fabry cardiomyopathy represents a frightening complication of the disease and one of major responsible of high morbidity and mortality in Fabry patients [38]. Clinical onset of cardiac involvement include arrhythmias, chest pain or infarction. Electrocardiographic study

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shows QRS and ST-T wave abnormalities and a short P-R interval. Echocardiography demonstrates an increased incidence of mitral insufficiency (MI), left ventricular hypertrophy (LVH) with increased interventricular septum thickness (ST), left ventricular posterior wall thickness (PWT), end diastolic diameter (EDD) and left ventricular mass (LVM) [38-40].The confirmation of heart substrate deposition is based on the ultra structural invasive recognition of sphingolipids in cardiomyocytes at cardiac biopsy. Moreover, sphingolipids deposition is also present in the tissue conduction cells and in the valves. About the effect of ERT on cardiomyopathy, in one open-label study 16 hemizygotes patients with severe LVH, elevated LVM but normal global LV function (as expressed by a normal EF) were investigated before and 12 months after ERT with standard dose of Agalsidase β. In all patients ERT resulted in a significant improvement of morphological parameters with reduction of LVH and LVM after Agalsidase therapy [41]. The effect of ERT on electrocardiographic alterations were also investigated: in a 46-year-old-female after a short course of agalsidase β the P-R interval returned to normal value and remained unchanged after 24 months of ERT [42].By contrast, in another study 16 Fabry patients with typical hypertrophic cardiomyopathy and a short P-R, although both LVH and LVM improved at 1 year control, no significant changes in P-R interval were recorded suggesting that Electrocardiographic alterations may not be a useful marker of ERT efficacy on cardiac involvement [43]. Besides, several single case reports have documented the lack of improvement of clinical arrhythmias after different and long period of ERT supposing that cardiac conduction tissue may be irreversible damaged by substrate deposition or the needing of a longer period of treatment with ERT to restore the conduction tissue function [22,38].

The Renal Involvement in Fabry Disease Fabry disease is often very difficult to elucidate and diagnose because signs and symptoms at the onset could be often misunderstood or underestimated. To date, the diagnosis of Fabry disease is more often in the hands of pediatrics, because of the early onset of the disease, and metabolists through a screening performed in selected study populations. In these cases the clinical evaluation, the detection of a low α−Gal A activity and the genotype study permit a definitive and non invasive diagnosis of the disease. However, the diagnosis of a renal involvement require the clinical evaluation and an histologic examination with the renal biopsy either to document the extension of the lesion or to define the efficacy of the therapy in patients who have started the ERT.

Diagnosis of Fabry Nephropathy Clinical presentation of renal involvement is most often heralded in childhood by the onset of microalbuminuria and proteinuria. Proteinuria is very often in a non nephrotic range and is associated with aminoaciduria, glicosuria, lipiduria, loss in the ability to concentrate and dilute urine, proximal tubular acidosis. With time proteinuria worsens but rarely it reaches nephrotic range. In a cohort study of 105 affected males proteinuria occurred in 100% of patients who had reached 52 years of age and the mean age of onset was 34 years. A

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nephrotic proteinuria was detected in only 18% of the study population while a complete nephrotic syndrome was present in only 20 % of patient with nephrotic proteinuria [16]. In another small study, the presence of a nephrotic proteinuria did not correlate either with the residual α−Gal A activity or the extension of lipids deposits and glomerular sclerosis [44]. In the presence of a proteinuria lead per se to the execution of a kidney biopsy with the aim to clarify the origin of urine abnormalities. Therefore, renal biopsy represents the golden standard for the diagnosis of Fabry nephropathy. However, a non invasive method for the diagnosis of the disease has been proposed through the ultrastructural evaluation of the urine sediment with the detection of the typical osmiophilic inclusion into the tubular cells in the sediment [45].

Renal Pathology Renal involvement is the consequence of accumulation of glycosphingolipid in the glomeruli and in the tubulointerstitium areas. All cells types are interested by substrate deposits but the entity of lipid storage is not uniform. Renal lesions are found in both males and females but in the latter the extension and the entity of histologic abnormalities are variable depending on the lyonisation of X-chromosome. Immunofluorescence do not show generally any lesion. In rare cases complement and immunoglobulin fractions have been documented at the immunofluorescence of renal biopsy of patients with Fabry disease. These aspects were indicative for a IgA nephropathy [46] or a Lupus nephritis [47] or a crescentic glomerulonephritis [48] that coexisted with the typical abnormalities of Fabry disease. In these cases, more than an incidental coincidence, a possible explanation of the coexistence of different lesions is that Gb3 deposit could be a chronic antigenic stimulation that may result in a pathologic autoantibodies formation [47]. At light microscopy the glomeruli appear enlarged and filled by evident lipid deposits. All kinds of glomerular cells are involved especially the visceral epithelial cells (podocytes) and less parietal epithelial cells, mesangial and endotelium cells giving a characteristic foamy appearing vacuoles. The reason why the largest amount of deposits are in podocytes is that these cells are postmytotic cells with a longer time of exposition to glycosphingolipid. Therefore, podocytes are involved in the early stages of the disease and are responsible for the early onset of proteinuria. Vacuolation is also present in the epithelium of proximal and distal tubular epithelium cells, including those of Henle’s loop and the collecting duct, in interstitial cells, in smooth muscle cells and in the endothelium of interstitial vessels. In advanced and severe disease focal and segmental sclerosis are prevalent in the glomeruli leading to the collapse of the glomerulare tuft. In interstitium areas the predominant aspects are those of tubular atrophy and interstitial fibrosis [49,50]. At electron microscopy the typical lesions in Fabry nephropathy are the myeloid or “zebra bodies”. They are round intracitoplasmatic electron-dense bodies with lamellated membrane structure localized in the lysosomes. Myeloid inclusions are localized in all cells but predominant in podocytes and in the distal tubular epithelium. In the early stages of the disease the glomerular filtration barrier, foot process and slit-diaphragm are unaffected [49,50]. This condition probably account for the non-nephrotic proteinuria that affected initially these patients [17,49]. With the progression of the disease there is a fusion of the

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podocytes and foot process while filtration membrane appears effaced [50]. Therefore, podocytes injury and matrix expansion and the consequent glomerular collapse represent the main cause of glomerulosclerosis that is responsible of the rapid progression of the disease to ESRD[49,51]. However, in several single cases reports a podocyte damage has been documented in males or females without the recognition of proteinuria or a renal failure [46,52,53]. Hence, other mechanisms more than the solely podocytes injury should be involved. The diffused and progressive substrate deposits in endothelium and smooth muscle cell of interstitium may contribute to the loss of renal function by a ischemic mechanism [49]. Furthermore, when tubular atrophy and interstitial fibrosis occur the glomeruli upstream may function poorly leading to hyperfiltration of other glomeruli that may contribute to segmental and global sclerosis [54].

Evolution of Renal Function In patients with the classic form of Fabry disease the onset of CRF may begin in the second-third decade of life. In the cohort study on 105 affected males, the 50 % of patients developed CRF within the 43rd year of age. Progression from CRF to ESRD was very rapid and occurred over a mean of 4+3 year corresponding to a mean rate of decline in GFR of 12.3 ml/min/year [16]. Such evolution is the same found in patients with diabetic nephropathy who did not assume anti-Renin-Angiotensin System (RAS) drugs and faster than other nephropathies [55]. In the same study a favourable correlation has been found between patients with an undetectable serum α−Gal A enzyme activity and the probability to develop CRF. Moreover, an indirect correlation has been demonstrated between the genotype and the probability to develop CRF: patients with conservative mutations had a low probability to develop CRF in respect to hemizygotes patients with non-conservative mutations [16]. This correlation suggested a possible genotype/phenotype correlation in Fabry disease. However, several single cases and recent cohort studies more than registries reports have decisively excluded this correlation, suggesting that other factors are probably involved in the phenotype expression of Fabry disease. By contrast, a significant correlation was observed in both hemi and heterozygotes between the level of proteinuria and the degree of renal dysfunction measured by estimated GFR [56]. This data confirm that proteinuria is a indicator of the stage of renal involvement and a predictor factor of progression of renal disease in both genders [57,58]. In heterozygous females, however, the picture is extremely variable because a normal and stable renal function can be observed after a long follow up either in nephrotic or in nonnephrotic proteinuric females [44]. Hypertension is not a common feature of Fabry disease, manifesting in only 30% of patient and in one third of them it compares before the onset of CRF . Therefore, it does not seems to contribute to the progression of CRF [16]. On the other hand, while the role of antihypertensive drugs in reducing the decline in GFR has been demonstrated in patients with diabetic e non-diabetic nephropathies [59-61], the same has not yet been established in proteinuric Fabry disease patients. ESRD is a common complication of Fabry disease due to the rapid progression of CRF and the mean age of patients initiating dialysis is 42 years[16]. Fabry disease is a rare cause of ESRD representing only 0,017 % of all causes of ESRD even if the prevalence of undiagnosed disease seems to be higher among patients on RRT [19]. The outcome of RRT in

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patient with Fabry disease has been previously analyzed by the European EDTA-ERA registry [18] and the American USRDS registry [19]. Patients with ESRD of European and American origin are similar despite genetic and geographic differences. In fact in both series the initial modality of RRT is similar (78 % for haemodialysis and 20% for peritoneal dialysis in European registry and 64% for haemodialysis and 34% for peritoneal dialysis in the US registry [18,19]. However, later studies performed on dialysis populations on RRT through a simple screening have documented an higher frequency of the disease among these patients, almost 10 times than reported in the two registries [12,62,63]. In heterozygous women ESRD is a rare complication (females represent about 10% of patients with Fabry disease on RRT in both the registries) and can manifest later in comparison with male patients [6,19]. In patients on maintenance dialysis treatment signs and symptoms of the disease such as pain, hypohidrosis, angiokeratomas, corneal opacities, hearing loss, gastrointestinal disturbances persist. Furthermore, morbidity is high due to cardiological and neurological complications resulting in a poor survival rate worse than in transplanted patients. Data from EDTA-ERA and USRDS registries demonstrated that the three-year survival among American patients on haemodialysis (63%) was similar to the three-year survival reported in European registry (60%) and the survival is significantly lower compared to non-Fabry non-diabetic controls [18,19]. Therefore, enzyme replacement therapy is mandatory to prevent the onset or the progression of severe extra-renal complications in patients on dialysis treatment. To date, limited published information is available: in a small cohort study 6 patients on dialysis treated with Agalsidase β at standard dose improved their quality of life and reduce the progression of cardiomyopathy after 24 months of treatment with ERT [64]. The possibility that extra corporeal dialysis treatment can interfere with Agalsidase modifying the normal pharmacokinetic profile of the drug led some authors to analyse the pharmacokinetic of Agalsidase β. Recent studies have reported no loss of the drug in the dialysate of the extracorporeal dialysis [65]. By contrast, limited information is available on the effect of the peritoneal membrane on agalsidase in patients on RRT with peritoneal dialysis but it has also been reported that there is no enzymatic loss from the peritoneal membrane in these patients [66].

The Outcome of Renal Transplant in Patients with Fabry Disease Early experiences on renal transplant in Fabry disease were published in the 80’s. In the first Maizel’s cohort study he reported a survival rate of 26% at 5 years due to the high incidence of death for sepsis in 4/8 allograft patients. He concluded affirming that “transplantation represents a significant risk… ” and consequently it should not be routinely employed in patients with Fabry disease [67]. However, in the same cohort study one patient was survived 8 years after transplantation. Moreover, in the same period several single case reports were published documenting patient survivals longer than 8 years [68-70]. Afterwards, an european experience reported a patient survival of almost 50% at 5 years and no death for lethal infections occurred in the study population [71]. The conclusion of this and subsequent reports was that renal transplantation permitted a good survival rate and should not be contraindicated but, on the contrary, encouraged and prompt in Fabry patients [71,72].

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10 years later, the EDTA Registry published the results of a questionnaire regarding the outcome of 33 renal transplant in patients with Fabry disease. In this study, the overall 3years graft and patient survival rates were 72 and 84% respectively and were similar to the to the rates documented in patients with other primary renal disease[18]. Similar results were found later by an analysis of the USRDS Registry that also documented an excellent outcome of renal transplantation in 93 allograft recipients with Fabry disease. In this cohort study, the 1, 5 and 10-years graft and patient survival rates in recipients with Fabry disease were statistically similar to the graft and patient survivals in transplanted patient without Fabry disease. For instance, 5-year graft survival was 76 % in Fabry patients and 67% in non-Fabry patients while patient survival was 83% and 82 % respectively [20]. These surprising results may be in part justified by the too small number of patients analyzed and, probably, by the lack of selection of patients in the control group. However, the favourable outcome of kidney transplant in Fabry disease will encourage renal transplantation as a valid alternative to dialysis in these patients. Reasons of this improvement on transplant outcome in Fabry’s allograft recipients could be the same that have improved the transplant survival in general. So, the effectiveness of new immunosuppressive drugs and a better employment in terms of doses, blood levels, more experiences in transplantation, a greater attention to extra-renal organ damage are all possible explanations of these good results. Kidney transplantation in Fabry disease corrects renal function and also improves extra renal symptoms like pain, sweating decrease and acroparesthesia [21,72-74]. It is unknown the mechanism by which just some months after the transplant there is an improvement of such symptoms correlated to the autonomic dysfunction. Some authors have attributed this result to the α−Gal A enzyme activity. Actually, early reports on renal transplantation in patients with Fabry disease have documented an increase of the circulating α−Gal A enzyme activity [75-78]. However, subsequent single cases and cohort studies did not confirm this data and no increase was any more found since the circulating α−Gal A enzyme concentration does not change following transplantation [21,72,79-81]. By contrast, the normal α−Gal A enzyme activity documented in the allograft tissue [73,78] permits the clearance of substrate from the graft tissue that is free of deposits. On the other hand, it is obvious that the normal α−Gal A enzyme activity of renal allograft tissue is not enough to supply the individual needs and therefore does not alter either the systemic substrate deposition and consequently the progression of the systemic complications of Fabry disease [82]. Therefore, even if cardiovascular and neurological morbidity and mortality are less severe and frequent in transplant recipients compared to patients on haemodialysis [18,20], in allograft recipients, heart and brain complications persist and progress representing the primary cause of death in these patients [69, 83]. Loss of the graft in transplanted patients with Fabry disease is generally attributed to acute or chronic rejection [84,85]. Furthermore, a possible graft sudden loss for allograft thrombosis has been reported in patients with Fabry disease and renal transplant. In a small cohort study a spontaneous post transplant allograft artery thrombosis occurred in one allograft recipient with a resistance to APC that was also documented in 3/5 transplanted patients. The prophylactic treatment with anticoagulant drugs of patients with resistance to APC permitted to exclude thrombotic events in these patients after a long post transplant follow up [34].

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On the other hand, loss of the transplant due to substrate deposition in the graft is very doubtful and controversial. Several reports have documented the recurrence of clinical signs and symptoms such as neuropathic pain at different ages after the transplant [22,71]. When an allograft biopsy was performed either because of the presence of a relapsed symptom or to confirm the hypothesis of a graft rejection, in some cases the typical intracitoplasmatic lamellar bodies at electron microscopy were documented [73,86-89] while in other patients, no histological signs of recurrence were found in the graft [69,90]. However, when documented, the substrate deposits were only small size glycosphingolipid deposits, prevalently localized in the vascular endothelial cells or in tubular epithelium but not in other cells. Therefore, these deposits could simply result from the exposition of endothelium and distal tubules by a large amount of circulating ceramides that arrives in the kidney and engulfs the endothelial and epithelial cells [73,88,89]. Moreover, chimerism or recolonization of the endothelium graft vessels through host endothelium could also explain why some cells from the host can be found in the graft [87]. A third hypothesis to explain the presence of deposits in the graft is the onset of a circulating α galactosidase A inhibitor [86]. On the other hand, it appears unjustifiable to define the presence of these deposits as a relapse of Fabry disease, considering that the graft maintains a normal α−Gal A enzyme activity and the lack of detection of Gb3 from the urine of transplant patients [22] As an alternative, the possible recognition of urinary Gb3 could be attributed to cell desquamation [91]. However, recurrence of Fabry disease in the renal allograft remains a controversial phenomenon that is still under discussion [92]. Most reported transplants on patients with Fabry disease have been performed from cadaveric donor. As with other hereditary renal disease living related donors for patients with Fabry disease and ESRD should be evaluated careful because of the risk of them developing renal failure from the same condition. Hemizygous males are obviously contraindicated for renal donation. Heterozygous females should also be careful examined for living donation. In the past there have been some reports of renal donation from females related to a recipient affected by Fabry disease. In a female carrier the disease was excluded because she was asymptomatic but when she underwent a living renal donation to her daughter affected by chronic glomerulonephritis a typical Fabry nephropathy in the graft was detected at the time of the transplant [93]. In another case an asymptomatic and clinically unaffected sister donated a kidney to her hemizygous brother [94]. In an heterozygous female a possible living renal donation was instead excluded after the performing of a renal biopsy that documented the typical renal lesions of Fabry disease [81]. Finally, in a young ESRD female receiving a HLA identical living related kidney from her sister, a typical Fabry nephropathy in the graft was detected [95]. Even if a long post transplant graft and patient survival is possible [96], in all these cases either the onset of proteinuria or a typical histological renal damage were documented in the recipients after the transplant. To date, the prevalent opinion is that the renal donation from a related heterozygous female should be avoided. Some authors have suggested that living related should always be excluded from heterozygotes [94]. Other authors have instead suggested that related female could be evaluated for a possible kidney donation but only after a renal biopsy that excluded a renal involvement of Fabry disease or after the recognition of a normal enzymatic activity [81]. However, considering the variability of phenotype and α−Gal A level in females, either a normal renal histology or a normal α−Gal A enzyme activity do not exclude the disease in a related female and so they are not

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sufficient to exclude a carrier condition in a related female. Only the genetic investigation with the genotype test, can definitely assess if a related female is affected or not, thus becoming a possible candidate to organ donation.

The Enzyme Replacement Therapy in Patients with Renal Transplant The development of ERT represents an attempt to correct Fabry disease through the restoring of a normal enzyme activity with the aim to correct the systemic disease. After the trial to infuse a purified α Gal A extracted from human liver and plasma [97] or to infuse high dose of galactose [98], the new recombinant human α−GA (r-h GA) agalsidase has been produced. To date, two different r-h GA enzyme formulations are available ; they can be distinguished by the cell type used for production, i.e. human fibroblast (agalsidase α−Replagal ®, TKT Inc.) and Chinese hamster ovary cells (agalsidase β-Fabrazyme® , Genzyme Corp). Preclinical studies performed in the mouse model have established the pharmacokinetics of r-h GA. These studied demonstrated that the tissue distribution of the infused enzyme was dose-dependent and that there was a different clearance of substrate from different tissues. So, the Gb3 deposits mobilization was higher from the liver, spleen, skin and less from kidney, heart [99]. Subsequent clinical trials have documented in humans the safety and efficacy of agalsidase in depleting the substrate deposits from several organs and tissues [14,15]. Both agalsidase formulations are similar regarding the glycosylation activity; however, they differ for the dose administered intravenously every two weeks that for Replagal® is 0.2 mg/kg b.w. and for Fabrazyme® is 1 mg/kg b.w. Several clinical studies have demonstrated the efficacy of ERT in improving symptoms such as neuropathic pain, gastrointestinal disturbances and quality of life [100,101]. In patients with Fabry nephropathy, both formulations seems to stabilize proteinuria that persist unchanged after 1 or 2 years of ERT irrespective of renal function at baseline [100,102]. Actually, proteinuria is a marker of podocytes injury; since the removal of Gb3 from podocytes is limited the proteinuria did not change in patients on ERT. In patients with normal or mild CRF prolonged treatment with ERT stabilized serum creatinine slowing the progression of CRF to ESRD [101,103-106]. By contrast, in patients with a severe CRF and a GFR <40 ml/min the progression of renal insufficiency was rapid to ESRD as observed in the patients not treated with ERT. Based on these results, these authors supposed the possible existence of a “point of no return” of serum creatinine so that the start of ERT beyond this level of renal function is not able to arrest or reverse the progression of CRF lacking a possible improvement of renal function [106,107] . Although the high number of studies regarding the use of the recombinant enzyme agalsidase in several renal function stages or renal conditions, to date only case reports or single centre experiences reported the efficacy and safety of ERT in patients with kidney transplantation. In our pilot clinical trial [22], we have demonstrated the safety and the efficacy of ERT in 3 kidney transplant patients with Fabry disease who had severe cardiac involvement (figure 1). These patients received Agalsidase β at a dose level of 1 mg/kg biweekly for a period of 18 months. In one patient, pain in the extremities had relapsed several years after transplantation and it was still present at baseline. The pain resolved within

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few months after the beginning of ERT. In another patient, proteinuria, present at baseline, had disappeared after 18 months of ERT. The renal function, as restored by kidney transplantation, was preserved and consequently no adjustment of the immunosuppressive regimen of the patients was required. Plasma Gb3 levels were determined by using the tandem mass spectrometric method [108] and showed considerable inter-patient variability at the baseline but a reduction of approximately 30 to 50 % in all patients while on ERT. At start of ERT, urinary Gb3 was not detectable in any of the patients which is probably explained by the absence of glycosphingolipid accumulation in the allograft, in particular in the tubular cells. As documented in other studies [101], cardiac response to agalsidase treatment was not uniform. Arrhythmia due to atrial fibrillation remained unchanged in one patient after 18 months of agalsidase β treatment. Echocardiography demonstrated an appreciable reduction in LVM in 2 of the 3 patients while in the other patient morphological parameters had worsened while on ERT (figure 2). As a possible explanations for the non-uniform cardiac responses, we argued either the highest Gb3 accumulation in cardiomyocytes compared to other tissues [15] or the existence of a “tipping point” at which Gb3 storage causes irreversible tissue damage or, finally, the lower dose of agalsidase administered to one patient. With regard to infusion duration, the infusion time at start of ERT was 4 hours, but after the first month the infusion time was reduced by 15 minutes every subsequent infusion. At 6 month of therapy, the infusion duration was 2 hours for all patients without the occurrence treatment-related adverse reactions or intolerance. None of the patients seroconverted at the end of the study probably because of the reduced immunoreactivity of allograft recipients due to the concomitant immunosuppressive therapy [82].

Figure 1. The electron micrograph of a section of myocardium (patient 1) shows a massive deposition of Gb3 in the cardiomyocytes

In another multi-centre clinical study we recruited 6 allograft recipients with Fabry disease with severe cardiac involvement. All patients were treated with agalsidase β at a dose level of 1 mg/kg every 2 weeks for a period of 1 year. At baseline mean serum creatinine was 2.1 mg/dl and proteinuria was detected in only 2 patients. All patients suffered from severe

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cardiomyopathy with high LVM. After 12 months of agalsidase β treatment mean serum creatinine remained unchanged ( 1,9 mg/dl) while proteinuria normalized in the 2 patients for whom it was reported at baseline. In 4 out of 6 patients, increased plasma Gb3 was detected at baseline and levels decreased with 30 to 50 % as measured at the most recent study visit. Urinary Gb3 was undetectable in all patients. Echocardiographic evaluation showed in all patients a slight while not significant improvements of LVM (figure 3). Arrhythmias, present at baseline in 3 out of 6 patients, remained unchanged after 1 year of ERT. In all patients, infusion time was progressively reduced to 2 hours at 6 months of therapy without the occurrence of any adverse reactions or symptoms suggestive of intolerance [109].

Figure 2. Statement of Left Ventricular Mass (LVM) at the baseline and after 18 months of enzyme replacement therapy. In Pts 1 and 3 LVM improved mildly to markedly while in Pt 2, after an improvement at 12 month control, LVM moderately worsened.

Figure 3. Left Ventricular Mass (LVM) at the beginning and after 12 months of ERT with Agalsidase β in 6 patients with Fabry disease and kidney transplantation

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At present, in Italy we are evaluating the effect of ERT in patients with Fabry disease on RRT. In this national, multicenter, open study we enrolled 31 Fabry patients ( 29 males and 2 females); 16 have a renal transplant and 15 are on dialysis treatment. At the moment, the mean ERT duration is 38 months (range18-52 months) for the patients with renal transplant and 35 months (range 12-56 months) for patients on dialysis (Table 1). The study is still in progress but from a preliminary evaluation it is possible to anticipate some important results. Regarding the patients on dialysis treatment the number of events (cardiac and cerebral) occurred after the beginning of the ERT seems to be lower in comparison with the events occurred before the beginning of ERT. Among the group of transplanted patients, first of all the graft survival rate is remarkable high with a mean of 102 months (range 20-212 months). Furthermore, no episode of acute rejection has been documented in all 16 allograft recipients during the ERT period. The monitoring of immunosuppressive therapy in a group of 9 patients among a period of 12 months demonstrated that neither the serum levels of calcinueurin inhibitor or the dose of immunosuppressive drugs has been significantly modified during this period [110] . Table 1. Study population of 31 patients with Fabry disease on RRT in Italy

Patients, # Sex, male/female Mean age, years (range) Mean age of Tx/D, months (range) Diagnosis -renal biopsy DBScreening ERT -Fabrazyme ® Mean Infusion time, min (range) Replagal ® Mean Infusion time, min (range) Mean ERT duration, months (range)

TX 16 15 /1 48 (30-62) 102 (18-210) 10 6 15 128 (40-180) 1 40 (40) 38 (18-52)

HD - PD 15 14/1 47 (26-65) 54 (12/188) 8 7 13 130 (45-240) 2 40 (40) 35 (12-56)

These accumulated clinical experiences demonstrate that ERT is safe and effective in kidney transplant recipients suffering from Fabry disease. ERT preserves renal allograft function, it improves extra-renal Fabry disease involvement and ERT also may prevent progression of severe cardiomyopathy which can be present in patients on RRT. However, studies in larger groups of patients and long-term follow-up will be required to confirm these preliminary clinical observations.

Conclusions and Future Objectives All these experiences can now definitely assess the favourable outcome of kidney transplantation in patients with Fabry disease. This result is attested by the improvement of sign and symptoms of the disease after the transplant, by the less number of neurological events, by a lower morbidity and mortality of the transplanted patients with respect the

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dialysis patients. Also the cumulative patient and graft survival of the allograft recipients with Fabry disease are encouraging being comparable to the same survival data of recipients with other nephropathies. These results solely prompt us to promote kidney transplantation in Fabry disease accelerating an early “allograft replacement therapy” in patients with Fabry nephropathy who reached the ESRD. Also in transplanted patients, the recombinant α−Gal A agalsidase is safe and effective improving or reversing signs and symptoms of Fabry disease , as demonstrated by several clinical experiences. To date, the only studies performed in Fabry allograft recipients have utilized the formulation β of agalsidase −Fabrazyme ® that has been shown to be safe and effective in ameliorating some extra renal symptoms, the Fabry cardiomyopathy and stabilizing graft function several years after the beginning of ERT. Obviously, a more substantial number of patients and a longer period of treatment are required to confirm the efficacy of the drug and to assess the real value of ERT. Nevertheless, as for other patients with Fabry disease, several issues have still to be elucidated. Even if there are no doubts that both heterozygous and hemizygous Fabry patients with renal transplant should be treated with ERT, some questions persist open. What dose should be used and what is the safest rate of infusion? Is it possible to reverse organ damage or does a “point of no return” really exist also for Fabry allograft recipients ? Will ERT completely restore organ histology and function or will it stabilize and prevent further organ deterioration ? When will gene therapy be available ? Continue research and collection of efficacy data will attribute to an increased knowledge about Fabry disease diagnosis and management with enzyme replacement therapy. An even better understanding of the specifics of the disease and its treatment will increase the likelihood that these questions will be answered .

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[20] Ojo A, Meier-Kriesche HU, Friedman G, Hanson J, Cibrik D, Leichtman A, Kaplan B. Excellent outcome of renal transplantation in patients with Fabry’s disease. Transplantation 2000; 69(11); 2337-9. [21] Mignani R, Gerra D, Maldini L, Bignardi L, Casanova S, Cambi V, Cagnoli L. Longterm survival of patients with renal transplantation in Fabry’s disease. In: Schieppati A, Daina E, Sessa A, Remuzzi G Eds: Rare Kidney Disease. Contrib Nephrol. Basel, Karger, 2001; 136, 229-33. [22] Mignani R, Panichi V, Giudicissi A, Taccola D, Boscaro F, Feletti C, Moneti G, Cagnoli L. Enzyme replacement therapy with agalsidase beta in kidney transplant patients with Fabry disease: A pilot study. Kidney Int, 2004; 65: 1381-5. [23] Lacomis D, Roeske-Anderson L, Mathie L. Neuropathy and Fabry’s disease. Muscle Nerve 2005; 31: 102-7 [24] Cable WJ, Dvorak AM, Osage JE, Kolodny EH. Fabry disease: significance of ultrastructural localisation of lipid inclusions in dermal nerves. Neurology 1982; 32:347-53. [25] Gold KF, Pastores GM, Botteman MF…….Quality of life of patients with fabry disease. Qual Life Res 2002; 11:317-27. [26] Ries M, Mengel E, Kutschke G, Kim KS, Birklein F, Krummenauer F, Beck M. Use of gabapentin to reduce chronic neuropathic pain in Fabry disease. J Inherit Metab Dis 2003; 26: 413-4. [27] Shiffmann R, Floeter MK, Dambrosia JM, Gupta S, Moore D, Sharabi Y, Khurana RK, Brady RO. Enzyme replacement therapy improves peripheral nerve and sweet function in Fabry disease. Muscle Nerve 2003; 28: 703-10. [28] Ries M, Schiffmann R. Fabry disease: angiokeratoma, biomarker and the effect of enzyme replacement therapy on kidney function. Acta Dermatol 2005; 141: 904-5. [29] Germain DP, Avan P, Chassaing A, Bonfils P. pateints affected with Fabry disease have an increased incidence of progressive hearing loss and sudden deafness : an investigation of twenty-two hemizygous male patients. BMC Med Genet 2002; 3: 10-5. [30] Hajioff D, Enever Y, Quiney R, Zuckerman J, MacDermot, Mehta A. Hearing loss in Fabry disease: the effect of Agalsidase alfa replacement therapy. J Inherit Metab Dis 2003; 26: 787-94. [31] Goodwin SE, Richfield L, Milligan A, Mehta A. gastrointestinal symptoms in patients with Fabry disease and their response to pancreatin supplements. Acta Paediatr 2003; 443:110. [32] Dehout F, Roland D, Treille de Granseigne S, Guillaume B, van Maldergem L. Relief of gastrointestinal symptoms under enzyme replacement therapy in patients with Fabry disease. J Inherit Metab Dis 2004; 27:499-505. [33] Utsumi D, Yamamoto N, Kase R. High incidence of thombosis in Fabry’s disease. Intern Med 1997; 36: 327. [34] Friedman G, Wik D, Silva L, Abdou J, Meier-Kriesche H, Kaplan B, Bonomini L, DeFranco P, Lyman N, Mulgaonkar S, Jacobs M. Allograft loss in renal transplant recipients with Fabry‘s disease and activated protein C resistance .Transplantation 2000; 69 (10): 2099-102. [35] Moore DF, Altarescu G, Herscovitch P, Schiffmann R. Enzyme replacement reverses abnormal cerebrovascular responses in Fabry disease. BCN Neurology 2002; 2: 4-11.

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[36] Jardim L, Vedolin L, Schwartz IVD, Burin MG, Cecchin C, Kalakun L, Matte U, Aesse F, Pitta-Pinheiro C, Marconato J, Giugliani R. CNS involvement in Fabry disease: clinical and imaging studies before and after 12 months of enzyme replacement therapy. J Inherit Metab Dis 2004: 27: 229-40. [37] Feligiebel A, Muller MJ, Mazanek M, Baron K, Beck M, Stoeter P. White matter lesion severity in male and female patients with Fabry disease. Neurology 2005;65: 600-2. [38] Kampmann C, Baehner F, Ries M, Beck M. Cardiac involvement in Anderson-Fabry disease. J Am Soc Nephrol 2002; 13: S147-S149. [39] Linhart A, Palcek T, Bultas J, Ferguson JJ, Karetova D, Zeman J, Ledviniva J, Poupetova H, Elleder M, Aschermann M. New insights in cardiac structural changes in patients with Fabry’s disease. Am Heart J 2000; 139: 1101-1108. [40] Goldman ME, Cantor R, Schwartz MF, Backer M, Desnick RJ: Echocardiographic abnormalities and disease severity in Fabry’s disease. J Am Coll Cardiol 1986;7:115761. [41] Weidemann F, Breuning F, Beer M, Sandstede J, Turschner O, Voelker W, Ertl G, Knoll A, Wanner C, Strottmann J. Improvement of Cardiac Function During Enzyme Replacement Therapy in Patients with Fabry Disease. A Prospective Strain Rate Imaging Study. Circulation 2003; 108: 1299-301. [42] Waldek S, Germain DP, Banikazemi M, Guffon N, Lee P, Linthorst GE, Wilcox W, Desnick RJ. Stabilization of renal function and long-term safety after enzyme replacement therapy in Fabry disease. Nephrol Dial Transplant 2003; 18 (supp 4) :630 (Abstract). [43] Maier SKG, Maass AH, Knoll A, Wanner G, Breunig F. Inconsistent electrocardiographic alterations in Fabry patients with 12 months of enzyme replacement therapy. Proc 4th European Round Table on Fabry Disease, Munich October 17-18 , 2003. [44] Oliveira JP, Valbuena C, Soares C, Bustorff M, Costa E. Renal pathology in Fabry disease: comparative analysis and clinical correlation of kidney biopsies of two nephrotic and one non-nephrotic proteinuric patients. Nephrol Dial Transplant 2004: 276 Abs) [45] Praet M, Quatacker J, Van Loo, Vanholder R, Lameire N, Ringoir S. Non-invasive diagnosis of Fabry’s disease by electronmicroscopic evaluation of urinary sediment. Nephrol Dial Transplant 1995; 10: 902-3. [46] Kawamura O, Sakuraba H, Itoh K, Suzuki Y, Doi M, Kuwabara H, Oshima S,Abe S, Warabi H, Yoshizawa N. Subclinical Fabry disease occurring in the contest of IgA nephropaty. Clin Nephrol 1997; 47(2): 71-5. [47] Rahman P, Gladman DD, Wither J, Silver MD. Coexistance of Fabry disease and systemic lupus erythematous. Clin Exp Rheumatol 1998; 16: 475-8 [48] Singh HK, Nickeleit V, Kriegsmann J, Harris A, Jennette JC, Mihatsch MJ.Coexistence of Fabry’s disease and necrotizing and crescentic glomerulonephritis.Clin Nephrol 2001;55:73-9 [49] Gubler MC, Lenoir G, Grunfeld JP, Ulmann A, Droz D, Habib R. Early renal changes in hemizygous and heterozygous patients with Fabry’s disease. Kidney Int 1978; 13 : 223-35. [50] Thurberg BL, Rennke H, Colvin RB, Dikman S, Gordon RE, Collins AB, Desnick RJ, O’Callaghan M. Globotriaosylceramide accumulation in the Fabry kidney is cleared

Renal Transplantation in Patients with Fabry’s Disease

[51] [52] [53]

[54] [55] [56] [57] [58] [59]

[60]

[61] [62]

[63]

[64]

[65]

[66] [67]

19

from multiple cell types after enzyme replacement therapy. Kidney Int 2002; 62 : 193346. Kriz W, Lemley KV. The role of podocyte in glomerulosclerosis. Curr Opin Nephrol Hypertens 1999; 8: 489-97. Farge D, Nadler S, Wolfe LS, Barre P, Jothy S. Diagnostic value of kidney biopsy in heterozygous Fabry’s disease. Arch Pathol Lab Med 1985; 109: 85-8. Tsutsumi O, Sato M, Sato K, Mizuno M Sakamoto S. Early prenatal diagnosis of inborn error of metabolism: a case report of a fetus affected with Fabry’s disease. Asia Oceania J Obstet gynaecol 1985; 11: 39-45. Alroy J, Sabnis S, Kopp JB. renal pathology in Fabry disease. J Am Soc Nephrol 2002; 13: S134-S138. Branton M, Schiffmann R, Lopp JB: Natural history and treatment of renal involvement in Fabry disease. J Am Soc Nephrol 2002; 13 (Suppl 2):S134-138. The Fabry Registry Aggregate Data Annual Report (RADAR) 2004. Genzyme corp. 2005 Brenner BM, Grunfeld JP. Renoprotection by enzyme replacement therapy. Curr Opin Nephrol Hypertens 2004; 13: 231-41. Warnock DG. Fabry disease: diagnosis and management, with emphasis on the renal manifestations. Curr Opin Nephrol Hypertens 2005; 14: 87-95 Lewis EJ, Hunsicker LG, Bain RP, Rohde RD. The effect of angiotensin-convertingenzyme inhibition on diabetic nephropathy.The collaborative study group. N Engl J Med 1993; 329;1456-62 The GISEN group: Randomised placebo-controlled trial of effect of ramipril on decline in glomerular filtration rate and risk of terminal renal failure in proteinuric, non-diabetic nephropathy. Lancet 1997; 349: 1857-6. Remuzzi G, Schieppati A, Ruggenenti P. Clinical practice. Nephropathy in patients with type 2 diabetes. N Engl J Med 2002; 346; 1145-51. Linthorst GE, Hollak CE, Korevaar JC, Van Manen JG, Aerts JM, Boeschoten EW. alpha-Galactosidase A deficiency in Dutch patients on dialysis: a critical appraisal of screening for Fabry disease.NephrolDialTransplant.2003Aug;18(8):1581-4. Kotanko P, Kramar R, Devrnja D, Paschke E, Voigtlander T, Auinger M, Pagliardini S, Spada M, Demmelbauer K, Lorenz M, Hauser AC, Kofler HJ, Lhotta K, Neyer U, Pronai W, Wallner M, Wieser C, Wiesholzer M, Zodl H, Fodinger M, SunderPlassmann G. Results of a nationwide screening for Anderson-Fabry disease among dialysis patients. J Am Soc Nephrol. 2004 ;15:1323-9. Pisani A, Spinelli L, Sabbatini M, Andreucci MV, Procaccini D, Abbaterusso C, Pasquali S, Savoldi S, Comotti C, Cianciaruso B. Enzyme replacement therapy in fabry disease patients undergoing dialysis: effects on quality of life and organ involvement. Am J Kidney Dis. 2005 ;46:120-7. Kosch M, Koch HG, Oliveira JP, Soares C, Bianco F, Breuning F, Rasmussen AK, SchaeferRM. Enzyme replacement therapy administered during hemodialysis in patients with Fabry disease.KidneyInt2004;66:1279-82. Siamopulos KC. Fabry disease: kidney involvement and enzyme replacement therapy. Kidney Int 2004; 65: 744-53 Maizel SE, Simmons RL, Kjellstrand K, Fryd DS. Ten years experience in renal transplantation for Fabry’s disease. Transplant Proc 1981; XIII: 57-9.

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[68] Helin I. Fabry’s disease. A brief review in connection with a Scandinavian survey. Scand J Urol nephrol 1979; 13: 335-7. [69] Bannwart F: Morbus Fabry: Licht- und Elektronenmikroskopischer Herzbefund12 jahre nach erfolgreicher nierentransplantation. Schweiz Med Wschr 1982 (48); 112:1742-7. [70] Shet KJ, Roth DA, Adams MB. Early renal failure in Fabry’s disease. Am J Kidney Dis 1983; 2:651-4 [71] Donati D, Navario R, Gastaldi L. Natural history and treatment of uremia secondary to Fabry’s disease: an European experience. Nephron 1987; 46: 353-9. [72] Peces R, Aguado S, Fernandez F, Gago E, Gomez E, Marin R. Renal transplantation in Fabry’s disease. Nephron 1989; 51(2):294-5. [73] Friedlaender MM, Kopolovic J, Rubinger D, Silver J, Drukker A, Ben-Gershon Z, Durst AL, Popovtzer MM. Renal biopsy in Fabry’s disease eight years after successful renal transplantation. Clin Nephrol 1987; 27(4): 206-11. [74] Erten Y, Ozdemir FN, Demirhan B, Karakayali H, Demirag A, Akkoç H. A case of Fabry’s disease with normal kidney function at 10 years after successful renal transplantation. Transplant Proc 1998; 30 (3): 842-3. [75] Clarke JTR, Guttmann RD, Wolfe LS, Beaudoin JG, Morehouse DD. Enzyme replacement therapy by renal allotransplantation in Fabry’s disease. New Engl J Med 1972;287, 1215-18 [76] Krivit W, Desnick RJ, Bernloko RW, Wold F. Enzyme transplantation in Fabry’s disease. N Engl J Med 1972; 287:1248-9. [77] Desnick RJ, Simmons RL, Allen KKY, Woods JE, Anderson CF, Najarian JS, Krivit W. Correction of enzymatic deficiencies by renal transplantation: Fabry’s disease . Surgery 1972; 72: 203-211 [78] Philippart M, Franklin SS, Gordon A. Reversal of an inborn sphingolipidosis (Fabry’s disease) by kidney transplantation. Ann Intern Med 1972; 77: 195-200. [79] Spence MW, MacKinnon KE, Burgess JK, d’Entremont DM, Belitsky P, Lennon SG, McDonald AS. Failure to correct the metabolic defect by renal transplantation in Fabry’s disease. Ann Int Med 1976; 84:13-6 [80] van den Bergh FA, Rietra PJ, Kolk Vegter AJ, Bosh E, Tager JM. Therapeutic implications of renal transplantation in a patient with Fabry’s disease. Acta Med Scand 1976; 200: 249-56. [81] Wuthrich RP, Weinreich T, Binswanger U, Gloor HJ, Candinas D, Hailemariam S. Should living related kidney transplantation be considered for patients with renal failure due to Fabry’s disease? Nephrol Dial Transplant 1998; 13: 2934-2936 [82] Desnick RJ, Banikazemi M, Wasserstein M: Enzyme replacement therapy in Fabry’s disease, an inherited nephropathy. Clin Nephrol 2002; 57(1):1-8. [83] Kramer W, Thormann J, Mueller K, Frenzeel H: Progressive cardiac involvement by Fabry’s disease despite successful renal allotransplantation. Int J Cardiol 1985; 7 (1):72-5. [84] Donati D, Sabattini MG, Capsoni F, Baratelli L, Cassani D, De Mario A, Martegani M, Gastaldi L. Immune function and renal transplantation in Fabry’s disease. Proc Eur Dial Transplant Ass 1984;21: 686-92. [85] Mignani R, Cagnoli L. Enzyme replacement therapy in Fabry’s disease: recent advances and clinical applications. J Nephrol 2004; 17 (3): 354-63.

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[86] Faraggiana T, Churg J, Grishman E, Stauss L, Prado A, Bishop DF, Schuchman E, Desnick RJ. Light- and electronmicroscopic histochemistry of Fabry’s disease. Am J Pathol 1981; 103: 247-262. [87] MacMahon J, Tubbs R, Gephhardt G, Steinmuller D. Pseudorecurrence of Fabry’s disease in renal allograft., Lab Invest 1986;54: 42A (Abs). [88] Mosnier JF, Degott C, Bedrossian J, Molas G, Degos F, Pruna A, Potet F. Recurrence of Fabry’s disease in a renal allograft eleven years after successful renal transplantation. Transplantation 1991; 51: 759-762. [89] Gantenbein H, Bruder E, Burger HR, Briner J, Binswanger U. Recurrence of Fabry’s disease in a renal allograft 14 years after transplantation. Nephrol Dial Transplant 1995; 10: 287-289. [90] Buhler FR, Thiel G, Dubach UC, Enderlin F, Gloor F, Tholen M. Kidney transplantation in Fabry’s disease. Br Med J 1973; ii: 28-9. [91] Chatterjee S, Gupta P, Pyeritz RE, Kwiterovich PO. Immunohistochemical localization of glycosphingolipid in urinary renal tubular cells in Fabry’s disease. Am J Clin Pathol 1984; 82: 24-8 [92] Peces R. Is there true recurrence of Fabry’s disease in the transplanted kidney? Nephrol Dial Transplant 1996; 11 (suppl 3): 561. [93] Grunfeld JP, Le Porrier M, Droz D, Bensaude I, Hinglais N,Crosnier J. La transplantation renale chez les subjets atteints de maladie de Fabry Transplantation du rein d’un sujet heterozygote a un sujet sain. Nouv Presse Med 1975;4: 2081-5. [94] Shweitzer BJ, Grachenberg CB, Bartlett ST. Living kidney donor and recipient evaluation in Fabry’s disease. Transplantation 1992; 54:924-6 [95] Puliyanda DP, Wilcox WR, Bunnapradist S, Nast CC, Jordan SC. Fabry disease in a renal allograft. Am J transplant 2003; 3: 1030-2. [96] Grunfeld JP, Lidove O, Joly D, Barbey F. Renal disease in Fabry patients. J Inherit Metab Dis 2001; 24: 71-4 [97] Desnick RJ, Dean KJ, Grabowski G, Bishop DF, Sweeley CC. Enzyme therapy in Fabry disease: differential plasma clearance and metabolic effectiveness of plasma and splenic -galactosidase A isozymes. Proc Natl Acad Sci USA 1979; 76: 5326-5330. [98] Frustaci A, Chimenti C, Ricci R, Natale L, Russo A, Pieroni M, Eng CM, Desnick RJ Improvement in cardiac function in the cardiac variant of Fabry’s disease with galactose-infusion therapy. New Engl J Med 2001; 345: 25-32 [99] Ioannou YA, Zeidner KM, Gordon RE, Desnick RJ. Fabry disease: preclinical studies demonstrate the effectiveness of alpha-galactosidase A replacement in enzymedeficient mice. Am J Hum genet 2001; 69 (1): 14-25 [100] Schiffmann R, Kopp JB, Austin HA, Sabnis S, Moore DF, Weibel T, Balow JE, Brady RO. Enzyme replacement therapy in Fabry’s disease. A randomized controlled trial. JAMA 2001;285(21):2743-9. [101] Eng CM, Guffon N, Wilcox WR, German DP, Lee P, Waldek S, Caplan L, Linthorst GE, Desnick RJ. Safety and efficacy of recombinant human -galacosidase A replacement therapy in Fabry’s disease. New Engl J Med 2001;345(1): 9-16. [102] Breunig F, Knoll A, Weidemann F, Strotmann J, Wanner C Effect of enzyme replacement therapy on clinical outcome in patients with fabry disease. Nephrol Dial Transplant 2005;20(5) :246 (abstract).

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[103] De Schoenemakere G, Chauveau D, Grunfeld JP. Enzyme replacement therapy in Anderson-Fabry’s disease: beneficial clinical effect on vital organ function. Nephrol Dial Transplant 2003; 18 (1): 33-5. [104] Wilcox WR, Banikazemi M, Guffon N, Waldek S, Lee P, Linthorst G, Desnick RJ, Germain DP. Long-term safety anf efficacy of enzyme replacement therapy for Fabry disease. Am J Hum Gen 2004; 75: 65-74. [105] Beck M, Ricci R, Widmer U, Dehout F, Garcia de Lorenzo A, Kampman C, Linhart A, Sunder-Plassmann G, Houge G, Ramaswami U, Gal A, Metha A. Fabry disease: overall effects of agalsidase alfa treatment. Eur J Clin Invest 2004 ; 34: 838-44. [106] Schiffmann R, Ries M, Timmons M, Flaherty JT, Brady RO. Long-term therapy with Agalsidase alfa for Fabry disease: safety and effects on renal function in a home infusion setting. Nephrol Dial Transplant 2005; : . [107] Schwarting A, Dehout F, Feriozzi S, Beck M, Mehta A, Widmer U, Sunder-Plassman G. Agalsidase alfa prevents the declinine in renal function in patients with Fabry disease. Nephrol Dial Transplant 2005; 20(5): 248 (Abstract). [108] Boscaro F., Pieraccini G., La Marca G., Bartolucci G., Luceri C., Luceri F., Moneti G.Rapid quantification of globotriaosylceramide in human plasma and urine: a potential application for monitoring enzyme replacement therapy in Anderson-Fabry disease. Rapid Comm. Mass Spectrom. 2002; 16(16): 1507-14. [109] Mignani R, Panichi V, Martinelli F, Giudicissi A, Pisani A, Barone R, Moneti G, Taccola D, Cianciaruso B, Puliatti C, Feletti C, Cagnoli L. Enzyme replacement therapy with agalsidase in Fabry’s disease patients with kidney transplantation: results after 1 year of treatment. Nephrol Dial Transplant 2003; 18 (suppl 4): 633 (Abstract). [110] Mignani R, Feriozzi S, Abaterusso C, Barbieri A, Bianco F, Cioni A, Comotti C, Cossu M, Demaria R, Di Vito R, Giudicissi A, Gotti E, Martinelli F, Panichi V, Pisani A, Ragazzoni, Ricci R, Savoldi S, Soliani F, Testi G, Cianciaruso B, Cagnoli L. Effects of ERT on patients with Fabry disease on RRT: A national survey program in Italy. In press.

In: Progress in Kidney Transplantation Editor: Dominick W. Mancuso, pp. 23-35

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter II

Can Estimated Renal Graft Function Serve as a Valid Endpoint in Clinical Trials? Christophe Mariat*, Eric Alamartine and François Berthoux Service de Néphrologie, Dialyse et Transplantation Rénale, et Laboratoire d’Explorations Fonctionnelles Rénales Hôpital Nord, Saint-Etienne, France

Abstract Chronic allograft dysfunction is at present, one of the main challenges in kidney transplantation, and many therapeutic strategies are emerging in order to improve longterm graft survival. In this respect, short-term endpoints commonly used in clinical trials progressively become meaningless and new surrogate markers of long-term outcome are warranted. Among them, the evaluation of graft function has recently received great attention. Renal function is at best described by the glomerular filtration rate (GFR) and numerous mathematic equations are routinely employed to estimate the GFR. However, several recent reports have strongly challenged the performance of these equations when applied to transplant patients, and thereby have raised some major concerns regarding their widespread use as a suitable tool to determine graft function in clinical trials. Herein, we review the recent studies that have lead to question the validity of these equations. We then, emphasize the potential dangers of predicting renal graft function by using these equations. Finally, we discuss what should currently be the standards for GFR evaluation in kidney transplantation trials and, what could be the possible future alternatives.

*

Service de Néphrologie, Dialyse et Transplantation Rénale, et Laboratoire d’Explorations Fonctionnelles Rénales. Hôpital Nord, CHU de Saint-Etienne, 42000, Saint-Etienne, France. [email protected]

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Introduction Over the past two decades, substantial improvements in short-term kidney transplant outcome have greatly limited our ability to assess the efficacy of newer therapeutic strategies according to conventional short-term end-points, namely the one-year graft/patient survival and the one-year acute rejection rate. For instance, with current figures of less than 15% of patients experiencing an acute rejection episode over the first year post-transplant, it has become logistically challenging if not clinically questionable, to test a new treatment on its ability to further prevent acute rejection (table 1). Meanwhile, these traditional end-points have failed to predict long-term survival, which is at present, a major challenge in renal transplantation. Identification of new, short-term surrogate markers capable to correlate with long-term graft outcome is thus necessary and should allow testing new immunosuppressive agents according to more meaningful criteria. Among other candidates, post-transplant graft function seems to be a particularly attractive surrogate marker for clinical trials, either used alone or incorporated as part of a composite end-point. [1-5]. Table 1. Fictional examples of the number of patients for each group that would be required in clinical trials using conventional short-term endpoints (two-sided test, type I error or risk α of 5%). POWER Improvement in 1 year-acute rejection rate from 15% to Improvement in 1 year-graft survival from 90% to

10% 7% 95% 98%

80% 683 236 432 134

90% 914 316 578 180

In many recent trials, comparison of renal graft function is already largely reported as the primary criteria for the evaluation of alternative immunosuppressive strategy [6, 7]. Obviously, before qualifying graft function as a valid surrogate marker, one must first make sure to use a valid measure of renal function. There are a number of ways to determine renal function. The glomerular filtration rate (GFR) is considered as the best overall index of renal function. Exact quantitative measurement of GFR requires the determination of renal clearance of a marker freely filtered by the kidney without undergoing any metabolism, tubular secretion or reabsorption and thus, rapidly secreted in the urine by glomerular filtration only. Inulin or synthetic inulin-like polyfructosans fulfill these criteria and since its introduction in 1935, inulin clearance has remained the gold standard for the measurement of GFR[8]. More precisely, the undisputed reference method is the renal clearance of inulin determined by the continuous infusion technique and with urine collected after bladder catheterization (in order to avoid any error due to incomplete emptying of the bladder). Other exogenous markers have been advocated for a direct GFR determination such as radiolabeled isotopes (51Cr EDTA, 99mTc DTPA or 125I Iothalamate) and non-radioactive contrast agents (Iothalamate or Iohexol). They are traditionally also used as reference methods of GFR measurement. Unfortunately, all these methods, including the inulin clearance are at different degrees, expensive, time-consuming and cumbersome and

Can Estimated Renal Graft Function Serve as a Valid Endpoint in Clinical Trials?

25

consequently not easily implementable in clinical trials. As an alternative, a number of easyto-use mathematical equations incorporating different anthropometrical variables in addition to biological parameters have been developed to predict, rather than directly measure GFR. These GFR predicting equations have been widely used to provide a bedside assessment of renal function, and tend now, to literally substitute for the aforementioned reference methods to evaluate renal graft function in clinical research. However, data have recently accumulated questioning the performance of these equations in renal graft function assessment, thereby suggesting that the unrestricted use of these equations in clinical trials might lead to flawed conclusions.

GFR Predicting Equations: Potential Candidates for Assessing Renal Graft Function A great number of mathematical equations have been developed over years in order to provide physicians with the best GFR estimate possible. More recently, several equations have been directly tested in renal transplant patients, and four of them eventually seem to emerge as the most deserving candidates, either because they do show a better predictive performance as compared to the others or alternately just because they have been consecrated by a more or less long usage. These four equations are: The Cockcroft-Gault formula [9] [(140 - age (years) ) X weight (kg) / (0.814 X serum creatinine (µmol/l) )] X 0.85 for women The Walser equation [10] For men: 7.57 X (serum creatinine (mmol/l) ) -1 - 0.08 X age (years) + 0.096 X weight (kg) 6.66 For women: 6.05 X (serum creatinine (mmol/l) ) -1 - 0.08 X age (years) + 0.08 X weight (kg) 4.81 The Nankivell equation [11] 6.7/(serum creatinine (mmol/l) X 0.0884) + 0.25 X weight 100/height (m) 2 + 35 (25 for women)

(kg)

- 0.5 X urea

(mmol/l)

-

The MDRD-study equations [12, 13] Equation 6: 198 X (serum creatinine (mg/dl) ) -0.858 X (age (years) ) -0.167 x (0.822 if patients is female) X (1.178 if patients is black) X (serum urea nitrogen concentration (mg/dl) ) -0.293 X (urine urea nitrogen excretion (g/day) ) 0.249 Equation 7: 170 X (serum creatinine (mg/dl) ) -0.999 X (age (years) ) -0.167 x (0.762 if patients is female) X (1.18 if patients is black) X (serum urea nitrogen concentration (mg/dl) ) -0.170 X (serum albumin concentration (g/dl) ) 0.318

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Christophe Mariat, Eric Alamartine and François Berthoux

Abbreviated Equation: 186 X (serum creatinine (mg/dl) ) -1.154 X (age (years) ) -0.203 x (0.742 if patients is female) X (1.21 if patients is black) Cockcroft and Gault first published their equation in 1976. This formula has been developed taking into account the relationship found between age and 24-hour creatinine excretion/kg in 236 adult patients, mainly males and aged from 18 to 92. In the original study, the Cockcroft-Gault formula was validated against measured creatinine clearance and was picked among other equations due to a better correlation with measured creatinine clearance (mean correlation coefficient of 0.83). Stated otherwise, the most popular GFR estimating formula which is still probably the most widely used GFR test in the clinical as well as in the research setting, only gives an indirect prediction of GFR. The validity of the Cockcroft and Gault formula has been extensively evaluated in different patient populations. However, until recently very few studies have specifically assessed its ability to assess renal graft function[14]. The Walser equation has been first published in 1993 and similarly to Cockcroft and Gault formula, includes only age, serum creatinine and weight, making it very attractive for a beside estimate. Unlike Cockcroft and Gault formula that gives an estimate of creatinine clearance, which in turn gives an approximation of GFR, the Walser equation provides a direct prediction of GFR. Indeed, this equation was developed in comparison to urinary clearance of 99mTc DTPA from a cohort of 85 patients with moderate to severe chronic renal failure (serum creatinine concentration ≥ 177 µmol/l, mean measured GFR of 13 ml/min). Note that in the original publication, the measured GFR was normalized not to 1.73 m2 of body surface area but to 3 m2 of height2. The Nankivell formula is the only one that has been computed from a renal transplant population (against a direct measure of GFR by plasma 99mTc DTPA clearance). For this reason, this equation has always been seen as more appropriate than any other one for assessing renal graft function and has been de facto deemed qualified for clinical research. As a matter of fact, the Nankivell formula was integrated in methods of large international trial, long before the first studies trying to confirm the initial promising data came up. Levey and colleagues derived different predictive equations from the 1628 patients included in the modification of diet in renal disease (MDRD) Study and undergoing renal clearance of 125I Iothalamate. Since their publication in 1999, the MDRD equations have been hyped as somewhat a new standard in GFR prediction and a considerable number of studies have looked at the added value these equations may actually provide[15-21].

How is Predictive Performance Evaluated? How Should it be? The methodology used to assess the performance of a given GFR predicting equation is far from being standardized and remains quite heterogeneous across studies. This severely limits the ability to compare the results and obviously contributes to blur the picture about the real value of the prediction. In addition, statistical methods used to analyze the equations' validity may sometimes be inappropriate from the standpoint of clinical research. In this particular situation, the key question that should be addressed is whether estimated GFR can safely substitute for GFR measured by reference methods.

Can Estimated Renal Graft Function Serve as a Valid Endpoint in Clinical Trials?

27

Correlation analysis is largely used in studies testing a predictive equation against a reference method and is certainly the most accessible criteria of performance. The correlation coefficient r, measures the strength of the relationship between predicted and true GFR and more precisely, analysis of r2 indicates how much variability of predicted GFR account for variability of measured GFR. However, a significant correlation only means that the null hypothesis of no relationship between the GFR predicting equation and the reference method can be rejected. We clearly need more information to assess performance of a test predicting GFR. In 2002, the National Kidney Foundation released clinical guidelines on the evaluation of Chronic Kidney Disease[13]. In this regard, was proposed a methodological framework to evaluate GFR predicting equations according to Bias, Precision and Accuracy. Bias expresses the systematic deviation from the gold standard measure of GFR (true GFR) and is given by the difference between the true and predicted values of GFR (absolute bias). The difference from the gold standard can also be expressed as a relative difference, e.g., percent difference from the measured GFR (relative bias). This has the advantage of allowing a more valuable evaluation throughout the whole spectrum of kidney function. Clinically this is relevant, as there is less concern about the difference between 100 and 130 mL/min/1.73 m2 than between 30 and 60 mL/min/1.73 m2. Precision expresses the variability (or dispersion) of prediction equation estimates around the gold standard GFR measure and corresponds to the standard deviation of the difference between the true and estimated GFR. Accuracy combines precision and bias and is measured by the proportion of estimates falling within a certain percent of the true GFR (for example, 30%-accuracy is the proportion of predicted GFR within ± 30% of the true GFR). Bias, Precision and Accuracy, as defined by the National Kidney Foundation are simple and reproducible criteria. They seem to be appropriate to give a fair picture of the predictive performance and, naturally tend to be more and more used in studies looking at the validity of GFR estimating equations. Agreement, which is best evaluated by the Bland and Altman method, should definitely be part of a standardized evaluation of the predictive performance, too [22]. Indeed, agreement directly indicates how well the equation will substitute for the reference standard itself. Thereby, it's probably the most informative way to describe the predictive performance and to make sure that the equation may safely replace the reference method in the specific setting of clinical trials.

Performance of Cockcroft-Gault, Walser, Nankivell and MDRD Equations in Predicting Renal Graft Function We analyzed the predictive performance of several equations from a cohort of 294 renal transplant patients for whom 500 inulin clearance measurements were available [23]. Cockcroft-Gault, Walser, Nankivell and MDRD equations were found to closely correlate with inulin clearances (r > 0.65 and P<0.0001, for each of them). However, all of them displayed considerable lack of agreement with limits of agreement over 40 ml/min/1.73m2

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apart. In this line, at least 70% of estimated GFR were found to differ from inulin clearance by ± 10%, 43% by ± 20%. Overall, none of these equations turned out to demonstrate a level of agreement with inulin clearance compatible with clinical research requirements. This is particularly true for clinical trials investigating a therapeutic strategy designed to improve graft function and which aim to compare at one point the mean predicted GFR between two groups. In such trials, it may appear relevant to detect an improvement of 10 ml/min in GFR. It is clearly not if GFR is estimated with one of these equations, since more than one third of the predicted values spontaneously differ from true GFR by more than 10 ml/min. Likewise, testing an hypothesis of superiority of 20% ("mean GFR under treatment B of 72 ml/min against 60 ml/min under treatment A", for instance) can seem reasonable; not by using an equation that falsely gives a value beyond this threshold for 43% of the estimated GFR. In our cohort, the MDRD and Walser equations exhibited better accuracy than CockcroftGault and Nankivell equations. The superiority of the MDRD equations over at least the Cockroft-Gault formula is kind of consistently found in recent studies[24-27](tables 2 and 3). Similarly, we and others have reported a better predictive performance of the Walser equation[23-25, 28]. However, the level of agreement with a reference method for both MDRD and Walser equations still fall far short of what should be required within the context of a clinical trial. Table 2. Comparison across studies of the 10% Accuracy of MDR, Walser, Cockcroft-Gault, and Nankivell equations in renal transplant patients

MDRD ∗ Walser Cockcroft Nankivell

Mariat C. et al. (1) Kidney Int.2004 30% 28% 24% 23%

Gaspati F. et al. (2) Am J Transplant.2004 44% 46% 31% 27%

Poge U. et al. (3) Am J Transplant.2005 25% NA 9.5% NA

Bosma R. et al. (4) Am J Transplant.2005 38% NA 36% 35%

The 10% Accuracy is defined as the proportion of predicted GFR falling within 10% of the true GFR. the Equation 7 was used by Mariat C et al., the abbreviated version by the others. (1) cohort of 294 patients; renal inulin clearance used as the reference method of GFR measurement (mean measured GFR = 49 ml/min/1.73m2; range: 8 - 122 ml/min/1.73m2) (2) cohort of 81 patients; plasma iohexol clearance used as the reference method of GFR measurement (mean measured GFR = 56.1 ml/min/1.73m2; range: 21.8 - 86.1 ml/min/1.73m2) (3) cohort of 95 patients; plasma clearance of 99m Tc-DTPA used as the reference method of GFR measurement (mean measured GFR = 36 ml/min/1.73m2; range: 11.8 - 71.0 ml/min/1.73m2) (4) cohort of 798 patients; renal clearance of 125 I-iothalamate used as the reference method of GFR measurement (mean measured GFR = 55 ml/min/1.73m2; range: 18 - 115 ml/min/1.73m2) NA: Not available

Surprisingly, Nankivell equation is never found to display a better GFR estimation and seems to provide an even worst prediction than Cockcroft-Gault formula in renal transplant patients. There is no clear explanation regarding the rather bad performance of the Nankivell equation, but this finding, that consistently comes up from one study to another, does not plead for the use of this equation to assess renal graft function, especially in clinical trials.

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Table 3. Comparison across studies of the 20% Accuracy of MDR, Walser, CockcroftGault, and Nankivell equations in renal transplant patients

MDRD ∗ Walser Cockcroft Nankivell

Mariat C. et al. (1) Kidney Int.2004 57% 53% 46% 43%

Gaspati F. et al. (2) Am J Transplant.2004 76% 80% 57% 50%

The 20% Accuracy is defined as the proportion of predicted GFR falling within 20% of the true GFR. the Equation 7 was used by Mariat C et al., the abbreviated version by Gaspari F et al. (1) cohort of 294 patients; renal inulin clearance used as the reference method of GFR measurement (mean measured GFR = 49 ml/min/1.73m2; range: 8 - 122 ml/min/1.73m2) (2) cohort of 81 patients; plasma iohexol clearance used as the reference method of GFR measurement (mean measured GFR = 56.1 ml/min/1.73m2; range: 21.8 - 86.1 ml/min/1.73m2)

In order to promote further methodological homogeneity, we analyzed our data through the analytical framework set up by the National Kidney Foundation in the 2002 K/DOQI recommendations[29]. These guidelines aimed to standardize the evaluation and classification of kidney disease in non-transplant patients and have defined a unifying terminology, the chronic kidney disease (CKD), based on the presence of kidney damage and/or impairment of kidney function, irrespective of diagnosis. Additionally, they have adopted a classification into five stages of CKD according to the level of renal function and have recommended for the evaluation of GFR to preferentially use the MDRD Study and Cockcroft-Gault equations. These recommendations stem from a systematic analysis of the performance of MDRD and Cockcroft-Gault equations based on their respective Bias, Precision and Accuracy in nontransplant patients. By using the very same analytical methodology, we sought to compare the validity of MDRD equations (equation 7 and the abbreviated MDRD equation) and Cockcroft-Gault formula in transplant patients and found the performance in predicting renal graft function of these equations was severely impaired. When considering the prediction in term of accuracy, the performance of MDRD equation 7, its abbreviated version and Cockcroft-Gault formula proved particularly poor. For the MDRD Study equations, at least 25% of the calculated GFR were found to give a prediction beyond 30% of the corresponding inulin clearance value. The figure was even worse for Cockcroft-Gault equation since only 59% of the estimates fell within 30% of the measured GFR. In comparison, the K/DOQI guidelines base their recommendation in non-transplant patient, on a 30%-accuracy of 75 and 90% for Cockcroft-Gault and MDRD equations, respectively. Importantly, the three estimates recommended by the K/DOQI guidelines in non-transplant patients, failed to efficiently categorize the transplant patients into the different stages of CKD as defined by the same guidelines. Overall, in our study population, less than two third of the transplant patients would have been well classified, and so, whatever the GFR estimates considered. Interestingly, when comparing the predictive performance of the three GFR estimates to each other, we still found that MDRD equations perform better than Cockcroft-Gault equation. But here again, their level of performance was found to be insufficient, at least far below the K/DOQI standards.

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Is There a Real Danger of Predicting Rather than Measuring Graft Function in Kidney Transplantation Trials? At present, the usual way to look at how a therapy may impact on renal graft function is to compare the mean GFR between two groups of patients. Intuitively, given the poor accuracy of all the equations, one may think that such a strategy is risky and potentially misleading. To directly address this issue, we performed several simulations from our cohort of 500 inulin clearances. This whole population was split into two groups of n=250 each, either according to the date of transplantation (sort by chronological order, simulation #1) or according to the recipient's age (sort by increasing age, simulation #2). For simulation #1, the mean true GFR significantly differed between the two groups, and so did the mean GFR estimated by MDRD equation. However, differences were no longer significant when Cockcroft-Gault, Nankivell or Walser equations estimated the GFR. In other words, had been these equations used in an hypothetical trial reproducing these conditions, the real beneficial effect of the tested therapy might have been missed (table 4). Conversely, in simulation #2, the mean true GFR was not different between the two groups, and neither was the mean estimate given by Nankivell and MDRD equations. But here, Cockcroft-Gault and Walser did give a significant difference between the two groups (table 5). In keeping with this finding, a recent trial has been reported on the effect of a new CTLA4 fusion protein (belatacept) in renal transplantation[7]. Among the secondary endpoints, the effect on the GFR at one year post-transplant has been analyzed. Interestingly, while belatacept was found to have a beneficial effect when GFR was measured by iohexol clearance (66,3 vs 53,5 ml/min/1,73m2 for the belatacept-based treatment group and the cyclosoprine A-based treatment group, respectively), no significant difference was reported between the two groups when GFR was estimated by MDRD equation (72,4 vs 68,0 ml/min/1,73m2 for belatacept and cyclosoprine A group, respectively). Table 4. Comparison of mean GFR between two fictional groups of transplant patients (simulation #1)

Inulin Clearance MDR Walser Cockcroft Nankivell

Group A mean ± standard deviation 45 ± 16 ml/min/1.73m2 47 ± 17 ml/min/1.73m2 43 ± 14 ml/min/1.73m2 52 ± 17 ml/min/1.73m2 56 ± 16 ml/min/1.73m2

Group B mean ± standard deviation 53 ± 22 ml/min/1.73m2 52 ± 22 ml/min/1.73m2 45 ± 17 ml/min/1.73m2 54 ± 20 ml/min/1.73m2 58 ± 20 ml/min/1.73m2

P-value (t-test) < 0.0001 = 0.014 = 0.092 = 0.125 = 0.067

An historic cohort of 500 inulin clearances (true GFR) is split into two groups of 250 clearances each, according to the date of transplantation. The difference in mean GFR (either the true GFR or the GFR estimated by different equations) between the two groups is evaluated using a Student t-test.

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Table 5. Comparison of mean GFR between two fictional groups of transplant patients (simulation #2)

Inulin Clearance MDR Walser Cockcroft Nankivell

Group A mean ± standard deviation 51 ± 19 ml/min/1.73m2 50 ± 19 ml/min/1.73m2 45 ± 15 ml/min/1.73m2 57 ± 19 ml/min/1.73m2 57 ± 16 ml/min/1.73m2

Group B mean ± standard deviation 47 ± 21 ml/min/1.73m2 49 ± 21 ml/min/1.73m2 43 ± 16 ml/min/1.73m2 48 ± 18 ml/min/1.73m2 57 ± 19 ml/min/1.73m2

P-value (t-test) =0.119 = 0.668 = 0.059 < 0.0001 = 0.978

An historic cohort of 500 inulin clearances (true GFR) is split into two groups of 250 clearances each, according to the recipient's age. The difference in mean GFR (either the true GFR or the GFR estimated by different equations) between the two groups is evaluated using a Student t-test.

Table 6. Comparison of the proportion of patients with a GFR ≥ 60 ml/min/1.73m2 in two fictional groups of transplant patients (simulation #1)

Inulin Clearance MDR Walser Cockcroft Nankivell

Group A 16% 21% 10% 28% 38%

Group B 29% 30% 14% 36% 45%

P-value (Chi 2-test) = 0.001 = 0.019 = 0.172 = 0.057 = 0.124

An historic cohort of 500 inulin clearances (true GFR) is split into two groups of 250 clearances each, according to the date of transplantation. The proportion of patients with a GFR (either the true GFR or a GFR estimated by different equations) beyond 60 ml/min/1.73m2 is compared between the two groups using a Chi 2-test.

Table 7. Comparison of the proportion of patients with a GFR ≥ 60 ml/min/1.73m2 in two fictional groups of transplant patients (simulation #2)

Inulin Clearance MDR Walser Cockcroft Nankivell

Group A 27% 26% 14% 41% 35%

Group B 22% 25% 12% 23% 38%

P-value (Chi 2-test) = 0.215 = 0.918 = 0.500 < 0.01 = 0.516

An historic cohort of 500 inulin clearances (true GFR) is split into two groups of 250 clearances each, according to the recipient's age. The proportion of patients with a GFR (either the true GFR or a GFR estimated by different equations) beyond 60 ml/min/1.73m2 is compared between the two groups using a Chi 2-test.

Finally, comparing mean GFR is tantamount to look for small differences that obviously are not reliably detected by the different predictive equations. Instead, one may think that it could be easier to compare proportion of patients in each group with a GFR beyond a certain

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threshold (in other words, to turn a quantitative continuous variable into a nominal dichotomous one)? When applied to our simulations, this option did not seem to provide great advantages over a classical comparison of means and did not prevent misleading conclusions (tables 6 and 7).

Are We Doomed to Exclusively Use Reference Methods to Assess Renal Graft Function in Clinical Trials? Despite the considerable number of formulas that have been developed over years, it seems that none of them (including the ones that are currently used in clinical trials) can boast an acceptable predictive performance to substitute for a reference method of direct GFR measurement, at least, in situations where an accurate assessment of renal function is required. Therefore, one may ask whether there is still some room to design an n-th equation. To answer this question we may need to first figure out why GFR prediction is so challenging in transplantation. There are probably several potential reasons. First, the majority of these equations has been initially developed from non-transplant patients and doesn’t include many factors specific to transplant recipients that may impact on their predictive performance. For instance, variables such as number of acute rejections, length of time spent on dialysis or cumulative steroids dose have been shown to be predictors of muscle mass index in transplant recipients, independently of body weight and other parameters usually included in the GFR estimates[11]. Second, the nephron mass transplanted to the recipient is never taken into account and yet, is very likely to directly influence the GFR measured after transplantation. So far, even in the equations specifically derived from a transplant cohort, the only anthropometrical indices that are computed come from the recipients. Since the renal mass is certainly correlated to the body habitus, some corrective variables relative to the donor characteristics might be required to enhance the performance of these estimates in the transplant setting. In this respect, it could be worth trying to compute a new, transplantationspecific GFR predicting equation incorporating some donor parameters. However, one has to keep in mind that other limiting factors exist and further jeopardize the reliability of these equations when applied not only to transplant patients but also to the general population. Among them, the choice and calibration of the assay used to determine the serum creatinine concentration has recently been pinpointed as a potential misleading factor[30, 31]. Similarly, patients’ demographics can affect the equation validity, and to some extent the predictive performance is closely dependent on the initial study population characteristics. For example, while the MDRD Study Equations have been developed and validated in a population exhibiting a mean GFR of 39.8 ml/min/1.73, several reports have found that their performance can be dramatically hampered in individuals with mild decrease in kidney function or normal GFR[21]. One alternative to this so far elusive " ideal equation" would be to improve the predictive performance of the existing equations. In fact, some attempts have already been made by

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trying to block the tubular secretion of creatinine with an oral administration of cimetidine. Indeed, the tubular secretion of creatinine increases with the degree of renal impairment and is likely to account at least partially for the overestimation of the prediction consistently observed with the equations. Many investigators have reported that the predictive performance of cimetidine-corrected creatinine clearance and cimetidine-corrected CockcroftGault formula substantially improved in patients with chronic kidney disease[32-34]. Recently, Kemperman and colleagues concluded that in renal allograft recipients, accuracy of Cockcroft-Gault formula 24 hours after 2400 mg of cimetidine was also significantly enhanced and proposed its use for routine follow-up of transplant patients[35]. In this line, the influence of oral cimetidine on the performance of naturally better predictor as Walser and MDRD equations, has to be definitely investigated. Finally, in clinical trials, we may still need the reference methods of GFR measurement for some time. Among all the methods available, the plasma clearance of iohexol, a non-ionic, low-osmolality radio contrast agent, appears particularly suitable and could be recommended as a high quality standard[36-39]. The renal clearance of iohexol has shown virtually identical results with renal clearance of inulin when a constant infusion technique is used. The plasma clearance of iohexol, following a bolus injection of the marker is well characterized and offers a valid alternative to the renal clearance. The clearance can be calculated from the plasma disappearance rate and the plasma concentration. The plasma clearance can, in a simple way be determined with only a few blood samples, drawn in the mono-exponential part of the plasma disappearance curve between 120 and 240 minutes (up to 600 minutes if the expected GFR is below 40 mL/min) according to a one-compartment model (corrected with the Brochner-Mortensen formula). This method has been validated against inulin clearance, is now well standardized and has already been used in clinical trials[7].

Conclusion Renal graft function is now a key criteria to evaluate new therapeutic strategies in kidney transplantation. Using a precise and reliable method to measure an endpoint in clinical trials is critical in order to avoid flawed and misleading interpretation. In this context, the GFR predicting equations do not guarantee an accurate assessment of renal graft function. The recent literature on kidney transplantation does not support the widespread use of any of these equations for clinical research. At present, reference methods of direct GFR measurement should be systematically considered while designing kidney transplantation trials.

References [1] [2]

Hariharan, S., et al., Surrogate markers for long-term renal allograft survival. Am J Transplant, 2004. 4(7): p. 1179-83. Kaplan, B., J. Schold, and H.U. Meier-Kriesche, Poor predictive value of serum creatinine for renal allograft loss. Am J Transplant, 2003. 3(12): p. 1560-5.

34 [3] [4]

[5] [6] [7] [8] [9] [10]

[11] [12]

[13] [14] [15] [16]

[17]

[18]

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[21]

Christophe Mariat, Eric Alamartine and François Berthoux Hariharan, S., M.A. McBride, and E.P. Cohen, Evolution of endpoints for renal transplant outcome. Am J Transplant, 2003. 3(8): p. 933-41. Lachenbruch, P.A., et al., Biomarkers and surrogate endpoints in renal transplantation: present status and considerations for clinical trial design. Am J Transplant, 2004. 4(4): p. 451-7. Meyers, C.M. and A.D. Kirk, Workshop on late renal allograft dysfunction. Am J Transplant, 2005. 5(7): p. 1600-5. Pascual, M., et al., Strategies to improve long-term outcomes after renal transplantation. N Engl J Med, 2002. 346(8): p. 580-90. Vincenti, F., et al., Costimulation blockade with belatacept in renal transplantation. N Engl J Med, 2005. 353(8): p. 770-81. Gaspari, F., N. Perico, and G. Remuzzi, Measurement of glomerular filtration rate. Kidney Int Suppl, 1997. 63: p. S151-4. Cockcroft, D.W. and M.H. Gault, Prediction of creatinine clearance from serum creatinine. Nephron, 1976. 16(1): p. 31-41. Walser, M., H.H. Drew, and J.L. Guldan, Prediction of glomerular filtration rate from serum creatinine concentration in advanced chronic renal failure. Kidney Int, 1993. 44(5): p. 1145-8. Nankivell, B.J., et al., Predicting glomerular filtration rate after kidney transplantation. Transplantation, 1995. 59(12): p. 1683-9. Levey, A.S., et al., A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med, 1999. 130(6): p. 461-70. K/DOQI clinical practice guidelines for chronic kidney disease: evaluation, classification, and stratification. Am J Kidney Dis, 2002. 39(2 Suppl 1): p. S1-266. Gault, M.H., et al., Predicting glomerular function from adjusted serum creatinine. Nephron, 1992. 62(3): p. 249-56. Zuo, L., et al., Application of GFR-estimating equations in Chinese patients with chronic kidney disease. Am J Kidney Dis, 2005. 45(3): p. 463-72. Froissart, M., et al., Predictive performance of the modification of diet in renal disease and Cockcroft-Gault equations for estimating renal function. J Am Soc Nephrol, 2005. 16(3): p. 763-73. Gonwa, T.A., et al., Estimation of glomerular filtration rates before and after orthotopic liver transplantation: evaluation of current equations. Liver Transpl, 2004. 10(2): p. 301-9. O'Meara, E., et al., The Modification of Diet in Renal Disease (MDRD) equations provide valid estimations of glomerular filtration rates in patients with advanced heart failure. Eur J Heart Fail, 2005. Mahajan, S., et al., Assessing glomerular filtration rate in healthy Indian adults: a comparison of various prediction equations. J Nephrol, 2005. 18(3): p. 257-61. Poggio, E.D., et al., Performance of the Cockcroft-Gault and modification of diet in renal disease equations in estimating GFR in ill hospitalized patients. Am J Kidney Dis, 2005. 46(2): p. 242-52. Poggio, E.D., 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, 2005. 16(2): p. 459-66.

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[22] Bland, J.M. and D.G. Altman, Statistical methods for assessing agreement between two methods of clinical measurement. Lancet, 1986. 1(8476): p. 307-10. [23] Mariat, C., et al., Assessing renal graft function in clinical trials: can tests predicting glomerular filtration rate substitute for a reference method? Kidney Int, 2004. 65(1): p. 289-97. [24] Gaspari, F., et al., Performance of different prediction equations for estimating renal function in kidney transplantation. Am J Transplant, 2004. 4(11): p. 1826-35. [25] Perico, N., F. Gaspari, and G. Remuzzi, Assessing renal function by GFR prediction equations in kidney transplantation. Am J Transplant, 2005. 5(6): p. 1175-6. [26] Poge, U., et al., MDRD equations for estimation of GFR in renal transplant recipients. Am J Transplant, 2005. 5(6): p. 1306-11. [27] Bosma, R.J., et al., Predictive performance of renal function equations in renal transplant recipients: an analysis of patient factors in bias. Am J Transplant, 2005. 5(9): p. 2193-203. [28] Goerdt, P.J., et al., Predictive performance of renal function estimate equations in renal allografts. Br J Clin Pharmacol, 1997. 44(3): p. 261-5. [29] Mariat, C., et al., Predicting Glomerular Filtration Rate in Kidney Transplantation: Are the K/DOQI Guidelines Applicable? Am J Transplant, 2005. 5(11): p. 2698-703. [30] Cirillo, M., P. Anastasio, and N.G. De Santo, Relationship of gender, age, and body mass index to errors in predicted kidney function. Nephrol Dial Transplant, 2005. 20(9): p. 1791-8. [31] Murthy, K., et al., Variation in the serum creatinine assay calibration: A practical application to glomerular filtration rate estimation. Kidney Int, 2005. 68(4): p. 1884-7. [32] Ixkes, M.C., et al., Cimetidine improves GFR-estimation by the Cockcroft and Gault formula. Clin Nephrol, 1997. 47(4): p. 229-36. [33] Roubenoff, R., et al., Oral cimetidine improves the accuracy and precision of creatinine clearance in lupus nephritis. Ann Intern Med, 1990. 113(7): p. 501-6. [34] Kemperman, F.A., et al., Follow-up of GFR estimated from plasma creatinine after cimetidine administration in patients with diabetes mellitus type 2. Clin Nephrol, 2000. 54(4): p. 255-60. [35] Kemperman, F.A., et al., Cimetidine improves prediction of the glomerular filtration rate by the Cockcroft-Gault formula in renal transplant recipients. Transplantation, 2002. 73(5): p. 770-4. [36] Brown, S.C. and P.H. O'Reilly, Iohexol clearance for the determination of glomerular filtration rate in clinical practice: evidence for a new gold standard. J Urol, 1991. 146(3): p. 675-9. [37] Gaspari, F., et al., Glomerular filtration rate determined from a single plasma sample after intravenous iohexol injection: is it reliable? J Am Soc Nephrol, 1996. 7(12): p. 2689-93. [38] Gaspari, F., N. Perico, and G. Remuzzi, Application of newer clearance techniques for the determination of glomerular filtration rate. Curr Opin Nephrol Hypertens, 1998. 7(6): p. 675-80. [39] Thomsen, H.S. and K. Hvid-Jacobsen, Estimation of glomerular filtration rate from low-dose injection of iohexol and a single blood sample. Invest Radiol, 1991. 26(4): p. 332-6.

In: Progress in Kidney Transplantation Editor Dominick W. Mancuso, pp. 37-53

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter III

The Role of ‘Statins’ in Renal Transplantation Martin Lee and Lavjay Butani* Department of Pediatrics; University of California Davis Children’s Hospital; Sacramento, CA

Abstract Chronic kidney disease is well recognized to be a state of aberrant lipid metabolism and inflammation, both of which contribute to the cardiovascular morbidity and mortality encountered in this population. Even after successful renal transplantation, both dyslipidemia and the inflammatory milieu persist, and lead not only to premature atherosclerosis, but also to the development of chronic allograft nephropathy and eventual graft loss. The independent adverse effects of, and also the complex interplay between, dyslipidemia and inflammation on vascular health will be discussed in the first part of this chapter. The statins are a group of agents that have potent lipid lowering effects and are being used commonly in the post-transplant period. More recently, the statins have been demonstrated to have immunomodulatory effects independent of their lipid-lowering action. These immunologic effects of the statins may turn out to be of much greater significance for patients with CKD and those with transplants. The latter part of this chapter will address the clinical data demonstrating the beneficial effects of statins in transplant recipients and also discuss the mechanisms by which the statins might mediate these benefits.

*

Address all correspondence to: Lavjay Butani, MD; Section of Pediatric Nephrology; Department of Pediatrics; University of California Davis Children’s Hospital; 2516 Stockton Boulevard, Ticon II, 3rd Floor; Sacramento, CA 95817, USA; Tel: 916-734-8118; Fax: 916-734-0629; E-mail: [email protected]

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Why Add Statins to the Existing Complex Drug Regimen of Transplant Recipients? Cardiovascular disease is a leading cause of death in the adult and even in the pediatric dialysis population [1, 2]. Although the risk of dying from cardiovascular causes decreases considerably after renal-transplantation (Tx), cardiac disease remains one of the most common causes of death in the post-transplant period in children and in adults [3]. A variety of factors have been associated with the development of cardiac disease in the patient population with chronic kidney disease (CKD). Some of the contributors are chronic anemia [4], hypertension [5], hyperparathyroidism [6], elevated homocysteine levels [7], and hyperlipidemia [8, 9]. There is growing concern that a high-risk lipid profile and the associated inflammatory state could promote allograft injury thereby contributing to the development and progression of chronic allograft nephropathy (CAN), the most common cause of graft loss [10]. As a consequence, interest in monitoring and attempting to prevent and treat hyperlipidemia in the post-transplant period has increased dramatically.

How Do Lipids Cause Graft Dysfunction? Dyslipidemia in CKD Hyperlipidemia is a common occurrence in renal transplant recipients with a reported prevalence of 30%-75%, even on long-term follow-up [11, 12]. In order to better comprehend the consequences of this dyslipidemia, one first needs to recognize that the uremic environment, to which most transplant patients are exposed, is inherently a state of aberrant lipid metabolism. The etiology of this is multifactorial, partly related to the decreased activity of the enzymes lipoprotein lipase and hepatic lipase that are involved in lipoprotein metabolism, and further worsened by the high glucose load to which the body is exposed during dialysis. Even after successful Tx, commonly used medications such as the calcineurin-inhibitors (CNI) and corticosteroids, add to the dyslipidemia by interfering with low-density lipoprotein (LDL) receptor functioning. In addition, CNIs reduce bile acid synthesis and steroids create a hyperinsulinemic state, both of which cause increased synthesis of triglyceride (TG) rich lipoproteins. Other lipid abnormalities including the appearance of smaller and denser LDL particles, increased Apolipoprotein (Apo)-C III, decreased Apo A I, increased lipoprotein (a) and the accumulation of partly metabolized TGrich particles called ‘remnant lipoprotein particles’ (RLPs) have all been attributed to the inflammation seen in the setting of CKD, as discussed below.

CKD-A Proinflammatory State Renal failure is now well recognized to be a state of enhanced inflammation. The current hypotheses link this causally to many factors such as the increased oxidative stress associated with renal failure, the uremic milieu itself, dialysis related factors, dyslipidemia, and chronic

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infections (such as vascular access infections, bronchitis and periodontitis) that are seen in the CKD population [13]. The state of heightened oxidative stress in CKD is caused by loss of antioxidants during dialysis (zinc, selenium, vitamin C and E), but is also present during the early stages of CKD due to a reduction in the activity of superoxide dismutase and glutathione peroxidase. These changes are evidenced by the increased malondialdehyde levels in red blood cells and increased plasma levels of F2 iso-prostanes, which are formed in vivo by the peroxidation of arachidonic acid via non-cycloxegenase pathways, encountered in patients with CKD [14]. This oxidative stress leads to increased pro-inflammatory cytokines such as IL-6, and IL-1β. Moreover, the ensuing inflammation causes even more oxidative stress as the oxidative burst of mononuclear cells and neutrophils enhances the oxidation of LDL via release of myeloperoxidase. Oxidized LDL then leads to more inflammation by mechanisms that will be discussed in a later section, thus creating a vicious cycle. Another mechanism of increased inflammation in CKD is the accumulation of advanced glycation end-products (AGE) such as pentosidine, which are normally filtered and metabolized via renal tubular uptake. AGEs activate mononuclear cells resulting in inflammation that further increases production of AGEs. Since AGEs are highly protein bound to albumin, dialysis does not result in a reduction in their levels. Lastly, dialysis membranes and dialysate contamination can also cause lymphocyte and complement activation, adding to the inflammatory burden in patients with advanced stages of CKD [15]. Recently it has been shown that the rise in serum inflammatory markers (C-reactive protein [CRP], white blood cell count, fibrinogen, and factor VII) is directly correlated with the decrease in glomerular filtration rate (GFR) as CKD progresses [16]. As might be expected, improvements in the GFR after Tx decrease both the markers of inflammation and of oxidative stress. However these levels do not return to normal [17]. The most probable contributors to continued inflammation in the post-transplant period are the allogeneic immune stimulation caused by the graft and the immunosuppressive drugs used [18].

Mechanisms of Vascular Injury It appears that both dyslipidemia and inflammation can mediate vascular damage. The mechanisms of vascular injury caused independently by dyslipidemia and by inflammation, as well as the complex interrelationships between these two systems, are discussed in detail below.

Dyslipidemia Causing Vasculopathy Hyperlipidemia induced graft dysfunction is related to the chemical properties of cholesterol. Cholesterol is a Janus-faced molecule that is critical to the composition of cell membranes and is a substrate for steroid/bile acid synthesis. It confers fluidity to cell membranes due to its absolute insolubility in water. However, this insolubility makes its transport via the blood stream to tissues impossible without a special carrier system, the lipoproteins [19]. A complex system of lipoproteins delivers cholesterol produced by the liver

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to tissues and returns excess cholesterol back to the liver. While the details of this system are complex, the basic architecture is simple. The lipoprotein carriers are named according to the amount of protein to cholesterol content, i.e. VLDL, LDL, intermediate-density, and highdensity lipoproteins (HDL). The VLDL particles contain the greatest amount of Cholesterol. LDL is the predominant lipoprotein in the body and consists of a solid core of Cholesterol esters and TG surrounded by a lipid monolayer that contains protein and non-esterified Cholesterol. As LDL courses through the blood stream the central core of Cholesterol esters is shielded by the outer lipid layer. However the non-esterified Cholesterol molecules found in the outer lipid monolayer move freely into the lipid bilayers of endothelial cells [20]. As levels of LDL in the blood increase, the deposition of Cholesterol into intimal layers of the vessel wall also increases. This aberrant deposition is believed to be the nidus for a series of changes that lead to the formation of atheromatous plaques and also contribute to the development of CAN. The first microscopic sign of this process is the presence of foam cells, which are macrophages that have become over laden with lipid and Cholesterol. As described by the American Heart Association classification nomenclature, the lesions progress through a series of predictable steps of increased lipid accumulation, smooth muscle changes and eventual surface defects which may then cause thrombosis [21].

Inflammation Mediated Vasculopathy The inflammatory response appears to augment the classic description of hyperlipidemia induced vascular changes described above. These changes occur after LDL enters the intima of susceptible regions and undergoes oxidation via the scavenger receptor pathway. These susceptible regions of endothelium differ in their expression of ApoB-100 retentive molecules such as biglycan and decorin [22]. This varied tendency of the vascular endothelium to accumulate LDL particles is an area of ongoing research. Once the lipoproteins find their way into the endothelium they undergo cell-mediated oxidation of Apo B-100 via macrophage lipoxygenases [23]. This oxidation gives the entire lipoprotein a greater net negative charge which then promotes further macrophage uptake and lesion progression [22]. Oxidized LDL in addition to being cytotoxic, directly increases the expression of adhesion molecules (including vascular cell adhesion molecule [VCAM] and intercellular adhesion molecule [ICAM]), which increase the adhesion of monocytes to the endothelium. Additionally it increases the expression of several oxidation-sensitive atherogenic genes (by binding to cell surface receptors or by shifting intracellular redox balance) such as NKκB, which increases monocyte and T-cell recruitment into the intima. Moreover, oxidized LDL decreases inducible nitric oxide synthase (NOS), resulting in increased vascular tone, and it also directly increases collagen and mesangial cell proliferation [24]. The lipid peroxidation products formed during the oxidation of the lipid core, such as malondialdehyde, react with proteins such as Apo B, which make the oxidized LDL particle highly immunogenic. Not only does this further promote macrophage uptake of the LDL particle and lesion progression (as mentioned above), but autoantibodies to oxidized LDL form and bind to the oxidized LDL. These are then presented with class II Major Histocompatibility Complex (MHC) molecules and cause further T-cell activation.

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In addition to the effect of oxidized LDL, the other lipid abnormities seen in CKD, described earlier, can also contribute to vascular injury. The increased lipoprotein (a), the RLPs and the smaller denser LDL all have an increased susceptibility to oxidation, and can penetrate the vascular intima more easily, making them highly atherogenic. In addition, as Apo A-I is a negative acute phase reactant protein its levels in CKD are decreased. Serum Amyloid Associated Protein (SAA), an acute phase reactant, displaces Apo-I from HDL, thereby altering its structure and increasing its TG content. This alteration in HDL structure increases the adherence of HDL to vascular endothelium and reduces its antioxidant properties. This then contributes to increased production of oxidized LDL [25]. Additionally, the inflammatory milieu in CKD causes the acute phase reactant proteins such as CRP and fibrinogen to rise. Both of these are procoagulants that cause an upregulation of VCAM, ICAM and P-selectin expression, which then increase the adherence of platelets to the endothelium and propagate damage to the vessel wall. Interestingly, hyperlipidemia induced vascular inflammation may directly beget more hyperlipidemia, adding fuel to the fire. A causal link between inflammation and dyslipidemia makes teleological sense. Since the occurrence of inflammation signals an increased metabolic requirement for injured cells in the vicinity of the inflammatory process, the appropriate compensatory response from the liver would be to increase production and delivery of cholesterol to the injured area [26]. In support of this theory, patients with chronic inflammatory diseases such as rheumatoid arthritis have been found to have altered lipid profiles (elevated LDL levels, and decreased HDL levels) which would maximize cholesterol delivery to tissues [27, 28]. The converse also appears to be true in that patients with high levels of anti-inflammatory cytokines, such as IL-10, have been shown to have higher levels of HDL which would promote reverse cholesterol transport [29]. The net result of the vascular injury resulting from the complex interplay between the metabolic derangements and inflammation is cardiovascular disease, CAN and death. Clinical studies support the paramount role of lipids/inflammation in affecting cardiac outcomes. Elevated Cholesterol has been shown to predict loss of renal function in type 1 and 2 diabetics [30] while high Apo B and low HDL levels have been shown to correlate with deterioration of renal function in CKD patients [31].

Intervention Strategies Although very little direct data exist on the long-term clinical significance of lipid levels in children, compelling indirect data in support of the harmful effects of dyslipidemia are available from the Pathobiological Determinants of Atherosclerosis in Youth (PDAY) study. This landmark study found that the development of fatty streaks in the coronary arteries (which are precursors of atherosclerotic plaques) was positively correlated with elevated LDL and low HDL levels in young adults [32], pointing to the importance of controlling sustained abnormalities in levels of atherogenic lipids. Since CKD is such an overwhelming state of inflammation and dyslipidemia, children with CKD are at great risk of developing adverse cardiovascular outcomes. This should prompt a concerted effort on the part of their healthcare providers to attempt to ameliorate the dyslipidemia by all possible means.

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Dietary Modification While dietary modification to reduce the intake of saturated fats, consistent aerobic exercise, and weight loss (for the overweight patient) should certainly be strongly recommended to all patients after renal-Tx, the practical benefit of these recommendations for life-style change is quite limited. Delucchi et al offered the Step II American Heart Association diet (containing a low fat and low saturated Cholesterol content) to 22 children with hyperlipidemia after renal-Tx; only about half of the eligible children agreed to participate in the study. Moreover, no patient demonstrated 100% compliance with the diet. Even in the setting of such a controlled study, the benefit of the diet in ameliorating the dyslipidemia was quite modest. No patient lost weight, nor was the mean BMI affected, and while the total Cholesterol and LDL did decrease, the magnitude of the decline was small (11% and 14% respectively at 12 weeks) [33].

Treatment with ‘Statins’ An alternative that is available to transplant nephrologists is the use of lipid lowering agents such as the HMGCoA-reductase inhibitors also referred to as ‘statins.’ The statins are a class of drugs that inhibit the conversion of HMGCoA to mevalonic acid. Since Cholesterol biosynthesis accounts for most of the circulating Cholesterol in humans, inhibition of this rate-limiting step leads to lower serum Cholesterol. This reduction in total Cholesterol results in an increase in the expression of surface LDL receptors, thereby causing a decrease in serum LDL levels. Beneficial effects of statins on the lipid profile have been demonstrated in adult renal transplant recipients in several studies; the mean reduction in total Cholesterol and LDL has been shown to be in the range of 20%-30% and 35-40% respectively [34, 35]. A few studies have also demonstrated a reduction in the total TG level with higher doses of the statins [34, 36]; no significant change in the HDL has previously been reported with these agents [34, 35, 37].

Clinical Data Supporting the Use of ‘Statins’ in Children Statins have also been used safely and successfully in improving the lipid profile in children and adolescents with familial hypercholesterolemia [38], but data on their use in pediatric transplant recipients are limited. Penson et al were the first to demonstrate the efficacy of pravastatin in ameliorating hypercholesterolemia in 21 pediatric and adolescent heart transplant recipients with no adverse consequences [39]. In recent years, other investigators have published data on the use of the statins in children with renal transplants. Argent et al, in a prospective study, showed that atorvastatin safely reduced total Cholesterol, LDL and serum TG by approximately 40%, 60% and 45% respectively, in 9 children with renal transplants who had persistent hyperlipidemia [40]. Our own study was quite different from the previously published pediatric data in that, based on our center’s favorable experience using pravastatin pre-emptively in adult renal transplant patients [41] we routinely started using pravastatin pre-emptively in all pediatric renal transplant patients from June

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1999 onwards and demonstrated a significant reduction the total serum Cholesterol and the incidence of post-transplant hypercholesterolemia [42]. Based on these preliminary data, we hypothesized that pravastatin would also favorably impact other lipid parameters. A standard fixed dose of pravastatin was used; children <10 yr of age received 10 mg once daily while those >10 yr were treated with 20 mg once daily. Pravastatin was initiated within 24–48 h of transplantation. Neither the baseline demographic characteristics nor the mean pre-transplant serum Cholesterol were significantly different between the historical control and treatment groups. The serum Cholesterol in the pravastatin group was significantly lower at all time points except at 1 month post-transplant. The repeated measures ANOVA test showed that in the pravastatin group there was a significant decline in the serum Cholesterol at 1, 3, 6, 9 and 12 months after Tx compared to the baseline value (p < 0.05 for each comparison); no such difference was seen in the serum Cholesterol in the control patients. A subsequent study from our center confirmed the findings of our pilot study in that the use of pravastatin preemptively in pediatric renal transplant recipients was able to significantly reduce the prevalence of hypercholesterolemia and hyperlipidemia. Based on a logistic regression analysis, the failure of the serum Cholesterol to decline in the immediate post-transplant period was likely due to the effect of high dose prednisone. By 3months after Tx, all the children were successfully weaned to a much lower steroid dose (0.2 mg/kg/day), allowing pravastatin to overcome the overwhelming dyslipidemic effect of the steroid. That a reduction in the steroid dose was not the only reason for the decline in the serum Cholesterol is made apparent when one compares the pravastatin-treated patients to the controls. In spite of identical steroid exposure in the 2 groups, the serum Cholesterol failed to change in the control patients with time, even as far out as 12-months after Tx, when the prednisone dose was very low. Another risk factor for hypercholesterolemia and hypertriglyceridemia found in our study was the pre-transplant Cholesterol, i.e. patients who were dyslipidemia pre-transplant had a higher risk of being persistently so even after Tx. Multivariable linear regression analyses also confirmed an independent effect of the pre transplant serum Cholesterol on the posttransplant Cholesterol (p=0.006). A similar finding was seen in a cohort of heart-transplant recipients [43], and points to the existence of host factors (environmental or genetic) that play a significant role in determining lipid levels in individuals. The study findings in relation to the HDL and 2 lipid ratios were surprising and concern us. The total HDL significantly declined after Tx, the change becoming apparent by 3months, corresponding to the time when the prednisone dose was being tapered most rapidly. This decline in the HDL was of a large enough magnitude that 8% of patients at 1-year had an HDL that was below the lower limit of normal. The 2 predictors for an HDL level < 35 mg/dl were a lower GFR and a lower steroid dose. Corticosteroid therapy has been shown to increase the HDL-level in patients with sarcoidosis [44]; similarly in one study in adult renaltransplant patients, steroid withdrawal was associated with a 14%-22% decrease in HDLlevels, although in none of the patients did the HDL fall into an abnormally low range [45]. In the latter study, some indicators of a dyslipidemic state improved after steroid withdrawal, such as Apo B and fasting insulin, while others (LDL and TG) remained unaffected. Shortterm clinical benefits after steroid-withdrawal included weight loss and a decrease in waist girth. Corticosteroids may raise the HDL by increasing Apo A-1 synthesis and by decreasing the activity of Cholesterol Ester Transfer Protein (CETP), the latter being an enzyme responsible for maturation of the HDL particle [46]. In addition to the associated

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inflammatory changes previously discussed, the association of HDL levels with renal function is linked to the role of the kidneys in catabolism of HDL [47]. Patients with CKD and those on dialysis have low levels of HDL and more importantly have a low HDL 2 to HDL 3 ratio [48, 49]. These changes have been linked to a down regulation in the enzyme lecithin cholesterol acyl transferase (LCAT) which is responsible for the HDL-mediated removal of excess Cholesterol from peripheral tissues [50, 51]. That the kidney has a causal role in mediating this down regulation of LCAT is confirmed by the observation that after renal Tx, HDL levels go back to normal in those patients with good renal function, while they remain low in those with poor graft function [49]. In our study, in addition to measuring the various lipid levels, we also elected to determine the values of 2 lipid ratios, the LDL/HDL and Cholesterol/HDL ratios, since both of these have been validated as reliable measures of the risk of adverse cardiovascular outcomes in adults. The rationale in using these ratios is that they reflect the balance between the atherogenic and antiatherogenic lipids and may give a better assessment of the net lipid milieu that the patient is exposed to compared to any one individual lipid level. The superiority of the predictive value of these ratios has been confirmed in population studies [52, 53]. In our study we found that there was a trend for the mean values of both of these ratios to increase with time, although this was not statistically significant. However, 8%-15% of children at the end of the study had abnormally elevated ratios. The 2 predictors for these were elevated pre-transplant Cholesterol (for the Cholesterol/HDL ratio) and a younger age (for both). We hypothesize that the latter was likely, again, due to the greater use of peritoneal dialysis in younger children, which would lead to a worse lipid profile, compared to the older patients. There are several limitations to our study, most importantly the small number of patients and the short follow-up. Moreover, since we did not have a concurrent control group that was not on pravastatin, we cannot determine the effect of pravastatin in contributing to the abnormalities in the HDL nor its effect on either of the 2 lipid ratios, although none of the previous adult studies have found an impact on HDL with the statins. There are, however, animal studies in which pravastatin has been shown to decrease HDL levels [54, 55]. The mechanism by which it does so is not clear, but it may be related to the transfer of cholesterol esters from HDL to VLDL particles. We did not perform measurements of HDL sub fractions nor did we check Apo levels, both of which have an impact on the risk of atherosclerosis. Nevertheless, our study is the only study to date that has evaluated the efficacy of the preemptive use of pravastatin in the post-transplant period, and demonstrated it to effectively reduce total Cholesterol, TG and LDL after Tx. Randomized trials using pravastatin versus placebo are needed to determine if the use of statins has any impact on the HDL abnormalities that were encountered in our population. Our study also re-emphasizes the importance of steroids in contributing to the development of an adverse lipid profile in children after Tx, in spite of the use of statins, and should be an added incentive to transplant centers to move to steroid-minimization protocols, data pertaining to which are limited at present to uncontrolled trials and abstracts [56].

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Immunomodulation by Statins: Myth or Reality? A burgeoning area of interest as well as ongoing research in the transplant literature relates to the immunomodulatory and anti-inflammatory effects of the statins, many of which are independent of the effect of the statins on lipids. Much of this interest has come from reanalysis of large clinical trials like the CARE trial that were originally designed to assess the cardioprotective effect of the statins through reduction of hyperlipidemia. Reanalysis has shown anti-inflammatory and cardioprotective effects unrelated to the degree of correction of the dyslipidemia [57]. This notion is further supported by the lack of temporal association between the effects of statins on LDL and on inflammatory markers. In one study, after 12 weeks of treatment with a statin, patients showed decreased human soluble VCAM-1 expression, increased tissue plasminogen activator (tPA) levels and decreased LDL levels. However, within three days of stopping therapy the levels of sVCAM-1 and tPA returned to their pretreatment levels while there was no change in the LDL levels. This suggests that lower LDL did not directly cause the anti-inflammatory and pro-fibrinolytic effects present during statin therapy [58]. These observations suggest that the statins have mechanisms of action apart from the intended Cholesterol lowering effect, for which they are most conventionally prescribed. Similar evidence exists for the transplant population. The use of pravastatin has been associated with improved 1-year allograft and patient survival, a lower incidence of severe cardiac rejection and a decreased incidence and progression of transplant coronary vasculopathy in a single study of cardiac transplant recipients [59]. Similarly, a 4-month randomized controlled pilot study in renal transplant recipients showed that the use of pravastatin lowered the incidence of biopsy-proven acute rejection by about 50%, and also reduced the incidence of recurrent rejection and the need for monoclonal antibodies for rejection reversal [41]. A recently published placebo controlled randomized double blind study of fluvastatin in hypercholesterolemic renal transplant recipients demonstrated a benefit of fluvastatin in reducing cardiac deaths or non-fatal myocardial infarctions after a mean follow-up of 5 years [60]. Although there was no improvement in graft survival in the overall population, a subsequent analysis of the data showed a benefit of fluvastatin in improving allograft survival in a subset of patients who had good blood pressure control. Our own study was small and uncontrolled and so we could not adequately assess the impact of pravastatin on graft function or rejection. Compared to the historical control group, however, the incidence of acute rejection at 1-year in the pravastatin treated patients was lower, but not statistically so (22% in the control group versus 6% in the pravastatin group; p=0.5, fisher’s exact test). Clearly long-term follow-up studies using a control group are required to determine the effect of treatment with the statins on acute rejection, graft loss and patient survival. Surrogate markers of an adverse outcome such as the development of coronary artery calcification and studies of carotid artery intimal thickness may be more realistic end-points for pediatric patients, and are being planned by us. As inhibitors of HMGCoA reductase, statins affect the synthesis of numerous compounds in addition to Cholesterol. Cholesterol is an end product of the mevalonate pathway, which uses acetyl CoA as a carbon source to make numerous hydrophobic membrane bound molecules including Cholesterol, ubiquinone, dolichol, vitamin D, as well as geranyl and farnesyl isoprenoid groups. The pathway begins by joining acetyl CoA particles together to make the six-carbon HMG CoA. This is subsequently reduced by HMG CoA reductase, the

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enzyme inhibited by statins, to yield mevalonate. Mevalonate is then turned into a more useable five-carbon isoprenoid structure from which the numerous membrane bound molecules are derived. The decreased production of the geranyl and farnesyl groups may explain much of the anti-inflammatory effects of the statins. Geranyl and farnesyl isoprenoid groups are used in the ‘prenylation’ of proteins. The prenylation reaction is a posttranslational modification that places isoprenoid membrane anchors on proteins targeted for membrane association. The carboxy terminal sequence of Cys-A-A, where A is an aliphatic amino acid, is both the signal and the site of attachment of the prenyl group [61]. The superfamily of Ras-related small GTPases uses these 10 carbon hydrophobic membrane anchors to attach to the cell membrane. This family of proteins affects numerous areas of cellular signaling, migration and proliferation. In fact, there have been over 50 different effector molecules shown to interact with this super-family of proteins [62]. The activity of all these proteins is affected by prenylation. The best understood example is that of the Ras oncogene. When functioning normally, this GTPase is anchored to the cell membrane by a covalently linked prenyl group and conveys signals from receptor tyrosine kinases that stimulate cell differentiation and proliferation. The mutant form of this gene, the Ras oncogene, causes uncontrolled cell proliferation and is found in about 30% of all cancers [63, 64]. Blocking the prenylation of the Ras oncogene in vitro, thus interfering with its membrane association, has been shown to reverse its effect on cell proliferation and is being actively studied as a potential chemotherapeutic strategy [65]. As there are numerous GTPases involved in the inflammatory response, statin mediated depletion of prenylation substrates may explain their antiproliferative and anti-inflammatory effects [66]. In fact, the immune-modulating effects of statins may be classified into those related to prenylation, and those independent of prenylation. Prenylation related observations: It has been shown in vitro that statins inhibit endothelial cell expression of tissue factor induced by monocytes. The increased expression of tissue factor occurs at the transcriptional level and is mediated by RhoA. The inhibition of expression of tissue factor appears directly related to prenylation depletion because it can be reversed by the addition of geranylgeranylpyrophosphate (a prenyl precursor) [67]. Other effects believed to be due to prenylation inhibition are decreased smooth muscle/mesangial cell proliferation (from decreased membrane accumulation of Rho A) which may help retard the intimal hyperplasia associated with vascular lesions and with CAN [21, 68, 69] and decreased platelet adhesion (mediated by decreased NF-κB). Yet another effect attributed to decreased prenylation is a decrease in mean Natural Killer (NK) cell cytotoxicity by about 50% that has been shown in animal models [70] and in human studies [41, 59]. Prenylation independent effects: Apart from the effects on the mevalonate pathway, statins appear to have independent immunomodulatory and anti-inflammatory effects. One of these direct effects demonstrates a fascinating link between the statins, oxidized LDL and nitric oxide (NO) and was described by Mehta et al. They demonstrated that oxidized LDL induces expression of LOX-1, a specific receptor for oxidized LDL which, when stimulated, inhibits production of endothelial nitric oxide synthase (eNOS). Pretreatment of endothelial cells in vitro with statins not only lowered oxidized LDL mediated expression of LOX-1, but also prevented the subsequent decrease in eNOS production [71]. Apart from the previously discussed benefits of decreasing oxidized LDL, increasing NO synthesis also appears

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beneficial since decreased production of NO has been implicated as a causative factor in the progression of atherosclerosis and abnormal progression of inflammatory states [72, 73]. Statins also affect the surface expression of numerous T-cell signaling molecules, including MHC I, MHC II and CD 3,4,8,28,40,54,80 and 86. Kuipers et al, have shown the likely mechanism for this to be alteration of cell membrane lipid rafts. Deficient intracellular transport to the proper areas of the cell membrane appears to be the principle cause but the precise mechanism of this is not well understood [74]. Another way statins can affect the immune response is by blocking the interaction between lymphocyte function-associated antigen-1 (LFA-1) and ICAM-1 [75]. This effect, which would decrease T-cell stimulation and adhesion, has been shown to be independent of the effect on HMG CoA reductase. Rather, it occurs through binding of statins to a novel allosteric site within LFA-1 [76]. Other potentially beneficial effects of the statins include an increase in apoptosis in vascular smooth muscle cells and renal tubular epithelial cells, a decrease in interstitial inflammation and fibrosis in CNI-associated nephropathy in synergy with angiotensinblockade [77], and a decrease in serum CRP levels (by decreasing IL-6). CRP is an important predictor of CAN and cardiovascular disease/death in CKD and in healthy populations by virtue of its proinflammatory effects mediated by complement activation, NK activation, increased IL-8 (via NF-κB), increased chemokine expression, and increased matrix metalloproteiniase-1 expression (resulting from binding of CRP to the Fcγ RII receptors CD32 and CD 64, followed by its internalization into endothelial cells, monocytes and neutrophils) [78]. CRP also causes increased uptake of oxidized LDL by cells, activates angiotensin-1 receptors, increases adhesion molecule expression in endothelium, decreases eNOS and increases plasminogen activator inhibitor-1, all of which are detrimental to the graft and to the patient. Another non-cholesterol related potential benefit of the statins that may apply to renal transplant patients is their effect on bone modeling and on proteinuria. Statins have been shown to decrease hip fractures in the elderly (71% risk reduction) by increasing expression of bone morphogenetic protein (BMP2) which stimulates osteoblast differentiation [79] this may turn out to be of immense benefit to the transplant recipient, who is at high risk of osteopenia and of fractures [80]. Statins have also been shown to reduce proteinuria and stabilize renal function in a recent prospective trial in proteinuric and hypercholesterolemic CKD patients [81], the effect being correlated with decreased urinary endothelin-1 excretion in treated patients.

Risks of Statins The main concern with the use of the statins, especially in the transplant population, relates to the potential for rhabdomyolysis. The mechanism underlying this myopathy is related to the inhibition of the mevalonic pathway by the statins in skeletal muscle cells. The lipophilic statins, such as lovastatin, by virtue of their ability to cross the cell membrane and gain access to the skeletal muscle cells in larger concentrations, are associated with the highest risk of rhabdomyolysis. This risk is compounded even further when other drugs that share the metabolic pathway of the statin in question are co-administered, resulting in very

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high plasma levels of the statin. Although in the past it had been presumed that the higher risk of rhabdomyolysis with the concomitant use of the CNIs (cyclopsorine and tacrolimus), was related to the fact that both these classes of drugs shared the cytochrome P450 3A (CYP 3A) system for metabolism, recent evidence suggests otherwise [82]. Lemahieu at al studied the pharmacokinetics of atorvastatin in a group of healthy volunteers and found that the coadministration of cyclosporine A resulted in no change in the overall CYP 3A activity but markedly suppressed the activity of the intestinal and hepatic P-glycoprotein efflux pump (which is also responsible for drug metabolism) resulting in a 15-fold increase in exposure to atorvastatin acid. Another adverse effect of the statins that physicians and patients need to be aware of is hepatotoxicity manifested by elevation of the transaminases.

Conclusion As we have come to learn more about the true link between dyslipidemia, inflammation and vascular injury, the future indications for the use of statins may become much broader than previously thought. Predicting the effects of statins in vivo is nearly impossible given their pleiotropic effects. However, renal transplant patients may uniquely benefit from statins should their immunomodulatory/anti-inflammatory effects prove to prolong graft and patient survival independent of their protective effects through lipid lowering. With our current understanding, the use of the stains is clearly beneficial in the renal transplant recipient who is at high risk of developing hyperlipidemia and accelerated atherosclerosis. However, using statins in patients with normal lipid profiles solely for their immunomodulatory, vasodilatory and anti-proliferative effects is not as well established in humans. The use of these agents is also not without risk, and thought should be put into the choice of the statin and the immunosuppressive agent to be used in a particular patient, in order to minimize drug interactions. Hepatic enzymes and creatine kinase levels need to be monitored, along with patient education on the signs and symptoms of possible adverse events, to enable early detection of toxicities. Only long-term prospective controlled trials will be able to determine if statins affect long-term reduction in cardiovascular morbidity and mortality, and whether allograft survival will be favorably impacted.

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[38] Knipscheer HC, Boelen CC, Kastelein JJ, van Diermen DE, Groenemeijer BE, van den Ende A, et al. Short-term efficacy and safety of pravastatin in 72 children with familial hypercholesterolemia. Pediatr Res 1996;39(5):867-71. [39] Penson MG, Fricker FJ, Thompson JR, Harker K, Williams BJ, Kahler DA, et al. Safety and efficacy of pravastatin therapy for the prevention of hyperlipidemia in pediatric and adolescent cardiac transplant recipients. J Heart Lung Transplant 2001;20(6):611-8. [40] Argent E, Kainer G, Aitken M, Rosenberg AR, Mackie FE. Atorvastatin treatment for hyperlipidemia in pediatric renal transplant recipients. Pediatr Transplant 2003;7(1):38-42. [41] Katznelson S, Wilkinson AH, Kobashigawa JA, Wang XM, Chia D, Ozawa M, et al. The effect of pravastatin on acute rejection after kidney transplantation--a pilot study. Transplantation 1996;61(10):1469-74. [42] Butani L, Pai MV, Makker SP. Pilot study describing the use of pravastatin in pediatric renal transplant recipients. Pediatr Transplant 2003;7(3):179-84. [43] Rudas L, Pflugfelder PW, McKenzie FN, Menkis AH, Novick RJ, Kostuk WJ. Serial evaluation of lipid profiles and risk factors for development of hyperlipidemia after cardiac transplantation. Am J Cardiol 1990;66(15):1135-8. [44] Salazar A, Mana J, Pinto X, Argimon JM, Hurtado I, Pujol R. Corticosteroid therapy increases HDL-cholesterol concentrations in patients with active sarcoidosis and hypoalphalipoproteinemia. Clin Chim Acta 2002;320(1-2):59-64. [45] Lemieux I, Houde I, Pascot A, Lachance JG, Noel R, Radeau T, et al. Effects of prednisone withdrawal on the new metabolic triad in cyclosporine-treated kidney transplant patients. Kidney Int 2002;62(5):1839-47. [46] Moulin P, Appel GB, Ginsberg HN, Tall AR. Increased concentration of plasma cholesteryl ester transfer protein in nephrotic syndrome: role in dyslipidemia. J Lipid Res 1992;33(12):1817-22. [47] Igel-Korcagova A, Raab P, Brensing KA, Poge U, Klehr HU, Igel M, et al. Cholesterol metabolism in patients with chronic renal failure on hemodialysis. J Nephrol 2003;16(6):850-4. [48] Shoji T, Nishizawa Y, Nishitani H, Yamakawa M, Morii H. Impaired metabolism of high density lipoprotein in uremic patients. Kidney Int 1992;41(6):1653-61. [49] Savdie E, Gibson JC, Stewart JH, Simons LA. High-density lipoprotein in chronic renal failure and after renal transplantation. Br Med J 1979;1(6168):928-30. [50] Guarnieri GF, Moracchiello M, Campanacci L, Ursini F, Ferri L, Valente M, et al. Lecithin-cholesterol acyltransferase (LCAT) activity in chronic uremia. Kidney Int Suppl 1978(8):S26-30. [51] Vaziri ND, Liang K, Parks JS. Down-regulation of hepatic lecithin:cholesterol acyltransferase gene expression in chronic renal failure. Kidney Int 2001;59(6):2192-6. [52] Kinosian B, Glick H, Garland G. Cholesterol and coronary heart disease: predicting risks by levels and ratios. Ann Intern Med 1994;121(9):641-7. [53] Manninen V, Tenkanen L, Koskinen P, Huttunen JK, Manttari M, Heinonen OP, et al. Joint effects of serum triglyceride and LDL cholesterol and HDL cholesterol concentrations on coronary heart disease risk in the Helsinki Heart Study. Implications for treatment. Circulation 1992;85(1):37-45. [54] Kume N, Kita T, Mikami A, Yokode M, Ishii K, Nagano Y, et al. Induction of mRNA for low-density lipoprotein receptors in heterozygous Watanabe heritable

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Martin Lee and Lavjay Butani hyperlipidemic rabbits treated with CS-514 (Pravastatin) and cholestyramine. Circulation 1989;79(5):1084-90. Miyazaki A, Koieyama T, Shimada Y, Kikuchi T, Ito K, Kasanuki N, et al. Pravastatin sodium, an inhibitor of HMG-CoA reductase, decreases HDL cholesterol by transfer of cholesteryl ester from HDL to VLDL in Japanese white rabbits. J Atheroscler Thromb 2004;11(1):22-8. Sarwal MM, Vidhun JR, Alexander SR, Satterwhite T, Millan M, Salvatierra O, Jr. Continued superior outcomes with modification and lengthened follow-up of a steroidavoidance pilot with extended daclizumab induction in pediatric renal transplantation. Transplantation 2003;76(9):1331-9. Ridker PM, Rifai N, Pfeffer MA, Sacks F, Braunwald E. Long-term effects of pravastatin on plasma concentration of C-reactive protein. The Cholesterol and Recurrent Events (CARE) Investigators. Circulation 1999;100(3):230-5. Lai WT, Lee KT, Chu CS, Voon WC, Yen HW, Tsai LY, et al. Influence of withdrawal of statin treatment on proinflammatory response and fibrinolytic activity in humans: an effect independent on cholesterol elevation. Int J Cardiol 2005;98(3):459-64. Kobashigawa JA, Katznelson S, Laks H, Johnson JA, Yeatman L, Wang XM, et al. Effect of pravastatin on outcomes after cardiac transplantation. N Engl J Med 1995;333(10):621-7. Holdaas H, Fellstrom B, Jardine AG, Holme I, Nyberg G, Fauchald P, et al. Effect of fluvastatin on cardiac outcomes in renal transplant recipients: a multicentre, randomised, placebo-controlled trial. Lancet 2003;361(9374):2024-31. Smeland TE, Seabra MC, Goldstein JL, Brown MS. Geranylgeranylated Rab proteins terminating in Cys-Ala-Cys, but not Cys-Cys, are carboxyl-methylated by bovine brain membranes in vitro. Proc Natl Acad Sci U S A 1994;91(22):10712-6. Jaffe AB, Hall A. RHO GTPases: Biochemistry and Biology. Annu Rev Cell Dev Biol 2005. Malumbres M, Barbacid M. RAS oncogenes: the first 30 years. Nat Rev Cancer 2003;3(6):459-65. Giehl K. Oncogenic Ras in tumour progression and metastasis. Biol Chem 2005;386(3):193-205. Sebti SM. Protein farnesylation: implications for normal physiology, malignant transformation, and cancer therapy. Cancer Cell 2005;7(4):297-300. Rolfe BE, Worth NF, World CJ, Campbell JH, Campbell GR. Rho and vascular disease. Atherosclerosis 2005. Ishibashi T, Sakamoto T, Ohkawara H, Nagata K, Sugimoto K, Sakurada S, et al. Integral role of RhoA activation in monocyte adhesion-triggered tissue factor expression in endothelial cells. Arterioscler Thromb Vasc Biol 2003;23(4):681-7. Kozai T, Eto M, Yang Z, Shimokawa H, Luscher TF. Statins prevent pulsatile stretchinduced proliferation of human saphenous vein smooth muscle cells via inhibition of Rho/Rho-kinase pathway. Cardiovasc Res 2005. Corpataux JM, Naik J, Porter KE, London NJ. The Effect of Six Different Statins on the Proliferation, Migration, and Invasion of Human Smooth Muscle Cells. J Surg Res 2005. Cutts JL, Scallen TJ, Watson J, Bankhurst AD. Role of mevalonic acid in the regulation of natural killer cell cytotoxicity. J Cell Physiol 1989;139(3):550-7.

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[71] Mehta JL, Li DY, Chen HJ, Joseph J, Romeo F. Inhibition of LOX-1 by statins may relate to upregulation of eNOS. Biochem Biophys Res Commun 2001;289(4):857-61. [72] Shaw CA, Taylor EL, Megson IL, Rossi AG. Nitric oxide and the resolution of inflammation: implications for atherosclerosis. Mem Inst Oswaldo Cruz 2005;100 Suppl 1:67-71. [73] Laufs U. Beyond lipid-lowering: effects of statins on endothelial nitric oxide. Eur J Clin Pharmacol 2003;58(11):719-31. [74] Kuipers HF, Biesta PJ, Groothuis TA, Neefjes JJ, Mommaas AM, van den Elsen PJ. Statins affect cell-surface expression of major histocompatibility complex class II molecules by disrupting cholesterol-containing microdomains. Hum Immunol 2005;66(6):653-65. [75] Weitz-Schmidt G, Welzenbach K, Dawson J, Kallen J. Improved lymphocyte functionassociated antigen-1 (LFA-1) inhibition by statin derivatives: molecular basis determined by x-ray analysis and monitoring of LFA-1 conformational changes in vitro and ex vivo. J Biol Chem 2004;279(45):46764-71. [76] Weitz-Schmidt G. Lymphocyte function-associated antigen-1 blockade by statins: molecular basis and biological relevance. Endothelium 2003;10(1):43-7. [77] Li C, Sun BK, Lim SW, Song JC, Kang SW, Kim YS, et al. Combined effects of losartan and pravastatin on interstitial inflammation and fibrosis in chronic cyclosporine-induced nephropathy. Transplantation 2005;79(11):1522-9. [78] Devaraj S, Du Clos TW, Jialal I. Binding and internalization of C-reactive protein by Fcgamma receptors on human aortic endothelial cells mediates biological effects. Arterioscler Thromb Vasc Biol 2005;25(7):1359-63. [79] Wang PS, Solomon DH, Mogun H, Avorn J. HMG-CoA reductase inhibitors and the risk of hip fractures in elderly patients. Jama 2000;283(24):3211-6. [80] Brandenburg VM, Westenfeld R, Ketteler M. The fate of bone after renal transplantation. J Nephrol 2004;17(2):190-204. [81] Bianchi S, Bigazzi R, Caiazza A, Campese VM. A controlled, prospective study of the effects of atorvastatin on proteinuria and progression of kidney disease. Am J Kidney Dis 2003;41(3):565-70. [82] Lemahieu WP, Hermann M, Asberg A, Verbeke K, Holdaas H, Vanrenterghem Y, et al. Combined therapy with atorvastatin and calcineurin inhibitors: no interactions with tacrolimus. Am J Transplant 2005;5(9):2236-43.

In: Progress in Kidney Transplantation Editor: Dominick W. Mancuso, pp. 55-87

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter IV

Pharmacogenetics of Immunosuppressive Drugs Eric Thervet* and Dany Anglicheau Service de Transplantation rénale, Hôpital Necker and INSERM UMR S490 Paris, France

Abstract Organ transplantation is the treatment of choice, if not the only treatment, for patients with end-stage organ failure. Recent data have shown a constant improvement in graft survival during the past decade. This improvement essentially results from the discovery of new immunosuppressive drugs which prevent the occurrence of acute rejection episodes. However, there are still some major concerns in this field. The first one is the need for organ donors, because of the growing discrepancy between the number of transplantations performed and the number of patients awaiting transplantation. The second concern is the absence of a major improvement in long-term graft survival, partly because of the chronic nephrotoxicity associated with the use of some immunosuppressive treatments. Lastly, one of the major causes of graft failure in the long-term is now the premature death of patients with a functioning graft, mainly due to cardiovascular disease. Other major causes include stroke, infections and malignancies.

Introduction Organ transplantation is the treatment of choice, if not the only treatment, for patients with end-stage organ failure. Recent data have shown a constant improvement in graft survival during the past decade [1]. This improvement essentially results from the discovery of new immunosuppressive drugs which prevent the occurrence of acute rejection episodes. *

Address of correspondence: Eric Thervet. Service de Transplantation Rénale. Hôpital Necker. 149 rue de Sèvres. 75015 Paris, France. Tel : +33144495432; Fax : +33144495430; E-mail : [email protected]

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However, there are still some major concerns in this field. The first one is the need for organ donors, because of the growing discrepancy between the number of transplantations performed and the number of patients awaiting transplantation. The second concern is the absence of a major improvement in long-term graft survival, partly because of the chronic nephrotoxicity associated with the use of some immunosuppressive treatments [2]. Lastly, one of the major causes of graft failure in the long-term is now the premature death of patients with a functioning graft, mainly due to cardiovascular disease. Other major causes include stroke, infections and malignancies [3]. Many of these complications are secondary to the use of immunosuppressive drugs. It is well recognized that different transplant recipients respond in different ways to immunosuppressive medication. Because there is no reliable monitoring of the immunologic status of patients receiving immunosuppressive drugs, many attempts have been made to optimize therapeutic drug monitoring by determining the blood concentrations of these drugs at trough level defined as the pre-dose concentration, or at different times after oral intake [4]. However, this monitoring can only be performed after transplantation, and is therefore not predictive of the individual response to a given drug during the early phase after transplantation. The inter-individual variations are greater than the intra-individual variations, a finding consistent with the notion that inheritance is a determinant of drug responses. In the general population, it is estimated that genetics accounts for 20 to 95 percent of the variability in drug disposition and effects [5]. Many other non-genetic factors, such as organ function, drug interactions and the nature of the disease, probably influence the effects of medication. The recent identification of genetic polymorphisms in drug metabolizing enzymes and drug transporters led to the hypothesis that genetic factors may be implicated in the inter-individual variability of the pharmacokinetic or pharmacodynamic characteristics of immunosuppressive drugs, major side effects, and efficacy. The promising role of pharmacogenetics and pharmacogenomics in trying to elucidate the inherited basis of differences between individual responses to drugs lies in the potential ability to identify the right drug and dose for each patient. There has been some confusion over the use of the term “Pharmacogenetics” and “Pharmacogenomics” in current practice. By definition, pharmacogenetics deal with the role of few candidate genes, whereas pharmacogenomics use a genome-wide approach for polymorphisms determination. Once a drug is administered, it is absorbed and distributed to its site of action, where it interacts with targets like receptors and enzymes, undergoes metabolism, and is then excreted. Each of these processes might involve clinically significant genetic variations. Transplant physicians have always been interested in clinical pharmacogenetics. Because of the widespread use of azathioprine after transplantation, the paradigm of thiopurine Smethyltransferase polymorphism is well known in the transplant community [6]. This polymorphism is one of the best models for the translation of genomic information in order to guide patient therapeutics. More recently, the discovery of polymorphisms in genes encoding for proteins involved in the metabolism and the transport of cyclosporine and tacrolimus, two of the major drugs used after transplantation has increased this interest. Many attempts have been made to evaluate the association of these polymorphisms with calcineurin inhibitors pharmacokinetics, in order ultimately to guide their use in clinical practice.

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General Overview of Drug Metabolism and Transporters The field of pharmacogenetics has began with a focus on drug metabolism, but it has been extended to encompass the full spectrum of drug disposition, including a growing list of transporters that influence drug absorption, distribution, and excretion.

Drug Metabolism Most of the families of drug-metabolizing enzymes in humans have genetic variants. Many of these variants will translate into functional changes in proteins encoded. Metabolism usually converts drugs to metabolites that are more water soluble and thus more easily excreted [7]. It can also convert prodrugs into therapeutically active compounds, and it may even result in the formation of toxic metabolites. Pharmacologists classify pathways of drug metabolism as either phase I reactions (i.e. oxidation, reduction and hydrolysis) or phase II reactions (e.g. conjugation by acetylation, glucuronidation, sulfation, and methylation). The name of these reactions does not refer to a specific order in which they act and phase II reactions can preceed phase I reactions, and often occur without prior oxidation, reduction or hydrolysis. The cytochrome P-450 enzymes, a superfamily of microsomal drug-metabolizing enzymes, are the most important of the enzymes that catalyze phase I metabolism. Many families of enzymes are implicated in phase II metabolism. For immunosuppressive drugs, we will review the evidence for the role of uridine diphosphate-glucuronosyltransferase (UGT) and thiopurine methyltransferase (TMPT).

Drug Transporters Transport proteins have an important role in regulating the absorption, distribution and excretion of many medications. Members of the adenosine tri-phosphate (ATP)-binding cassette family of membrane transporters are among the most extensively studied transporters involved in drug disposition and effects. A member of the ATP-binding cassette family, Pglycoprotein, is encoded by the human ABCB1 gene (also called MDR1). A principal function of P-glycoprotein is the energy-dependant cellular efflux of substrates, including several immunosuppressive drugs. The expression of P-glycoprotein in many normal tissues suggest that it has a role in the intestinal barrier and in the excretion of xenobiotics and metabolites into urine or bile. It may play also a role in the blood-brain barrier and the regulation of foeto-maternal exchange through the placenta. Another member of the ATPbinding cassette family, MRP2 may also play a role in the transport of some immunosuppressive drugs, more specifically for the role of interactions between various drugs.

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Phase I Enzyme : Cytochrome P450 3a Polymorphisms Among the cytochrome P450 (CYP) enzymes, the members of the CYP3A subfamily are the most abundant and important drug-metabolizing enzymes in humans. They are present in liver, small intestinal mucosa and kidneys [8], and therefore contribute significantly to the absorption, oral first-pass metabolism, and systemic elimination of drugs [9, 10]. Of the four members of the CYP3A subfamily identified in humans, CYP3A4 and CYP3A5 are the main enzymes responsible for drug metabolism in adults [11, 12]. CYP3A4 is the major enzyme expressed in adult liver and small intestine in all the populations examined, with relatively uncommon sequence variations and limited functional alterations [12]. CYP3A5 expression is highly polymorphic, characterized by its presence in some but not all adults and by its variation with racial ancestry [12]. The four CYP3A genes are localized on chromosome band 7q21-q22.1 [13]. Regarding CYP3A4 gene, five promoter single nucleotide polymorphisms (SNPs) have been identified, and about twenty SNPs have been identified within the coding region (http://www.imm.ki.se/CYPalleles/). The most extensively studied polymorphism is the mutation of the CYP3A4*1B (-392A>G transition) in the 5’ regulatory region [14]. This polymorphism was initially called V(variant) allele. The frequency of this mutation is 4 % in a white population, and 67 % in black subjects, but no such variants have been found neither in Chinese subjects [15], nor in Japanese subjects [16]. The effect of the CYP3A4*1B variant allele on CYP3A4 activity is still controversial. From clinical studies, it was postulated that its presence reduces enzymatic activity [14]. Although the results of subsequent microsomal studies did not confirm that the CYP3A4*1B allele had any effect on enzymatic function [17], increased transcription was demonstrated in vitro, which theoretically results in higher protein expression and enzymatic activity in vivo [18]. Another explanation for CYP3A4*1 function may be secondary to its linkage with a CYP3A5 polymorphism [19]. As regards the CYP3A5 gene, what is considered as the wild-type form is designated as CYP3A5*1. The coding variants identified include CYP3A5*2, *4, *6, *8, *9 and *10 , which are present in various exons, especially exons 7 and 11 [20, 21]. Intronic SNPs are also present, some of which affect mRNA splicing and are an important cause of functionally defective alleles. CYP3A5*3 is the most common and functionally important variant allele in all the ethnic populations studied [20]. This non-coding SNP creates a cryptic consensus splice site in the pre-mRNA, resulting in the production of improperly spliced mRNA and a small amount of properly spliced mRNA [22]. The insertion causes a frame shift. The encoded protein is truncated, with loss of enzyme activity. The recent publication of the sequence for the entire CYP3A gene locus revealed the presence of two CYP3A pseudogenes, CYP3AP1 and CYP3AP2 [23]. Several SNPs have been reported within these pseudogenes. One SNP of CYP3AP1 was particularly explored regarding its impact on pharmacokinetic characteristics of CYP3As substrates. Because of a close association between the CYP3AP1 -44A>G SNP and CYP3A5 protein expression, Kuehl et al. identified the causal mutation within intron-3 of the CYP3A5 gene [22]. Subjects who carry the -44 G variant in the CYP3AP1 pseudogene display a higher level of CYP3A5 expression than those who do not.

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CYP3A5 genetic variants exhibit marked ethnic diversity. Pooling the information derived from different studies, it is possible to show that there is a significant difference in the frequency of the CYP3A5*3 allele in different populations. Thus, its estimation is 32 % in African-Americans, but 90-93 % in Caucasians [20]. The CYP3A5*1 allele is more frequent in populations of African descent, resulting in a higher CYP3A5 enzyme expression and function in those populations. The functional consequences of specific CYP polymorphisms may be difficult to demonstrate. Thus, it is hard to define the relative roles of CYP3A4 and CYP3A5, and to differentiate between their effects on drug metabolism, as their protein structure, function and substrates are similar. The situation is even more problematic in vivo due to the extensive overlap in the substrate specificities of CYP3A4/CYP3A5 and of the protein encoded by the ABCB1 gene. CYP and P-gp function synergistically to reduce intracellular concentrations of their substrates. Metabolism trough CYP3As is therefore affected by the presence and functional activity of P-gp, which is also genetically determined [24]. With regards to the functional consequences, the findings of studies with the use of specific in vivo probes, such as midazolam and nifedipine, do not support the notion that genetic origin may be important [25, 26].

Phase II Enzyme : Uridine Diphosphate Glucuronosyl Transferase Polymorphisms Glucuronidation is an important process of metabolism and detoxification performed by the UDP-glucuronosyl transferase (UGT) Supergene family [27]. UGTs are resident in the endoplasmic reticulum and catalyze the conversion of hydrophobic substrates to usually inactive hydrophilic glucuronides, which subsequently undergo renal and biliary elimination. Compounds targeted for glucuronidation include dietary constituents, therapeutic drugs, endogenous metabolites, hormones, as well as environmental carcinogens. The human UGT genes are differentially regulated in a tissue specific fashion in hepatic and extrahepatic tissues of the gastrointestinal tract [28-30]. Human UGTs have been divided into the UGT1 and UGT2 multigene families [31]. The human UGT1A gene locus is located on chromosome 2, which encodes at least 9 functional UGT1A proteins and 3 pseudogenes [32]. Four exons are located at the 3’ end of the UGT1A locus, which are combined with one of a consecutively numbered array of first exon cassettes towards the 5’ end of the gene locus to form individual UGT gene products. The tissue specific expression of the UGT1A gene locus has been well characterized and has been suggested to define tissue specific glucuronidation activity in the human digestive system [28]. An analysis of liver tissue led to the characterization of UGT1A1 [33], UGT1A3 [34], UGT1A4 [33], UGT1A6 [35], and UGT1A9 [36] cDNAs. Studies examining the human extrahepatic gastrointestinal tract have led to the identification of 3 extrahepatic UGT1A transcripts: UGT1A7 which is expressed in stomach and esophagus [28, 29], UGT1A8 which is expressed in colon and esophagus [28, 37, 38], and UGT1A10 which is expressed in gastric, esophageal, biliary and colonic tissue [28, 30, 39, 40]. In contrast to the UGT1A gene locus, the UGT2B and UGT2A genes have been mapped to chromosome 4, are individually encoded and comprise 6 exons [41, 42]. Transcripts have been identified for UGT2B4, UGT2B7 [19],

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UGT2B10 [43], UGT2B11 [44], UGT2B15 [45], UGT2B17 [46, 47] and UGT2A1 [42]. Except for UGT2B17 and UGT2A1, hepatic expression was detected for all UGT2B transcripts. Extrahepatic UGT2B expression has been shown for UGT2B7 in intestine, kidney and brain [48, 49], UGT2B10 and UGT2B15 in esophagus [29], as well as UGT2B10, UGT2B11, UGT2B15 and UGT2B17 in steroid sensitive tissues such as the mammary gland and the prostate [47, 50]. One report indicates that UGT2B4 is not expressed in the gastrointestinal tract [49]. The genetic multiplicity of the UGTs and their wide range of substrate specificities suggests that UGTs play an important role in human homeostasis and metabolism.

Phase II Enzyme : Thiopurine Methyltransferase Polymorphisms Partially purified Thiopurine Methyltransferase (TPMT) was initially characterized by Woodson and Weinshilboum in 1983 [6]. TPMT is encoded by a 27 kb gene on human chromosome 6p22.3 [51-53]. TPMT has ten exons, eight of which encode the 28 kDa protein. Exon 2 was observed in 1/16 human liver cDNA samples during initial cloning but has not been detected in most analyses. The hereditary nature of the TPMT deficiency in humans was initially identified by Weinshilboum and Sladek in a study of TPMT activity in red blood cells (RBC) [54]. This and subsequent studies determined the distribution of TPMT activity in RBC to be trimodal: ~ 90% of persons have high activity 10% have intermediate activity 0.3% have low or no detectable enzyme activity [52, 53] Patients with intermediate activity are heterozygous at the TPMT gene locus and the TPMT deficient subjects are homozygous for low activity alleles, as determined by molecular genetic and familial studies. Altered TPMT activity predominantly results from SNPs [55, 56]. Several TPMT alleles have been identified, including three alleles (TPMT*2, TPMT*3A and TPMT*3C) which account for 80–95% of intermediate or low enzyme activity cases [5559]. The mutant allele TPMT*2 is defined by a single nucleotide transversion (G238C) in the open reading frame (ORF), leading to an amino acid substitution at codon 80 (Ala>Pro) [60]. In a yeast heterologous expression system, this mutation led to a > 100-fold reduction in TPMT activity relative to wild type cDNA, despite a comparable level of mRNA expression [57]. The second and more prevalent mutant allele, TPMT*3A, contains two nucleotide transition mutations (G460A and A719G) in the ORF, leading to amino acid substitutions at codon 154 (Ala >Thr) and codon 240 (Tyr > Cys). When heterologously expressed in yeast or COS-1 cells, TPMT*3A had > 200-fold lower TPMT activity and immunodetectable protein compared to wild type cDNA [57]. Heterologous expression in yeast established an enhanced rate of proteolysis of mutant TPMT proteins encoded by TPMT*2 and TPMT*3A alleles, with degradation half lives of 15 min for both mutant proteins compared with 18h for the wild type protein [57]. Subsequent studies also established that TPMT*3B and TPMT*3C proteins have an enhanced rate of proteolysis when expressed in mammalian cells [61], consistent with the

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lower protein levels in individuals who inherit these alleles [62]. The mutant alleles TPMT*48 have also been identified during clinical genotype-phenotype analysis but the molecular mechanism(s) underlying the low activity has not been thoroughly evaluated for these variants. TPMT*4 has a G>A transition at the intron 9–exon 10 junction, which disrupts the final nucleotide of the intron at the 3′-acceptor splice site sequence [56, 59]. TPMT*5 was identified as a T146C transition in a heterozygous individual of undefined ethnicity who had intermediate TPMT activity [56]. This mutation results in a Leu > Ser amino acid substitution at codon 49. TPMT*6 was identified in a Korean subject with intermediate activity [56]. This A539T transversion in exon 8 results in a Tyr > Phe substitution at codon 180. TPMT*7 was identified in a single European subject with intermediate TPMT activity [58]. This allele contains a T681G transversion in exon 10, which results in a His > Glu amino acid substitution at codon 227. Lastly, TPMT*8 contains a single nucleotide transition (G644A) leading to an amino acid change at codon 215 (Arg > His) [63]. This allele has been identified in one heterozygous African American individual with intermediate activity. Based on the population genotype-phenotype studies performed to date, assays for the molecular diagnosis of TPMT deficiency have focused on the following alleles TPMT*2, TPMT*3A and TPMT*3C [55]. By using allele-specific polymerase chain reaction (PCR) or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to detect the three signature mutations in these alleles, a rapid and relatively inexpensive assay can be performed which will identify 80–95% of all mutant alleles [55, 56]. Population studies in Caucasian, East and West African, African-American, Chinese, Japanese, Thai and Southwest Asian populations have demonstrated the utility of this approach [55, 63-66] [67, 68]. However, the frequency and pattern of mutant TPMT alleles is different among various ethnic populations. For example, Southwest Asians (Indian, Pakistani) have a lower frequency of mutant TPMT alleles and all mutant alleles identified to date are TPMT*3A [65]. This is in contrast with East and West African populations where the frequency of mutant alleles is similar to Caucasians but all mutant alleles in the African populations are TPMT*3C [64, 66]. Among African-Americans, TPMT*3C is the most prevalent but TPMT*2 and TPMT*3A are also found, reflecting the integration of Caucasian and African-American genes in US populations [63]. More data on inter-ethnic variations in TPMT polymorphisms continue to emerge and accordingly the genotyping strategies of the future are likely to take into account such variations. The relationship between TPMT genotype and phenotype has been most clearly defined for TPMT*2, TPMT*3A and TPMT*3C in patients with leukemia and normal volunteers [55, 69]. TPMT*2 is the least common of the three alleles, representing 0.2–0.5% of all alleles in Caucasian populations [55, 65, 66, 69-71]. In Caucasian subjects, TPMT*3A is the most common of the three alleles, with a frequency of 3.2–5.7%, while TPMT*3C has an allele frequency of 0.2–0.8% [55, 66, 69-71]. The presence of TPMT*2, TPMT*3A, or TPMT*3C is predictive for phenotype, patients heterozygous for these alleles all have intermediate activity and homozygous subjects are TPMT deficient [55]. In addition, compound heterozygotes (TPMT*2/*3A, TPMT*3A/*3C) are also TPMT deficient [55], as would be expected. Most studies to date have used RBCs as surrogate tissues for measuring TPMT activity. A recent study demonstrated that TPMT genotype also influences TPMT activity in blast cells from leukemia patients [69], as would be predicted from previous studies of TPMT activity in these two tissues [72]. The median TPMT activity among 50 children and adults with ALL, who were homozygous for a wild type genotype, was 0.25 nU/mg protein compared with 0.1

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nU/mg protein in the five patients heterozygous for TPMT*3A [69]. However, a high degree of variability in TPMT activity was observed within both the homozygous wild type and heterozygous patient groups. In addition to a high degree of variability in TPMT activity within both the homozygous wild type and heterozygous groups, some individuals with a heterozygous genotype exhibit high activity whereas some homozygous wild-type subjects exhibit an intermediate phenotype. Such discrepancies are due to the fact that the SNPs discussed so far are not the only factors regulating catalytic activity. Population genetic studies have shown that the genotype at the ‘major locus’, which regulates TPMT activity accounted for only approximately two-thirds of the total variance in the level of RBC enzyme activity [73]. Other factors such as promoter polymorphisms, drug interactions, diagnosis, environment etc. could also play a role. Recent research has focused on a polymorphic tandem repeat initially described within a GC-rich area in the 5’-flanking region of the human TPMT gene [74]. The repeat elements consist of 17/18 base pairs (bp) and five alleles were initially reported that varied from 4–8 repeats in length [70]. The most common variable number tandem repeat (VNTR) alleles in the original Caucasian population sample had 4–5 repeat elements (alleles *V4 and *V5) [70]. Subsequent transient expression studies showed that these VNTR modulate the level of TPMT activity (to a much smaller extent in comparison to the effects of ORF-based SNPs) and demonstrated a decrease in reporter gene expression with increasing repeat numbers [74]. Yan et al. confirmed the modulatory role of the TPMT VNTR and described two additional VNTR alleles, *V3 and *V9 [75]. Linkage disequilibrium between the VNTR allele *V5 and TPMT*3A was noted [75]. A direct inverse relationship between RBC TPMT activity and the sum of repeat element number on both alleles, described in earlier studies, was not confirmed in this study, since the genotype *V4/*V4 had a lower activity than *V4/*V5 [75]. More recently, Alves et al. postulated that the number of particular motifs within the VNTR internal structure, rather than the sum of the undiscriminated number of repeats, is the potential causative factor affecting TPMT activity [76].

Drug Transporter : P-Glycoprotein The multidrug resistance gene, MDRI or ABCB1, encodes for the P-glycoprotein (Pgp), which belongs to the large adenosine triphosphate (ATP)-binding cassette (ABC) protein family, which includes various membrane molecules, all of them possessing ABC domains. Most ABC transporters are composed of two transmembrane domains (TMDs). Each TMD contains six membrane-spanning helices. Cytoplasmic nucleotide-binding domains generate energy for the transport process by hydrolysis of ATP. P-glycoprotein is one of the most thoroughly studied proteins among the ABC family, and a significant amount of information has been acquired regarding the structure and function of ABC transporters, based on analyses of Pgp. The MDR1 gene was initially discovered as the precursor to a protein associated with a major problem of cancer chemotherapy: failure caused by cross-resistance of tumors to many different cytotoxic agents. This phenotype, confirmed by experimental analyses in vitro (over-expression of MDR1 causes resistance in cultured tumor cells), is shared by other

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members of the transporter family that are closely related to MDR1 (multidrug resistance associated genes, MRP1-5) [77-79]. These transporter molecules protect cells against many drugs that are transporter substrates because they act as efflux pumps for xenobiotics, providing a barrier against the entry of various substances. Despite the high expression of Pgp in many cancers, where it poses a severe problem because it mediates cells that are resistant towards many chemotherapeutic agents, the physiological function of Pgp is not restricted to tumors. MDR1 is expressed in many normal tissues. One physiological function of Pgp could be in the adrenal cortex and might involve the metabolism of steroids [80]. In other tissues, Pgp acts as a cellular efflux pump to control the intracellular concentration of substances. Its cell- and organ-specific distribution, and its capacity to transport a broad range of compounds, might render Pgp an effective cellular protector against toxic substances that are Pgp substrates. Significant levels of Pgp are found in some key organs. For example, in the lower gastrointestinal tract (jejunum, ileum and colon), Pgp is found on the surface of epithelial cells, influencing intestinal drug absorption and, in some cases, constraining oral drug bioavailability [80-83], as well as possibly facilitating excretion across the intestinal mucosa. It is probable that Pgp has a protective function at the luminal surface of capillary endothelial cells at the blood brain barrier (BBB) and placenta [84, 85], controlling the amount of substances entering the brain or the fetus, respectively. In the liver and the kidney, Pgp is expressed in the biliary canalicular membrane of hepatocytes and in the brush-border membrane of proximal tubules [83], respectively. This distribution supports its role in the biliary and renal excretion of substances. The current model of the structure of Pgp and its mode of action is that two homologous halves, each with six transmembrane domains and one ABC-domain, recognize substrates, interact with each other and use energy (ATP) for transport. This is the result of the analyses of many variant Pgp molecules that have been recombinantly produced. Using these recombinant technologies, it has become clear that substrate specificity is affected by mutations in transmembrane domains 5, 6, 11 and 12. This indicates the presence of a drugbinding site in this region [86-91]. Also, intact ATP domains and the interaction of these with drug-binding sites are needed for transport function [92-94], (see [88] for a overview). One important question is whether hereditary variants of MDR1 account for the interindividual variability in the pharmacokinetics and pharmacodynamics of drugs. Mickley et al. have reported the first evidence of the presence of polymorphisms in the human MDR1 gene [95]. SNPs in Exons 21 and 24 (G2677T and G2995A) were observed in a population of tumor patients, in drug-resistant cell lines, in cells from refractory malignant malignomas and in healthy volunteers. A screen of the entire MDR1 gene for the presence of additional SNPs was undertaken by Hoffmeyer and coworkers [96], and led to the detection of 15 SNPs. Whether certain SNPs might be of functional consequence can be predicted, to some degree, from their position within the gene and protein. For example, SNPs that change amino acids, and thus possibly have an effect on protein function, are located at position A61G (replacement of Asn with Asp at position 21 of exon 2 of Pgp), a Phe–Leu change in position 103 next to the second transmembrane domain close to a glycosylation site, and a G1199A SNP in exon 11, which causes a Ser–Asn size- and charge-change close to the first ATPbinding domain. Nevertheless, no correlation between these SNPs with altered function or with Pgp activity has been reported, to date. However, another SNP, a C3435T change at a wobble position in exon 26, has been shown to have pharmacological consequences. The

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MDR1 genotype at the 3435 position has been shown by Hoffmeyer et al. to correlate with Pgp expression in the intestine, influencing the uptake of the orally administered Pgp substrate digoxin [96]. However, this association has not been found by all studies. The Tallele, particularly if homozygous, is associated with low intestinal expression of Pgp. Conversely, the corresponding C-allele is associated with increased Pgp levels. Individuals that carry the homozygous low-expressor (T)-allele, approximately 25% of the Caucasian population [97], show increased digoxin plasma-levels because of increased uptake.

Drug Transporter : MRP2 Multidrug resistance associated protein 2 (MRP2/ABCC2) is an ATP-dependent active transporter from the ABC family of drug transporters. MRP2 gene is composed of 32 exons encodes by an ∼ 45 kb gene located on chromosome 10q24 [98]. It consists of 1545 amino acids residues, with a molecular mass of ∼ 190 kDa of glycosylated proteins. Originally referred to as canalicular multispecific organic anion transporter (cMOAT), it is expressed on the bile cancalicular membrane, on the apical membrane of enterocytes, and renal tubules. It is establish that MRP2 plays a primary role in the biliary excretion and in restricting the oral absorption of its substrates including anionic drugs and glutathione and glucuronide conjugates of xenobiotics [98]. It is possible that the interindividual difference in the function of MRP2 affects the drug disposition. MRP2 deficience has been shown to be responsible from the Dubin-Johnson syndrome in humans, characterized by a conjugated hyperbilirubunemia. MRP2 is also important from a pharmacological point of view [98]. Firstly, MRP2 is responsible for the intracellularly formed glucuronide and reduced glutathione (GSH)-conjugates of clinically important drugs [98]. Secondly, MRP2 is also involved in the biliary excression of non-conjugated anionic drugs. These drugs released into the duodenum are then re-absorbed. Thus, efficient biliary excretion by MRP2 plays an important role in the entero-hepatic circulation, wich is responsible for maintening significant plasma concentrations of the drug. MRP2 is also expressed on the apical membrane of enterocytes. Along the intestinal tract, the MRP2 expression is higher in the proximal segments [99]. It is possible that MRP2 affects the oral absorption of its substrates as suggested with several compounds [98]. MRP2 is expressed on the apical membrane of the renal proximal tubule epithelia [100]. Its role in the urinary excression of anionic compounds needs to be clarify. It is also expressed on the apical membrane of syncytiotrophoblast in human placenta [101] and gall bladder epithelia [102], and on the luminal membrane of cerebral endothelial cells [103]. Inter-individual differences in MRP2 expression and/or function have been reported. Using normal tissues from patients undergoing surgical resection of liver metastasis, the MRP2 mRNA level was determined by te semi-quantitative RT-PCR method [104]. It was found that there is an approximatively 15-fold difference in the expression of MRP2 among 13 subjects [104]. Inter-individual differences in the expression level of MRP2 has also been reported in the small intestine. Using duodenal specimens taken by biopsy, it was found that mRNA and protein levels of MRP2 showed 6-fold and 8-fold difference among 16 subjects [105]. Many non-genetic factors have been reported as modulators of the MRP2 expression in vitro or in vivo in animal models, such as hepatic diseases, cholestasis, renal failure,

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cytokines, antioxydants, AhR ligants, CAR ligants, PXR ligants… However, some genetic factors may also contribute to these inter-individual variations. SNP analysis of MRP2 has been performed in 48 healthy Japonese subjects [106]. Six kinds of SNP have been identified. The allele frequencies of frequently observed C-24T (promoter) , G1249A (exon 10), and C3972T (exon 28) SNPs are 18.8%, 12.5%, and 21.9% respectively. Among them, only G1249A is associated with amino acid alterations, from Val to Ile at amino acid position 417. Only one respective heterozygote subject was observed from the 48 volunteers for C2302T (exon 18, Arg768Trp), C2366T (exon 18, Ser789Phe), and G4348A (exon 31, Ala450Thr), and their allele frequency was calculated to be 1%. Recently, the consequences of the SNPs that modify the amino acid sequence were characterized in vitro [107]. It was suggested that the most frequently observed V417I substitution may not affect the in vivo function of MRP2, whereas the much less frequently observed S789F and A1450T may be associated with the reduced in vivo function.

Azathioprine Clinicians have known for more than two decades that a subset of patients are intolerant to thiopurine therapy. TPMT activity polymorphism is an interesting paradigm in the field of pharmacogenetics. It has been demonstrated that azathioprine and mercaptopurine toxicity may be more frequent in genetically TPMT deficient patient [71, 108-110]. The cellular accumulation of TGN is inversely proportional to TPMT activity, since high TPMT activity shunts more drugs down the methylation pathway, leaving less for activation to cytotoxic TGNs [110, 111]. Conversely, TPMT-deficient patients accumulate very high TGN concentrations in tissues, including RBC. The use of standard doses of thiopurine drugs in patients with complete TPMT deficience could be fatal as in the tragic case of a 65-year-old heart transplant recipient with very low TPMT activity who died from neutropenic sepsis after receiving the usual doses of AZA [112]. In a recent evaluation of patients referred to St. Jude Children’s Research Hospital with a history of acute intolerance to thiopurine drugs, TPMT deficiency or heterozygotsy was found to be significantly over represented (65.2% of patients) when compared to the general population (10%) [113]. A review of their clinical course indicated that significant dosage adjustments for TPMT-deficient (e.g., 10- to 15-fold decrease) and heterozygous patients (e.g., 2-fold decrease) allow for successful treatment with thiopurines without acute dose-limiting toxicity [113]. There is also evidence that in patients with very high levels of TPMT activity, AZA efficacy is decreased. This may be related to its rapid metabolization [108, 114, 115]. It has been reported that in children with acute lymphoblastic leukemia treated with mercaptopurine, the outcome was significantly worse in patients with higher TPMT activity [110]. After renal transplantation, Chocair et al has suggested the same phenomenon [114]. It is thought that a higher TPMT activity should decrease 6-TGNs concentrations. 6-TGNs are the active and toxic products. Phenotypic characterization and molecular genetic methods have been developed to diagnose these patients [55, 58]. In addition, we have shown that the evolution of TPMT activity correlates with both short and long term results after renal transplantation [115]. We already reported that the use of azathioprine after renal

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transplantation is associated with a TPMT induction [115, 116]. TPMT induction is associated with a lower incidence of clinical acute rejection. TPMT activity induction may be secondary to various azathioprine metabolism. Azathioprine is converted into 6-MP. If azathioprine is only poorly metabolized, 6-MP levels will be low and 6-MP will not induce TPMT activity. On the contrary, if azathioprine is well converted into 6-MP, 6-MP levels will be high and TPMT activity will be induced. Therefore TPMT activity may be induced by the rise of the concentration of its substrate, 6-MP. The metabolism of azathioprine is not fully understood. It has been reported that glutathion-S-transferase is implicated in the first enzymatic step of azathioprine liver metabolism. In vitro inhibition by ethacrynic acid of GST-related azathioprine metabolism in a liver microsome model supports this hypothesis (personal data). Of note, GSTM1-1 polymorphism is not involved in the metabolism of azathioprine. The variations in TPMT activity are attributed to genetic polymorphisms within the TPMT gene. TPMT*3A, the most common variant allele responsible for low TPMT activity in Caucasians, encodes a protein with two single nucleotide polymorphisms (SNPs), G460A in exon 7 and A719G in exon 10, leading to modifications in the amino acid sequence. The phenotypic test for TPMT activity determination in red blood cells and, subsequently, DNAbased tests, were among the first pharmacogenetic tests to be used in clinical practice. A recent paper has investigated the possible prediction by TPMT genotyping for the incidence of AZA-induced myelotoxicity in renal transplant recipients [117]. Identification of the TPMT*2 variant allele was performed using a PCR method to detect the G238C mutation. The presence of TPMT*3 variant alleles was determined using a PRC-RFLP method to detect G460A and A719G mutations. This restrospective study demonstrated that in kidney transplant recipients with a non-funtional mutant allele the was a reduction of hemoglobin and hematocrit. Because of this predictive potential of TPMT for the azathioprine-induce toxicity for leukocytes, neutrophils and red blood cells, prospective TPMT genotyping has been made and phenotyping have been advocated by drug manufacturers to assist to identifying individuals with low or absent TPMT activity who are at risk for sever, life-threatening myelosuppression. Recently, the US Food and Drug Administration heard arguments to address TPMT genetic variability in product package information to address patients with poor or intermediate TPMT enzymatic activity and to encourage TPMT status testing. It was also suggested that labeling could be improved by additional information indicating that patients with poor or intermediate TPMT enzymatic activity may tolerate only 1/10th to ½ of the average 6MP dose. To optimize patients care pharmacogenomic evaluation of TPMT genotype coupled with phenotypic TPMT enzyme activity monitoring or TGN concentrations measurement should be used to increase regular hematological monitoring.

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Tacrolimus Tacrolimus and CYP3As Polymorphisms CYP3A4*1B Polymorphism In one study, the role of the CYP3A4*1B SNP was investigated in 64 renal transplant recipients treated with tacrolimus [118]. Among these patients, 10.9 % were heterozygous and 3 % were homozygous for the variant CYP3A4*1B allele. After 3 months of tacrolimus treatment, a trend was observed toward a lower dose-adjusted trough level in patients heterozygous or homozygous for the CYP3A4*1B allele than in those with the CYP3A4*1/*1 genotype. When patients carrying the CYP3A4*1B allele were compared with those carrying the CYP3A4*1/*1 genotype, a significant difference in the tacrolimus dose-adjusted trough level was found, and this difference was still significant at 12 months. Because tacrolimus trough levels were similar in both groups at months 3 and 12, the differences observed in tacrolimus dose-adjusted trough levels were due to a significant greater tacrolimus requirement in patients with the CYP3A4*1B allele than in those with the CYP3A4*1/*1 genotype. In the same study, 2 patients carried the CYP3A4*3 allele. However, because of the small number of patients, it was not possible to draw any conclusions. The authors simply observed that in these 2 patients, the tacrolimus dose-adjusted trough level was higher than that in patients with the wild-type genotype. CYP3A5*3 Polymorphism Convincing data has been obtained regarding the association between the CYP3A5*3 SNP and tacrolimus dose requirements. Thus, in studies with tacrolimus in renal, liver, heart or lung transplant recipients, higher dose-adjusted trough levels were reported in homozygous CYP3A5*3/*3 patients than in individuals who were heterozygous or homozygous for the wild-type allele suggesting that tacrolimus bioavailability is higher in the former [16, 118122]. In addition, the results of a study of the association between the tacrolimus dose requirement and the -44 G SNP in the pseudogene CYP3AP1 (termed CYP3AP1*3), which is linked to CYP3A5*3 and lower expression of hepatic CYP3A5 were consistent with the results of studies using the blood trough concentration as the phenotype [123]. In a cohort of pediatric heart transplant recipients, a significant difference in tacrolimus dose-normalized blood levels was found between CYP3A5 expressors (i.e. CYP3A5*1 carriers) and non-expressors (i.e. CYP3A5*3 carriers) [120]. Thus, the former required a higher tacrolimus dose to maintain the same blood level. In the study by Hesselink et al., CYP3A5*1/*1 was observed in 4.7 % of 64 tacrolimus-treated patients, whereas 25 % were heterozygous and 70.3 % homozygous for the CYP3A5*3 variant allele [118]. The tacrolimus dose requirement and dose-adjusted trough level differed significantly in these 3 groups. Significantly lower tacrolimus dose-adjusted trough levels were found in patients carrying a CYP3A5*1 allele than in those carrying the CYP3A5*3/*3 genotype, both at 3 and 12 months of tacrolimus treatment. This difference was due to a higher dose requirement for patients expressing the CYP3A5 enzyme. We observed the same results in a group of 80 French transplant recipient population [119], when we determined the frequency of the different CYP3A5 genotypes. In this series, we found that the frequency of patients homozygous for the *1 allelic variant (CYP3A5*1/*1) was 5 % (4/80), whereas 11 % (9/80) were heterozygous,

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and 84 % (67/80) were homozygous for the CYP3A5*3 allele. One month after tacrolimus introduction, the CYP3A5 genotype was significantly associated with the tacrolimus daily dose, with a gene-dose effect. The mean dose required to obtain the targeted trough concentration was significantly lower in patients with the CYP3A5*3/*3 genotype than in those with the CYP3A5*1/*3 and CYP3A5*1/*1 genotypes. The average concentration/dose ratio, indicating the exposure to tacrolimus, also correlated with the CYP3A5 genotype suggesting that the tacrolimus dose needed to obtain the targeted trough concentration was higher in patients expressing the CYP3A5 enzyme. Note that all but one of our patients with the CYP3A5*1/*1 genotype were black, and this allele is indeed more common in African Americans as reported by Xie et al. [20]. Therefore, the higher frequency of the CYP3A5*1/*1 genotype in black subjects may at least partly explain the worse outcome after renal transplantation observed in this population if the tacrolimus dose is not correctly adjusted from the very beginning of treatment. Haufroid et al. have confirmed the association between the CYP3A5*3 SNP and tacrolimus dose requirements in a population of 50 tacrolimus-treated renal transplant patients [122]. It is not known if the CYP3A polymorphism influence drug metabolism because of their influence on gut or liver metabolism. In one study, there was no difference in the half-life of tacrolimus between African and European Americans [124]. The difference in bioavailability, likely to be due to differential CYP3A5 expression, was not seen with intravenous administration. These latter observations suggest that the intestine may be the more important site in determining heterogeneity. CYP3AP1*3 Polymorphism These findings are also in agreement with those of MacPhee et al. They observed a correlation between the presence of - 44A>G SNP in the CYP3AP1 pseudogene and the tacrolimus dose requirement [123]. Subsequently, they evaluated, in the same population, the relationship between the presence of the CYP3AP1 genotype and early episodes of acute rejection [125]. Among the 178 patients included in their study, they found that most of those with the CYP3AP1*3/*3 genotype achieved the target concentration within the first 2 weeks of tacrolimus treatment, significantly earlier than those with the CYP3AP1*1/*1 genotype. The overall proportion of patients experiencing a biopsy-confirmed episode of acute rejection was not different in the genotype groups during the first 3 months after transplantation. However, the timing of rejection was significantly different, with earlier episodes in individuals with the CYP3AP1*1 allele than in those with CYP3AP1*3/*3 genotype. On the day of acute rejection, the tacrolimus blood concentration was significantly lower in the CYP3AP1*1/*1 and CYP3AP1*1/*3 groups than in the CYP3AP1*3/*3 group. The timing of the acute rejection episode may therefore be determined by the lack of the required trough level in a specific population.

Tacrolimus and MDR1 Polymorphisms Tacrolimus is also a substrate for P-gp [126]. A study of liver transplant recipients treated with tacrolimus demonstrated that intestinal mRNA expression of the MDR1 gene is inversely correlated to the concentration/dose ratio, which expresses the tacrolimus dose needed to

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obtain a given blood concentration [127]. In this study, patients with strongly expressed MDR1 required higher tacrolimus doses to achieve adequate trough concentrations. Significant interindividual variations in the expression and function of P-gp may be due to genetic factors. Among 80 renal transplant recipients, we found that tacrolimus dose requirements were lower in patients who had one or two mutant alleles in their exon 12, 21, and 26 SNPs, with a gene-dose effect [128]. The most important relation was noted for the exon 21 2677G>(T,A) SNP. The tacrolimus dose requirement was 40% higher, and the concentration/dose ratio 36% lower, in homozygous than wild-type carriers, suggesting that for a given dose, the tacrolimus blood concentration is lower in the wild-type patients. However, Goto et al. found no association between 10 SNPs of the MDR1 gene and the tacrolimus concentration/dose ratio during the first postoperative days after liver transplantation [16] and MacPhee et al reported only a weak association between the exon 26 3435C>T SNP and the tacrolimus dose requirement [123]. Taken into account the exon 12, 21, and 26 SNPs, we performed a haplotype analysis. We analyzed our results according to the 2 most frequent haplotypes, and showed a 61% increase in tacrolimus dose requirements in patients homozygous for the wild-type allele in the 3 SNPs.

Cyclosporine Most of the studies performed have demonstrated the absence of a relationship between the cyclosporine dose requirement or dose-adjusted trough blood concentrations and the presence of the CYP3A4*1B or CYP3A5*3 allele in stable renal transplant recipients [118, 129-131].

CYP3A4*1B Polymorphism The CYP3A4*1B mutation has not been found to be associated with cyclosporine exposure. Rivory et al., who studied normal individuals (n=100) and renal transplant recipients (n=117), found no significant difference between the pseudo-clearance of cyclosporine in subject with different CYP3A4 genotypes [132]. The authors therefore concluded that genotyping for the CYP3A4*1B polymorphism would not be useful for optimizing drug therapy. This conclusion was supported by Von Ahsen et al, who studied 124 stable Caucasian renal transplant recipients [129]. However, in another study of 14 healthy volunteers, the results were contradictory, as the cyclosporine AUC/dose ratio was affected by the CYP3A4*1B genotype [131]. Note, that in 11 of the 14 subjects (79 %) were African American in this study and the authors admitted that this might have skewed their results. To date, the largest series studied is the one from Hesselink et al., who analyzed 110 renal transplant recipients treated with cyclosporine [118]. For the CYP3A4*1B allele, they observed that the wild type genotype CYP3A4*1/*1 was present in 87.1 % of the patients, whereas 8.3 % were heterozygous and 4.6 % were homozygous for the CP3A4*1B allele. At 3 and 12 months after transplantation, no significant differences in cyclosporine dose, trough level or dose-adjusted trough level were observed among individuals with the CYP3A4*1/*1, CYP3A4*1/*1B or CYP3A4*1B/*1B genotype.

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CYP3A5*3 Polymorphism With respect to the CYP3A5 genotype, Hesselink et al. reported the results in their population [118]. Firstly, they found that most patients treated with cyclosporine (71.6 %) were homozygous for the CYP3A5*3 variant allele and were thus expected to lack CYP3A5 activity. Secondly, they observed that 26 of the 110 patients carried one CYP3A5*1 allele, and 5 (4.6 %), the CYP3A5*1/*1 allele. There was no significant difference between the 3 CYP3A5 genotypes as regards the cyclosporine dose, trough levels or dose-adjusted trough levels, 3 and 12 months after transplantation. They also showed that, when considering only Caucasians patients taking ciclosporin, a significant influence of CYP3A5 genotype on cyclosporine dose requirement was found at month 12 after transplantation, whereas this difference was not present for the whole study population. One major drawback of this work and of the most other published investigations is that only trough levels were studied, and these are only a crude marker of cyclosporine pharmacokinetics. The trough cyclosporine concentration is indeed known to be one of the worst pharmacokinetic parameters for predicting the total drug exposure, as measured by the 12-hour area under the curve [133]. The CYP3As enzymes and P-gp transporter are involved in various aspects of drug absorption, metabolism, and excretion, and it is important to evaluate their respective roles using more complex pharmacokinetic parameters and exposure indices. We performed a study to explore the associations between the CYP3A5 and MDR1 genetic polymorphisms, and complete individual cyclosporine pharmacokinetic parameters, in a large population of 106 stable Caucasian renal transplant patients [130] using a maximum a posteriori Bayesian estimation [134]. This estimation allows the determination of pharmacokinetic parameters and exposure indices by a limited sampling strategy, thus giving a more accurate evaluation of cyclosporine pharmacokinetics. The cyclosporine pharmacokinetic parameters studied were the peak drug concentration (Cmax), time to Cmax, mean absorption time, and terminal half-life. We also studied the association between these SNPs and exposure indices (area under the concentration-time curve over the 12-hours administration period and over the first 4 hours). In this population, we identified 9 (8.5%) CYP3A5 expressors (i.e. CYP3A5*1 carriers) and 97 (91.5%) CYP3A5 non-expressors. None of the pharmacokinetic parameters studied was associated with the CYP3A5 genetic polymorphism. Yates et al. recently performed another study with unexpected results [135]. They demonstrated that oral cyclosporine clearance was lower in CYP3A5 expressors than non-expressors. However, this observation was suggested to be an artifact due to a linkage in the very small population studied (n=10) between CYP3A5 non-expressors and MDR1 3435T allele carriers. Lastly in a recent study of 50 Caucasian renal transplant recipients, a slight association was detected between the CYP3A5*3 SNP and the cyclosporine dose-adjusted trough level, but it was only significant when blood concentrations were adjusted to the last cyclosporine dose and not to the total daily dose [122]. The CYP3A genotype determinations in the different studies are unlikely to be useful for cyclosporine dose optimization in clinical practice. The discrepancy found between the role played by CYP3As polymorphisms in the effects on tacrolimus and cyclosporine metabolism is not well understand but may be explained by the more complex metabolism of ciclosporin. Few data are available regarding the CYP3A5-dependent cyclosporine metabolism, and the respective roles of CYP3A4 and CYP3A5 enzymes in this metabolism have not yet been defined. CYP3A5 enzyme may also play a less important role in cyclosporine metabolism

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than CYP3A4. Since the effects of CYP3A4*1B variant is still controversial, this may explain that no convincing data exist for a clinically relevant role of CYP3A polymorphism and cyclosporine metabolism. Finally the relative role of P-gp is also unknown despite the results from Lown et al [136].

Cyclosporine and MDR1 Polymorphisms Regarding MDR1 gene and CsA, the results are also conflicting. A recent report failed to show an association between CsA pharmacokinetics and MDR1 polymorphisms [118]. Conversely, Yates et al [135] have shown an association between CsA oral bioavailability and MDR1 3435C>T polymorphism in a population of 10 patients (6 African Americans and 4 Caucasians). Surprisingly, in MDR1 3435T carriers, oral CsA clearance was higher, and the CsA AUC0-12 lower than in individuals with the wild-type CC genotype. Confounding factors such as ethnic origin might be responsible for different distributions of numerous genetic variants, including the MDR1 polymorphism, among individuals. In a study of 14 heart transplant patients [137], the authors reported that there was no association between the exons 12, 21, and 26 MDR1 SNPs, and either CsA pharmacokinetic parameters or CsA doses. Haplotype analysis including these three SNPs, revealed a non-significant trend toward higher CsA exposure in carriers of the T-T-T haplotype (considered to signify deficient P-gp expression and/or function) than in C-G-C (wild-type) haplotype carriers. When we studied the effects of four MDR1 SNPs with extensive pharmacokinetic parameters in 106 stable Caucasian transplant patients on CsA therapy, we found an association between the doseadjusted Cmax and the dose-adjusted AUC0-4 and the MDR1 C1236T SNP [130]. However, this association was weak, and haplotype analysis showed only a trend towards higher exposure to CsA and better intestinal absorption in the mutated haplotype. MDR1 SNP determination may therefore not be relevant in clinical practice for CsA treated patients. These results compared with tacrolimus data may be explained by the fact that CsA is a potent inhibitor of the P-gp function [138]. Pharmacologic inhibition might thus limit the potential impact of the genetic variations. Conversely, the tacrolimus doses needed to inhibit P-gp exceeded those normally required for the prevention of rejection following organ transplantation [139]. One very interesting approach is the role of enzymes or transporter donor genotype for the metabolism or the toxic effect of immunosuppressive drugs. This has been first demonstrated for liver transplant, since after transplantation, the hepatic enzymes are of donor origin. Goto et al. previously found that recipients of a CYP3A5*1/*1-carrying graft liver tended to have a lower C/D ratio of tacrolimus than those with a CYP3A5*3/*3-carrying graft liver 3 weeks after living donor liver transplantation, but the difference was not statistically significant [16]. More recently, the role of MDR1 donor genotype has been explored for the incidence of nephrotoxicity. P-glycoprotein could be a factor of major relevance for this complication. In animals, an inverse relationship among periglomerular and interstitial fibrosis, CsA deposits in renal tissue, and the level of P-gp expression in proximal tubular cells was observed. In agreement it has been shown in a histopathologic study that low expression of P-gp in renal parenchymal cells was associated with the occurrence of CsA nephrotoxicity, namely chronic interstitial fibrosis, arteriolopathy, and tubular vacuolization. Because CsA is a P-gp substrate, these data indicate that factors

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that modulate P-gp expression could have an impact on CsA nephrotoxicity as a result of an accumulation of CsA within renal cells in patients with low expression of P-gp. These findings conducted to test the hypothesis of whether patients with the P-gp low expressor genotype are more susceptible to developing CsA nephrotoxicity. Because recipient’s and donor’s genotype can be discordant and the donor’s genotype seems to be the relevant predictor for P-gp expression in the renal graft, genotyping for ABCB1 was performed in recipients and their corresponding donors. This assumption is corroborated by an earlier study that demonstrated clearly that the transplanted kidney itself rather than the recipient determines the susceptibility to CsA nephrotoxicity [140]. The authors found that the common polymorphism C3435T in the ABCB1 gene encoding for the renal epithelial efflux transporter P-gp was associated with an increased risk for CsA nephrotoxicity in patients after renal transplantation. Specifically, the 3435TT genotype of renal organ donors was significantly overrepresented in a group of 18 recipients with CsA nephrotoxicity who were subsequently switched to a calcineurin inhibitor–free regimen that contained either azathioprine or mycophenolate mofetil, which are not P-gp substrates. Approximately 40% of all organ donors with the TT genotype showed CsA nephrotoxicity approximately 2.5 yr posttransplantation in comparison with allografts with the CT or the CC genotype and an incidence of only 10% CsA nephrotoxicity. These data suggest a dominant role of the donor’s ABCB1 genotype for developing CsA nephrotoxicity because no association with the recipient’s genotype was observed.

Sirolimus Sirolimus (SRL), a macrocyclic lactone, is a novel immunosuppressive drug used to prevent allograft rejection after renal transplantation. SRL displays the characteristics of a critical-dose drug. Its blood concentration could not be predicted by a standard body or demographic measure, or by dose, and it shows high degrees of intra- and inter-individual variability in its pharmacokinetic characteristics [141]. Exposure to SRL is known to be closely associated with the acute rejection rate and the occurrence of side effects such as hypertriglyceridemia, thrombocytopenia, and leukopenia [141]. These associations between the systemic exposure and safety and efficacy profiles require a close therapeutic drug monitoring of SRL. Like cyclosporine and tacrolimus, SRL is a substrate for the CYP3A enzyme subfamily and for the multidrug efflux pump P-gp [142]. Regarding SRL, these proteins have been suggested to explain the low availability of SRL after oral administration, due to an extensive intestinal [142] and hepatic [143] metabolism by the CYP3A and to counter-transport by the P-gp in the intestine [142, 144]. After avoiding the pharmacokinetic interactions with steroids and calcineurin inhibitors, there is a highly significant association between SRL concentration/dose ratio and the CYP3A5*1/*3 SNP [145]. A lower SRL concentration/dose ratio was observed in the CYP3A5 expressors (i.e. CYP3A5*1 cariers) than in the CYP3A5*3/*3 carriers non expressing CYP3A5, suggesting that CYP3A5 nonexpressors require a lower sirolimus daily dose to achieve adequate blood concentration. Patients with the CYP3A5*1/*1 genotype are more likely to have a high rate of liver and intestinal metabolism, and therefore require a higher daily dose to achieve adequate blood sirolimus levels. This pharmacogenetic effect may be partially abrogate by pharmacokinetic

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interactions. There is also an association between the CYP3A4*1B polymorphism and the SRL concentration/dose ratio [145]. However, the functional significance of this SNP on the enzyme activity is still a matter of debate. Even though, SRL is also a substrate for P-gp, the MDR1 gene product, no association between the MDR1 SNPs and the SRL concentration/dose ratio was demonstrated for the time-being.

Mycophenolate Mofetil Mycophenolate mofetil (MMF), an ester prodrug of mycophenolic acid (MPA) that acts by inhibiting the synthesis of purines, has been advocated as a novel drug for the prevention of acute graft rejection. It can specifically suppress proliferation of T and B lymphocytes, theoretically leaving haemopoiesis and polymorphonuclear neutrophil number and activity unchanged [146]; this feature has been presented as a major advantage over azathioprine. Mycophenolate mofetil reduced acute rejections of organ transplantation in animals and in people in open-label studies that also included cyclosporine. Three large registration trials found that mycophenolate mofetil reduced acute rejection by 30 to 50% compared with azathioprine [147, 148] or placebo [149] at 6 months after transplantation. MMF is part of standard treatment for preventing rejection of transplanted kidneys and, more recently, of heart, liver, lung, and bone marrow (Review in [150]) Nowadays, this drug is used by most transplant centres worldwide as part of maintenance immunosuppression regimens. MMF is a pro-drug that is rapidly and almost completely absorbed from the gut where it is de-esterfied to form MPA. MPA is extensively glucuronidated by several UGTs into an inactive 7-Oglucuronide (MPAG) and, to a lesser extent, to the pharmacologically active acylglucuronide (AcMPAG) [151, 152]. The latter metabolites are excreted via the kidney, at least in part by MRP2–mediated tubular transport [153]. MPA and MPAG are subject to enterohepatic circulation and recirculation. The biliary excretion of MPA/MPAG and subsequent distal absorption and reabsorption require several transport mechanisms including organic anion transporting polypeptides and multidrug resistance related proteins and the active involvement of UGTs. In the gut, bacterial deconjugation transforms MPAG back into MPA, which is absorbed from the colon. Because of this enterohepatic circulation, there is a second peak of MPA plasma concentration, occuring 6-12 h after oral administration. Finally, the majority of the absorbed MMF is eliminated by the kidney as MPAG [151]. Interindividual polymorphic regulation of the UGT1 and UGT2 genes has been already reported for the intestine, but not for the liver [27]. The UGT genes subject to such regulation, UGT1A1, UGT1A6, UGT2B4 and UGT2B7 contribute to MPA glucuronidation. As a consequence of the interindividual polymorphic UGT gene regulation, the rate of metabolism and the therapeutic efficacy or toxicity of drugs may be individually influenced by glucuronidation during or prior to resorption in the small intestine. With respect to MPA metabolism, this fact is of importance, since the phenolic metabolite MPAG is not pharmacologically active, while the acyl glucuronide (AcMPAG) is both able to inhibit IMPDH in vitro and may also possess toxic potential. In a study using cDNA-expressed enzyme in COS-7 cells, Mackenzie reported that three closely related UGTs (UGT1A8, UGT1A9 and, UGT1A10) have the capacity to glucuronidate MPA [154]. Of these, UGT1A8

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[28] and UGT1A10 [30] are present only in the gastrointestinal tract and hence can not be responsible for MPA glucuronidation in liver and kidney. In contrast, UGT1A9 gene products are found in the liver, but more abundantly in the kidney, and are therefore likely to play a role in MPA glucuronidation by this organ. A recent study with the aim to identify the UGT isoforms involved in the MPA phase II metabolism showed that UGT1A9 is the key UGT responsible for glucuronidation of MPA to its inactive 7-O-glucuronide (MPAG) and is predominantly active in the liver, kidney, and intestine [155]. UGT2B7, predominantly active in the liver, is responsible for generation of the active AcMPAG metabolite which has been associated with clinical side effects of MMF. UGT1A8 is mainly located in the gastrointestinal tract and has also been implicated in the metabolism and first-pass effect of MPA whereas under normal circumstances, UGT1A9 is responsible for approximately 40% to 50% of intestinal MPAG production [155]. In human liver microsomes, UGT1A9 expression varies by 17-fold, and this correlates with specific SNPs in the gene promoter region (T-275A, C-2152T) that result in significantly higher glucuronidation rates of MPA compared with those in wild-type individuals [156]. In clinical settings, a recently published study evaluated the impact of specific SNPs of the UGT1A9 gene that respectively resulted in increased in vitro glucuronidation activity (T275A and C-2152T) and decreased enzymatic activity (UGT1A9*3 mutation) in 95 de novo renal transplant recipients [157]. The T-275A and C-2152T SNPs of the UGT1A9 gene promoter have been found to be associated with significantly lower MPA exposure in renal recipients treated with 2 g mycophenolate mofetil daily, and part of this effect has been suspected to be secondary to the interruption of enterohepatic recirculation of MPA. Moreover, it has been shown in recent meeting that one UGT2B7 SNP (G-840A) may increase the blood concentrations of AcMPAG metabolite and that the C-24T MRP2 gene promoter SNP increases MPAG biliary excretion.

Interactions Interactions between CYP3A Enzymes and P-glycoprotein As already stated, the situation is problematic in vivo due to the extensive overlap between the substrate specificities of CYP3A4/CYP3A5 enzymes and the protein encoded by the MDR1 gene. An interesting approach would be to propose a multigenic approach to explore the relative roles of CYP3As and MDR1 genetic polymorphisms. MacPhee et al. analyzed their results using this approach [123]. As already mentioned, they found that the CYP3AP1 pseudogene genotype -44A>G was strongly associated with the blood concentration achieved by a given dose of tacrolimus, with a weaker association for the MDR1 C3435T genotype [123]. The association AATT (AA for the CYP3AP1*1B/*1B genotype and TT for the MDR1 genotype at position 3435) was chosen as the reference genotype against which to compare the others. This genotype may be considered to have the lowest CYP3A and P-gp activity and therefore to be associated with the highest bioavailability of tacrolimus. When the whole group was analyzed, the AGCC, AGCT, AGTT, GGCC, GGCT, and GGTT genotypes of the CYP3AP1 pseudogene and MDR1 gene differed significantly from AATT. Possession of a

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CYP3AP1*1 (G-44) allele was associated with 1.85 fold and 2.25 fold mean reductions for AG and GG respectively with the blood concentration of tacrolimus achieved by a given dose. Possession of the MDR1 CC genotype resulted in a mean 1.44 fold reduction in this concentration. Another study has used a multiple regression analysis to quantify the role of each polymorphism in dose requirement [122]. Using this approach, the authors have shown that CYP3A5*1/*3 polymorphism explained up to 45 % of the variability in dose requirement in relation with tacrolimus use. A very similar approach was recently used by Goto et al [16, 160]. They evaluated the role of MDR1 and CYP3As genotypes in donors and recipients of liver transplantation, as well as the influence of polymorphisms on mRNA expression and the tacrolimus dose-adjusted trough concentration. They showed that during the week after liver transplantation, the pharmacokinetics of tacrolimus are affected by flux via P-gp in the intestine, but that after the first week, it is mostly liver metabolism that contributes to the excretion of tacrolimus. The same authors found that carriers of the CYP3A5*1/*1 genotype require a high dose of tacrolimus to achieve the target concentration [16]. It is known that there is a close relationship between these proteins. MDR1 may limit CY3A metabolism in vivo [13]. Moreover, we found in Caucasians that the CYP3A5*3 allele carriers were more likely to possess the MDR1 3435T allele [159]. Differences in the CYP3A activity in the liver and in the P-gp function in the gut have been suggested to determine approximately 80% of the interindividual variability in oral CsA disposition. In a study of 25 stable renal transplant patients treated with the Sandimmum® formulation of CsA, Lown et al reported that liver CYP3A4 accounted for 56% of the variability in the apparent oral clearance of CsA, whereas the enterocyte content of P-gp accounted for 17% [136].

Interactions between Various Immunosuppressive Drugs Another problem is the interaction between immunosuppressive drugs. Some of these pharmacokinetic interactions may be related to enzyme or transporter. One good example is the interaction between cyclosporine and MMF. Several authors have shown that MPA exposure is significantly lower in CsA compared with tacrolimusbased immunosuppressive regimens. The reported differences is as high as 30-40 % and clinically relevant. In a rat study, Hesselink et al. have shown in a rat model that in the case of the absence of the drug-transporting protein Mrp2, the pharmacokinetics of MPA and MPAG are comparable between rats receiving either CsA or tacrolimus as co-medication [161]. These results demonstrated in vivo that Mrp2 is the transporter mainly responsible for the excretion of MPAG into bile and that inhibition of Mrp2 by cyclosporine is one mechanism which can explain the interaction between cyclosporine and MPA. In human, there are no published data regarding this finding. However, Marquet et al. have reported that the C-24T SNP located in the promoter region of the MRP2 gene might increase the bilary transport of MPAG (personal communication). Cyclosporine may interfere with this transport. These preliminary results have still to be confirmed.

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Perspectives in Pharmacogenomics and Solid Organ Transplantation Pharmacogenomics has an enormous potential ability to yield a powerful set of molecular diagnostic methods that will become routine tools enabling clinicians to select medications and drug doses for individual patients. Contrarily to phenotypic tests (e.g. enzymatic activity) whose results may vary with environmental factors, age, drug interactions, or food, a patient's genotype only needs to be determined once for any given gene, because it does not change, except for rare somatic mutations. Genotyping may therefore be performed during the pretransplant evaluation, with results available at the time of transplantation. However, several critical issues must be considered as strategies are developed to elucidate the inherited determinants of drug effects and before pharmacogenomics can be applied to clinical practice.

Pharmacogenomic Approaches The first one is that the inherited component of the response to drugs is often polygenic. Approaches to elucidate the polygenic determinants of this response include the use of anonymous SNPs maps to perform genome-wide searches for polymorphisms associated with drug effects, or candidate-gene strategies based on existing knowledge of a medication's mechanisms of action, metabolism and disposition. This latter strategy has the advantage of focusing resources on a manageable number of genes and polymorphisms that are likely to be important. Immunosuppressive drugs are good candidates for this approach, because their metabolic pathways are well known, due to the importance of drug interactions. High-density maps of the SNPs in the human genome might allow these SNPs to be used as markers of drug responses, even if the drug target remains unknown, thus providing a “drug response profile” associated with contributions from multiple genes to a drug response phenotype.

Phenotype Characterization Another important challenge in defining pharmacogenetic traits is the need for wellcharacterized patients who have been uniformly treated and systematically evaluated, which makes it possible to quantify drug responses objectively. In solid organ transplantation, therapeutic monitoring of immunosuppressive drugs is already used routinely in clinical practice and can be of great value. Genomic DNA should be obtained from all patients enrolled in clinical drug trials, with appropriate consent to permit pharmacogenetic studies. Because of marked population heterogeneity, pharmacogenomic relations must also be validated in different ethnic groups to avoid confounding factors. To prove the usefulness, prospective clinical studies must show that genotype determination before transplantation allows better use of a given drug regarding pharmacokinetics, and improves the safety and clinical efficacy.

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Future Trends Another approach for pharmacogenetics/pharmacogenomics is to apply this technique to drug targets. Apart from transplantation, such approaches have already been described for many examples, including angiotenin-converting enzyme (ACE) polymorphism and the renoprotective effects obtained with ACE-inhibitors, or the progression or regression of coronary atherosclerosis and fluvastatin use (reviewed in [5]). However, data for immunosuppressive drugs are still lacking for this exciting approach. Finally, increasing technological advances in molecular biology such as the combined use of SNPs and DNA chip technology, and biocomputing, will allow the screening and analysis of thousands of SNPs in one assay. The use of these automated techniques might also allow the determination of data regarding pharmacogenomics, immunogenetics (e.g. CCR5, TGF-β, cytokine, etc.), or identification of the genes involved in specific complications (e.g. glutathione-S-transferase [162] or IL10 promoter polymorphisms [163] and skin cancer). The ultimate goal could be the determination of the genetic background during the pre-transplant assessment for a given individual in order to determine his specific profile. Discoveries based on emerging information, and knowledge acquired by the genomic and genetic sciences will then greatly contribute to achieving personalized medicine.

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In: Progress in Kidney Transplantation Editor: Dominick W. Mancuso, pp. 89-104

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter V

Brain-Death-Induced Gene Regulatory Networks in Donor Kidneys Frans Gerbens1, Rutger J. Ploeg2 and Theo A. Schuurs*2 1

Department of Genetics, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands 2 Department of Surgery, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

Abstract Brain death has been shown to affect hormone regulation, hemodynamic stability and inflammatory reactivity. In transplant models, kidneys, livers and lungs retrieved from brain-dead (BD) rats do suffer from increased primary non-function and deteriorated graft survival. However, the mechanism(s) by which brain death leads to these processes have remained unclear, yet. To further unravel these mechanisms we performed DNA microarray studies with RNA isolated from kidneys from BD rats. Kidneys from sham operated animals were used as controls. Oligonucleotide arrays were manufactured using the Sigma/Genosys Rat Oligonucleotide Library harbouring 4854 unique, gene-specific rat sequences. In kidneys from normotensive donors 63 genes were identified that were either up (55) or down (8) regulated. By using PubMed searches and GeneOntology, genes were assigned to different functional clusters: Metabolism and Transport (including water channel Aqp-2), Inflammation and Coagulation (containing the largest number (16) of up regulated genes), Growth/Regeneration and Fibrosis (including genes as KIM-1 involved in tubular regeneration) and Defense and Repair (with the cytoprotective genes HO-1, Hsp70 and MnSOD2). Also genes encoding transcription factors and proteins involved in signal transduction (such as Pik3r1) were identified. In addition, Pathway AssistTM software was used for further interpretation of our microarray data in the context of pathways, gene regulatory networks and protein interactions. These type of analyses *

Adress of correspondence: Dr. Theo A. Schuurs; University Medical Center Groningen; University of Groningen; Department of Surgery; CMC V, Y2144; Hanzeplein 1; 9713 GZ Groningen; The Netherlands; Phone: (+31) 50 3619369; Fax: (+31) 50 3632796; E-mail: [email protected]

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Frans Gerbens, Rutger J. Ploeg and Theo A. Schuurs will allow us to create a better understanding of the brain-death-related biochemical pathways which are either induced or repressed. Ultimately, these approaches will help us to design specific interventions in the brain dead donor to better maintain or even repair organ viability and protect against ischemia /reperfusion injury.

Introduction The majority of kidneys used for transplantation are retrieved from brain dead (BD) donors. In the past years it has become evident that brain death in the donor should be regarded as a major risk-factor affecting organ viability, post-transplant function and graft survival in organ transplantation [1]. In the USA, a 5-year graft survival of 72% with a halflife of the graft of 13 years is seen after mismatched living transplantation [2]. In contrast, a 5-year graft survival of 62% and a transplant half-life of 9 years only has been recorded in the cadaveric transplantation setting. These differences cannot be fully attributed to HLAmatching, since the HLA-matching grade in cadaveric transplant combinations are frequently even better than in the living unrelated combination. Also, a shorter cold ischemia time in the living transplantation setting is unlikely to be the only explanation for superior results since cold ischemia times leading to delayed graft function after kidney transplantation play mainly a part when they are beyond 24 hours [3-5]. Clinical data and studies in animal brain death models have shown that donor brain death influences the hemodynamic stability, hormone regulation and in addition, the inflammatory reactivity in donor organs-to-be: Elevated expression of inflammatory mediators such as cell adhesion molecules and cytokines is associated with an increasing number of infiltrating macrophages, polymorphonuclear cells (PMN’s) and T-cells in liver, kidney, heart and lung [6-10]. Consequently, these deleterious changes will ultimately lead to histological damage, decreased function and lower graft survival [11, 12]. Although the finding that brain death affects the quality of future donor organs is nowadays generally accepted, the exact mechanism that is responsible for the brain-death induced organ damage has not been unraveled yet. To further identify the molecular changes induced by brain death we have performed DNA microarray studies with RNA derived from kidneys of BD rats [13]. Gene expression profiling allows for identification of individual differentially expressed genes but also enhances overall insight in activated or repressed pathways. By screening almost 5000 unique rat gene-specific sequences, involved in a wide range of biological pathways, 63 genes were identified as being differentially expressed. Most genes were categorised in different functional groups: Metabolism and Transport, Inflammation and Coagulation, Growth/Regeneration and Fibrosis, and Defense and Repair. The microarray experiments and subsequent classification of differentially expressed genes in functional groups has given us new and broader insights in brain-death-related processes potentially affecting donor organ viability and the balance of injury and repair. In addition, we have applied the Pathway AssistTM software in an attempt to further obtain a better interpretation of our microarray data. This type of analysis will allow us to identify regulatory key-players and yield information on the upstream regulatory processes. Ultimately, these approaches will enable the design of specific interventions to better

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maintain or even repair organ viability already in the brain dead donor and protect against ischemia /reperfusion injury during the transplantation cascade.

Materials and Methods Animals and Experimental Design Brain Death Adult male Wistar rats (HSD.Cpb:WU, 300-350 g) were used. Animals received care in compliance with the guidelines of the local animal ethics committee according to Experiments on animals Act (1996) issued by the Netherlands Ministry of Public Health, Welfare and Sports. Rats were randomly divided into three groups. All animals were anaesthetized and intubated. A frontolateral trepanation was made and a balloon catheter inserted in the extradural space. Inflating the catheter during one minute led to increased intracranial pressure and induced rapid brain injury leading to immediate brain death, simulating a condition comparable to acute isolated cerebral trauma in man. Animals (n=9) received hemodynamic support to achieve normotension after brain death induction. After brain death induction animals were ventilated for 6 hrs with O2/air. Control animals (n=9) were sham-operated and ventilated for 6 hours. Kidneys were harvested and flushed with UW preservation solution to remove blood cells that could interfere with the expression analyses, and snap frozen. Detailed description on anesthesia and ventilation, surgical procedures, induction of brain death and donor management have been presented before [14].

RNA Isolation and Microarray Experimental Set-Up Frozen kidney tissue was homogenized in liquid N2, using mortar and pestle. Total RNA was isolated using the SV Total RNA isolation kit (Promega, Leiden, The Netherlands) according to the manufacturer’s protocol. Integrity of total RNA was analyzed by gel electrophoresis and RNA samples were verified for absence of genomic DNA contamination by performing RT-PCR reactions in which the addition of the reverse transcriptase enzyme was omitted. Each experimental condition (control and normotensive BD rats) was divided into two groups. In addition, to minimize eventual biases due to biological variation of the individual animals, each group contained equal amounts of pooled RNA from 4-5 rat kidneys. Furthermore, each sample was analyzed in two-fold either being labeled with Cyanine 3 or Cyanine 5 fluorophores (Amersham Biosciences, Roosendaal, The Netherlands).

Microarrays Microarrays contained the complete Rat oligonucleotide library (Sigma, Zwijndrecht, The Netherlands) printed in triplicate on GAPSII slides (Corning Life Sciences BV, SchipholRijk, The Netherlands). The rat oligonucleotide library consists of 4854 65-mer oligo’s representing genes from a diverse range of functionalities. Positive and negative controls

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were also represented on the arrays. Microarrays were obtained from the Department of Anthropogenetics at the University of Nijmegen, The Netherlands.

Probe Construction, Hybridization and Data Acquisition Labeling of cDNA molecules was performed essentially according to a protocol described at http://pga.tigr.org/sop/M004_1a.pdf. Briefly, for each RNA sample, first strand amino-modified cDNA was synthesized by oligo-dT primed reverse transcription from 20 μg total RNA using Superscript II reverse transcriptase (Invitrogen, Breda, The Netherlands) in the provided buffer and in the presence of 0.5 mM of dATP, dCTP, dGTP, 0.3 mM dTTP and 0.2 mM amino-allyl dUTP (Sigma). After 16 h incubation at 42°C the RT reaction was stopped and cDNA was purified as described [15]. cDNA was fluorescently labeled with Cyanine 3 or Cyanine 5 fluorophores (Amersham), as described. Labeled cDNA was purified using Microcon YM-30 columns (Millipore BV, Amsterdam, The Netherlands). Each probe was put together by mixing the labeled cDNA reactions from the appropriate samples together with 15 μg poly-dA DNA (Qiagen, Hilden, Germany) and 7.5 μg human Cot-1 DNA (Invitrogen) and was heated at 95°C for 3 minutes, cooled to room temperature, mixed with an equal volume of preheated (42°C) hybridization buffer to a final concentration of 25% formamide, 5x SSC and 0.1% SDS. Hybridizations were performed under lifterslips (Erie Scientific, Portsmouth, UK) within hybridization chambers (Telechem, Sunnyvale, USA) in a waterbath at 42° C for 16 h, simultaneously. After hybridization the slides were washed, dried and scanned at 10 μm resolution in a GMS 428 laser scanner (Affymetrix, Santa Clara, USA). Image intensity data for each array-feature was extracted by ImaGene 4.2 software (BioDiscoveries, Marine Del Rey, USA).

Data Analysis For each array, raw median signal intensity data for each spot and each fluorophore was collated, filtered to exclude irregular and empty spots as determined by the ImaGene software. Initial data reduction analysis using principal component analysis (SPSS version 10) revealed no dependence between single color readings from both fluorophores and therefore these readings were treated separately. For each contrast, respective single color data from one sample was combined with its respective single color data from another sample. To account for dye bias, resulting data was normalized by intensity dependent regression (lowess) of the log transformed ratios by BRB ArrayTools version 3.0.1 as developed by Dr. Richard Simon and Amy Peng Lam http://linus.nci.nih.gov/BRBArrayTools.html). The resulting log transformed ratios for each gene were analyzed by the statistical package SAM [16]. Only genes were analyzed with no more than 25% missing values. For each array triplicate observations for each gene were averaged. For each contrast 4 individual observations per gene were collated. Missing data was imputed using the K-nearest neighbor method and complete permutation of the data was performed to determine the false discovery rate. One class response analysis was performed to identify genes significantly regulated

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between both classes and in total 24 permutations of the samples were performed to determine the false discovery rate. A list of genes significantly up or down regulated encompassing less than one median false positive gene was used for validation experiments and further analysis. Significant genes were assigned to different functional clusters based on known or putative biological function of the encoded proteins, as determined by searches on PubMed and by using GeneOntology (Gene Ontology Consortium) classifications.

Genetic Network Building The 63 significantly differentially expressed genes were used to identify gene regulatory networks using Interaction Explorer™ Software PathwayAssist (Stratagene). A genetic network was constructed based on information from the embedded ResNet 3.0 database (Ariadne Genomics), which contains 500,000 functional links for about 50,000 proteins, extracted from 4.5 million MEDLINE abstracts and full text articles (as of February 26, 2005). Interaction Explorer provides a method for searching objects individually by keyword, string, or attributes such as expression, regulation, binding, biological process, cellar localization, and others. From the 63 significantly differentially expressed genes, 56 could be converted to Entrez Gene Identifiers and were included in the analysis. The ResNet database had no information for another 22 genes resulting in a final genetic network of 34 genes. The total number of connections (1000+) was subsequently manually curated leaving 800+ connections.

Results Hybridization Oligonucleotide Arrays When expression profiles of kidneys from BD rats were compared with control rats, regarding a twofold change in expression as the cut-off point of a sequence being differentially expressed, we were able to identify 63 genes that were either up or down regulated. The expression of 56 genes had differential values between 2 and 5, whereas 7 genes ranged between 5 and 10. By far, most of the genes [55] were up regulated.

Brain-Death-Responsive Genes The differentially expressed genes were clustered according to the putative or known biological function of their encoded proteins, as shown in Table 1. The most frequent changes were observed in expression of pro-inflammatory and coagulatory markers, such as E- and Pselectin, alpha- and beta-fibrinogen, Il6, Ccl2, Mob-1 and MGSA. These changes primarily reflect a massive and intense immune activation of BD donor kidneys. Another important group of genes differentially expressed in BD donor kidneys is the transcription factor cluster. A relevant number of these transcription factors belong to the

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group of so called immediate early genes (IEGs): Egr-1, Egr-3, ZFP36, Atf-3 and Zf-9. Although signal transduction cascades primarily act via activation or phosphorylation of the proteins involved, we also found a number of genes encoding signal transduction proteins to be differentially expressed (mostly up regulated). In contrast to the deleterious processes mentioned above, changes in gene expressions involved also defense- and repair mechanisms (e.g. the heat-shock genes HMOX1 and Hspa2). In addition, elevated mRNA levels for genes like SPP-1 (osteopontin), KIM-1 or GADD45a (assigned to other gene-clusters) were not only just markers for cell damage, but strongly suggest that repair processes have already started in BD damaged kidney tissue. Table 1. Expression changes in brain-dead donor kidneys given as fold-variation compared to controls. Only those genes were included when 2 fold up or down regulation was measured and when significance was reached. Genes are grouped based on known or putative function as determined by searches on PubMed and by using Gene Ontology classifications Gene ID

Gene description

1. Metabolism and transport AF144756 Fatty acid binding protein 4, adipocyte (FABP4; ALBP) K01594 5S ribosomal RNA X13295 Lipocalin (LCN2) AF157026 Type IIb sodium phosphate transporter NM_012909 Aquaporin 2 (AQP2) 2. Immune activation and coagulation M86536 Chemokine (C-X-C motif) ligand 1 (Cxcl1; MGSA; KC protein) U17035 Chemokine (C-X-C motif) ligand 10 (Cxcl10; Mob-1) AF058786 Chemokine (C-C motif) ligand 2 (Ccl2; JE/MCP-1) NM_012589 Interleukin 6 (Il6) L18891 S100 calcium binding protein A8 (calgranulin A) (S100A8; MRP8) M98820 Interleukin 1 beta (Il1B) M80367 Guanylate nucleotide binding protein 2 (GBP2) NM_012881 Secreted phosphoprotein 1 (Osteopontin) (SPP1) M35601 Fibrinogen, alpha polypeptide (FGA) M35602 Fibrinogen, B beta polypeptide (FGB) U05675 Fibrinogen, B beta polypeptide (FGB) NM_012794 Glycosylation dependent cell adhesion molecule 1 (GLYCAM1) L25527 Selectin, endothelial cell (SELE) NM_013114 Selectin, platelet (SELP) NM_019153 Fibulin 5 (FBLN5) 3. Signal transduction AF239157 DEXRAS1, dexamethasone-induced 1 (RASD1) NM_013005 Phosphatidylinositol 3-kinase, p85 (PIK3R1)

Brain Dead/ Control 0.30 0.46 4.3 2.7 0.40 8.5 5.5 7.3 5.0 2.2 2.5 2.6 5.5 6.5 2.9 2.1 2.9 3.1 2.1 0.48 3.3 2.7

Brain-Death-Induced Gene Regulatory Networks in Donor Kidneys Table 1 Continued Gene ID

Gene description

3. Signal transduction (Continued) AF205438 Tribbles homolog 1 (Drosophila) (TRIB1) M74295 Ras homolog gene family, member B (RhoB) NM_012911 Arrestin, beta 2 (ARRB2; Beta-Arr2) NM_019292 Carbonic anhydrase 3 (CA3) 4. Transcription factors NM_017086 Early growth response 3 (EGR3) NM_012551 Early growth response 1 (EGR1) NM_012912 Activating transcription factor 3 (ATF3) X63369 Zinc finger protein 36 (ZFP36; TIS11) NM_019242 Interferon-related developmental regulator 1 (IRFD1; PC4) AF009330 Basic helix-loop-helix domain containing, class B2 (BHLHB2) NM_012591 Interferon regulatory factor 1 (IRF1) AF001417 Core promoter element binding protein (COPEB; ZF9) NM_013086 cAMP responsive element modulator (CREM) NM_012603 Myelocytomatosis viral oncogene homolog (avian) (MYC; cMYC) 5. Growth/regeneration and fibrosis L32591 Growth arrest and DNA-damage-inducible 45 alpha (GADD45a) AB020978 Growth arrest and DNA-damage-inducible 45 gamma (GADD45g) AF035963 Kidney injury molecule 1 (HAVCR1; KIM-1) M12201 Procollagen, type I, alpha 2 (COL1A2) Z78279 Collagen, type 1, alpha 1 (COL1A1) NM_017259 B-cell translocation gene 2, anti-proliferative (BTG2; TIS21) NM_019216 Growth differentiation factor 15 (GDF15) 6. Defense and repair AF075383 Suppressor of cytokine ignalling 3 (SOCS3) NM_012580 Heme oxygenase (decycling) 1 (HMOX1) L16764 Heat shock 70kD protein 1B (HSPA2) NM_012620 Plasminogen activator inhibitor-1 (SERPINE1; PAI-1) NM_017051 Superoxide dismutase 2, mitochondrial (SOD2) 7. Proteases AF149118 A disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type 1 motif, 1 (ADAMTS1) D87336 Bleomycin hydrolase (predicted) (BLMH_predicted) AF198087 Adrenal secretory serine protease precursor (AF198087) L05175 Granzyme M (lymphocyte met-ase 1) (GZMM)

Brain Dead/ Control 2.6 2.4 2.1 0.48 2.8 2.6 2.5 3.6 3.8 2.3 2.7 2.1 2.2 2.1

3.0 2.6 2.8 0.49 0.48 2.9 2.1 3.3 4.2 4.1 3.6 2.3 4.6 2.4 4.6 2.4

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Gene ID

Gene description

8. Miscellaneous M26758 Major acute phase protein (alpha1-MAP) L37380 Rat apical endosomal glycoprotein AF091577 Olfactory receptor 1370 (predicted) (OLR1370_predicted) NM_012752 CD24 antigen (CD24) NM_017180 Pleckstrin homology-like domain, family A, member 1 (PHLDA1) NM_017184 Troponin I, skeletal, slow 1 (TNNI1) V01224 Actin, alpha 1, skeletal muscle (ACTA1) J02705 Oncomodulin (OCM) X95094 Parathyroid hormone regulated sequence NM_013043 Transforming growth factor beta 1 induced transcript 4 (TGFB1I4) S74327 Clone E512, estrogen induced gene

Brain Dead/ Control 2.4 2.1 4.7 2.5 2.3 5.9 2.5 2.0 2.8 3.3 0.47

Gene Regulatory Networks All 63 genes recognized as being differentially expressed due to brain death (Table 1) were used to identify gene regulatory networks in rat donor kidneys. Recently, we identified the transcription factor NFκB to be activated (phosphorylated) upon brain death induction [17]. Moreover, many genes that were found to be differentially expressed contain NFκB binding sites in their upstream promoter sequences [18], which makes it very likely that NFκB plays a central role in brain-death-induced regulatory processes. Therefore, also NFκB was included in our analyses. By using the parameters expression, regulation, binding and promoter binding, PathwayAssist analyses was carried out. Using these parameters 34 out of the imported 64 genes appeared to be part of a gene regulatory network. To allow for better interpretation of the data genes were grouped based on the cellular localization of their encoded proteins. Figure 1 shows the complex network that exists between these genes. Each individual connection indicates a relation between genes or their encoded proteins whether this induction or repression of expression, physical binding, binding to promoter regions or a combination of these. Although a complex network exists it is evident that at least three key players can be identified: Il-6, Il-1β and NFκB.

Central Key Players Due to the complexity of the data we decided to position the individual key players, one by one, in a central location of the graph. In addition, all connections between the other key players, other than their relation to the centrally placed key player were omitted, giving the graphs a clearer overview. Figures 2a, b and c confirm the idea that Il-6, Il-1β and NFκB are

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true key players in the transcriptional changes in kidneys derived from brain dead rats, each of them are associated with 19, 19 or 20 genes, respectively.

Figure 1. Regulatory network for genes differentially expressed in donor kidneys upon brain death in rats. Proteins coded by the differentially expressed genes (Table 1) and all connections based on the attributes expression, regulation and binding are displayed. Proteins are positioned in the figure according to their cellular localization. Numbers indicate the number of references the respective connection is based upon. Red and green color indicates induced or repressed expression in the brain dead donor kidneys compared to controls.

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A

B

Figure 2 continued on next page

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C

Figure 2. Gene centralized reduced complexity regulatory networks. To emphasize the key-players Il-6 (A), Il-1β (B) and NFκB (C), all connections from the other two key-players other than their direct connection with the emphasized key-player were omitted. Numbers indicate the number of references the respective connection is based upon. Red and green color indicates induced or repressed expression in the brain dead donor kidneys compared to controls.

Conclusion In clinical organ transplantation, brain death in the donor has been shown to affect posttransplant function and graft survival. Brain death leads to systemic changes in the donor such as hemodynamic instability, hormonal and metabolic changes and increased inflammatory reactivity. Over the past years several groups have found in experimental models that organs retrieved from BD rats, suffer from increased primary non-function and poor graft survival after transplantation compared to the outcome with healthy non-brain-dead control animals [19]. Despite increasing research efforts, still little is known about the causes and the initiating mechanisms of brain death. These were important considerations to perform DNA microarray experiments using total RNA isolated from brain-dead donor-kidneys. In our study we found that in kidneys derived from BD donor rats the expression of 63 genes were significantly changed when compared to control animals. One group of genes that is seriously affected by the deleterious processes of brain death are genes involved in immune activation and coagulation. In recent years some groups have focused on the effect of brain death on the innate immune respons. Adhesion molecules and cytokines were found to be

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clearly up regulated in BD donor kidneys from rats [9, 10, 20, 21] and man [22-24]. Our results confirm these findings and, in addition, indicate that immune activation is massive and even more intense, containing the highest number [15] of up regulated genes. It is obvious that immunological activation, which is associated with recruitment of leukocytes will have profound deleterious effects on organ quality and function before and after transplantation. Therapeutical intervention, preferably in the donor, aiming at the prevention of up regulation of these genes, is therefore a worthwhile approach to decrease immunogenicity and increase graft viability [25, 26]. The outcome of the microarray experiment has given us a better insight in some parts of brain-death-related processes. However, it did not provide data on the different pathways that were activated or gene regulatory networks that exist in brain dead donor kidneys. Recent developments in specific software, designed to enable further interpretation of microarray data, has prompted us to identify brain-death-associated pathways, gene regulatory networks and biological key players. PathwayAssistTM analyses led to the identification of a large gene regulatory network: 33 of the 64 included genes were connected to each other in a complex pattern involving induction or repression of gene expression or physical interactions of the encoded proteins. The finding of such a large number of interacting genes/proteins instead of unrelated, individual genes further supports the validity of our microarray data. As illustrated by Figure 1, most of the encoded proteins of the differentially expressed genes are functionally located either at (the border of) the extracellular space or in the nucleus and only few in the cytoplasm. Apparently, these are ‘the places to be’ when it comes to adapting to changing conditions: In the nucleus genes necessary for changing conditions are transcribed whereas the changes primarily occur in the extracellular space, e.g. inflammatory components. Remarkably, four out of five cytosolic (or mitochondrial) proteins (HMOX1, SOD2, SOCS-3 and Hspa2) involved in the regulatory network have cytoprotective properties and can be regarded as proteins involved in cellular defence against various forms of stress [27, 28]. The PathwayAssistTM approach clearly identified Il-6, Il-1β and NFκB as regulatory key players in the brain-death-associated renal changes. Il-6 is well known for its involvement in inflammation and as an inducer of the acute phase response, in particular in the liver but also in kidney [29, 30]. According to our analyses Il-6 is directly connected to 19 other differentially expressed genes. At least eight of these genes/proteins are involved in inflammation and five can be regarded as acute phase proteins (Fibrinogen-alpha and -beta, HMOX1, SOD2 and Serpine1) [30-32]. Many of the interactions shown act (directly or indirectly) in both ways. An interesting example is Il-6 and SOCS-3 (suppressor of cytokine signaling-3): Il-6 induces SOCS-3 whereas SOCS-3 renders cells, exposed to high levels of cytokines such as Il-6 or Il-1β, less susceptible against their deleterious effects by inhibiting Il-6 or Il-1β signalling [33, 34]. The validity of Il-6 as a key-player is further supported by previous findings by our research group and others: Real-time PCR indicated that the Il-6 gene is early and massively expressed in kidneys from BD donor rats whereas high levels of Il-6 are found in human and rat sera [17, 35]. Another potential key player Il-1β gives rise to an almost similar set of interactions as Il6. This is not surprising regarding their activities, although Il-1β is primarily regarded as a pro-inflammatory cytokine. Il-1β is involved in the upregulation of many other inflammatory mediators such as chemokines, cytokines and adhesion molecules (Figure 2B) in a wide range

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of different cell types [36]. In addition, Il-1β, in contrast to Il-6, has a close relation with another key-player NFκB. Il-1β cytokine is capable of activating NFκB via the classical NFκB signaling route [37]. Briefly, upon binding to its receptor Il-1β induces a signaling cascade ultimately resulting in phosphorylation and ubiquitin-dependant degradation of IκB which normally keeps NFκB in an inactive state by sequestering it in the cytoplasm. This releases the NFκB dimer (p50 and p65) to translocate into the nucleus where they potentially can activate transcription of at least 150 target genes [18]. Therefore, NFκB is nowadays regarded as a central regulator of stress responses and it is therefore not surprising that in a recent study NFκB was shown to be activated in kidneys from brain dead rats [17]. Whether this is due to increased levels of Il-1β remains to be established, since the list of putative inducers of NFκB is almost as long as the number of genes it regulates [18]. Moreover, Il-1β cytokine was not found in increased levels in serum derived from brain dead rats, in a recently established slow-induction model of brain death [38]. However, in contrast to a systemic contribution, local renal Il-1β production could contribute to NFκB activation since early up regulated Il-1β gene expression was seen in the same model (unpublished results). One other potential candidate needs to be mentioned here: MGSA (KC-protein) mRNA transcripts are not only elevated in kidneys from brain dead donors it was also the sole cytokine/chemokine that had significant elevated serum levels, already 0.5 hr after brain death [38], as was assessed using a Luminex assay in which 14 different cytokines/chemokines were simultaneously analysed. Recently, MGSA was found to activate NFκB through the MEKK1/p38 mitogen-activated protein kinase pathway [39]. Therefore, MGSA, a chemokine synthesized during wound repair and inflammation, could be one of the earliest triggers of NFκB activation ultimately leading to differential expressions of a large number of genes involved in inflammation, survival and proliferation. So in summary, using PathwayAssistTM, we have been able to describe gene-regulatory networks that exist between genes that were identified as differentially expressed using microarray analyses. The results clearly identified the involvement of at least three keyplayers taking part in the brain-death-induced transcriptional changes: Il-6, Il-1β and NFκB. Especially in terms of developing specific interventions to prevent brain-death-related organ injury, these key-players may appear to be highly significant targets. Although rather detailed and complex, the network that we have visualised is probably just a tip of the iceberg and many more interactive changes are taking place. Another point that should be mentioned is that at this moment it is still unclear what kind of changes do occur in which cells. To obtain a better understanding of brain-death-related changes, further study on the relevant cell types will be a challenge for future research in this field.

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In: Progress in Kidney Transplantation Editor: Dominick W. Mancuso, pp. 105-121

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter VI

Kidney Transplant: The Search for Better Quality of Life Claire Terezinha Lazzaretti1 and José Miguel Rasia2 1

From the Division of Kidney Transplant and Unit of Psychology of University Hospital of the Federal University of Paraná – Brazil. 2 From the Departments of Sociology of the Federal University of Paraná – Brazil

Abstract The aim of kidney transplants, besides preserving life, permits patients to live their life as normally as possible, offering a better life expectancy, if not equal, at least close to nontransplanted patients of the same age. Patients put all their hope in an organ transplant to stay alive. However, their perspectives on their health problems are generally part of an individual conceptual model, with roots and meanings coming from their cultural context. With that point of view, the illness becomes part of the psychological, moral and social dimension of a particular culture, and this should be considered to understand the way patients interpret and answer their health problems. Starting from here, it is possible to think that the individuals who submitted themselves to a kidney transplant can adapt themselves to the adverse life circumstances, since they feel satisfied with the life they live, even though it is necessary to transform the concept of normality. This chapter attempts to measure the dimension of how chronicle renal diseases affect the life of people and investigates on how the transplant, a highly innovative modality of medical science, can contribute to the rescue or reconstruction of the identity, giving back their “freedom” of life. A hundred kidney transplanted patients participated in a life quality study using the WHO questionnaire. In this sample, twenty-two patients were randomly selected to be interviewed following a non-directive methodology to analyze the subjective conditions on their perception of life quality. The results of the self-answered questionnaire as well as the results of the qualitative results were analyzed. Although the majority of the individuals show that the transplant increased their quality of life, we can conclude that the disease and the post-transplant situation may lead to a particular meaning to the subject, frequently sheltering a subjective position of unhealthy. This situation shows that the concept of health escapes from the statistic calculations which

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Introduction In chronic renal failure, differently from other chronic diseases, the treatment favors the inability. For the patient’s survival, besides the systematic attendance to medical consultations, it is usually necessary to go on dialysis, a procedure that can maintain life per years after the loss of the renal function. Therefore it creates serious physical, social and psychological problems, so much for the patient as for his family. From the social and affective point of view, the relationships of those patients become conflicting because of the limitations they have to live with. These limitations go from feeding and ingestion of liquids to sexual impotence. The transplant, then, comes as an alternative procedure for a better quality of life, liberating the patient from the dialytic treatment which causes complex reactions for having an uncertain prognostic and meaning continuous dependence, and also configures an opportunity of rescuing the condition of "normality". Due to specific characteristics of the chronic renal disease, those patients, in most of the cases, as if the stigmatizing connotation given by the physical weakness and socioprofessional limitations was not enough, are put by the treatment itself (dialysis, consultations and systematic medications) in a group where all the members, submitted to the medical order regarding the prescriptions of the treatment, have in common typified and repeated actions. This is a favorable context for the identifications that create and sustain forms of sociability in the group, making possible the maintenance or permanence of the identification with the unhealthy. In a group, the individual not only incorporates the roles and attitudes of the others, but assumes their world, because the norms can mold and define their functions, making him a prisoner of certain representations, defined by a group of preexistent restrictions and reinforced by the partners' expectations. This process can influence the patient's behavior. The maintenance of relationships with other bearers of chronic renal failure, in the beginning, can make the adaptation with the traumatic factor caused by the diagnosis easier. However, it must be considered that, in the situation of post-renal transplant, it can become a difficulty in the retaking of the autonomy, because being the man a social being, the elements of his daily life are structured according to the convenience, in other words, in relation to the elements that have importance for the others. In that sense, it is important to verify to which extent the renal transplant can be considered as an innovative high complexity therapeutic resource in the tireless itinerary of the medical science in winning the disease. However, for involving components that are beyond the biological dimension and the success of the kidney graft, the degree of psychosocial rehabilitation reached by the individual in the post-renal transplant cannot be forgotten [1,2,3]. The renal transplants are not projected to transform a bedridden patient in a semi-disabled person, otherwise it would be senseless and of little effect in the social recovery. They are projected to provide a tolerable and even harmonious and balanced life for the patient [4]. In

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other words, while there is not a cure for a chronic disease, as the renal failure, the essential objective of the medicine should be to maximize the quality of life of those patients. In this search, countless researches about quality of life, with broader evaluation instruments, are accomplished yearly. Except for the clinical contraindications, all the patients with chronic renal failure are candidates to a renal transplant, and, in most cases, this is the best clinical and social costbenefit option. With no doubt, it is a consolidated procedure. But, how do these patients live? The demand for quality of life evaluation after the transplant, as already said, is intense, not only due to the high cost of the procedure, but also and mainly because of the increase of the patient's survival after the transplant [5,6,7,8]. So, the concern with the patient’s quality of life is a result of the recognition by the medical science that, besides evaluating the morbidity level and physical functionality, it would be also necessary to add the evaluation of individual and family psychosocial factors, because these are also affected by the chronic disease. Consequently, to reach this objective, more sensitive measures adding data of the subjective perception of the life post-transplant became necessary. A variety of instruments are available for evaluating quality of life, in their majority, are questionnaires structured with multidimensional subjects about the medical aspect, family, friendship, religious faiths, work, financial situation and other life circumstances. The answers given in the questionnaires vary with the social, educational and economical status, among others, and, besides, they are influenced by a variety of the patient's psychological factors. For instance, some people have prejudice against giving answers they consider not socially acceptable or favorable. Even understanding that the questionnaires are valid instruments, they still are not sensitive to the factors of the individual subjectivity. In the daily practice at University Hospital, it is verified that, besides the answers they gave about quality of life, the patients report situations of uneasiness with the diagnosis of chronic renal failure. They experience their life projects becoming obscure, because the diagnosis of a disease, mainly when it is a chronic disease, is always an abrupt event that is imposed and transforms the individual's daily life. The diagnosis of chronic renal failure imposes, besides the treatment, the redefinition of objectives and adaptation to a new way of life, the option presented at the beginning is to follow the medical prescription, in other words, a certain submission to the medical speech. Helman presents an important distinction to be reminded at the moment of acceptance of the diagnosis and treatment: "the term illness refers to 'what the patient feels when he goes to the clinic’ and disease to 'what he has when he goes back home, from the clinic'. Disease, therefore, is what the organ has; illness is what the man has" [9]. The fact that the diagnosis of a disease does not necessarily correspond to the feeling, the meaning that the patient attributes to it, was also observed in the post-transplant. The patient's perspective of his health problems are generally part of an individual conceptual model, with roots and meanings coming from their cultural context [3]. The disease is an integral part of the psychological, moral and social dimensions of a specific culture, and these should be considered to understand how the patients interpret and respond to their health problems. Starting from this understanding, it is possible to think that the individuals that underwent renal transplant can adapt to very adverse life circumstances if they feel satisfied with their way of life, even if for that it is indispensable to transform the concept of normality.

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Quality of life measures related to health are essential, mainly in groups of individuals that underwent a high complexity procedure as the renal transplant. However, a diversity of psychosocial effects associated or not to the physical limitations, hinders the process of the patients' adaptation. This reason is, by itself, enough to justify the interest and concern of health professionals about the patients' quality of life However, although it is agreed that quality of life in its multidimensional sense is important, to accomplish the objective of this chapter it is necessary to give one more step to reach the subjective significance of the course of life before and after the transplant for each individual, because the subjectivity of each one gives him the originality. This means to go beyond statistical and epidemic data, because if we believe that the individuals are social products and producers, their representations of the disease are extremely important. In other words, the concept of health and disease hold the individual's subjective interpretation.

Kidney Transplantation: A Course of Challenges The bearers of other chronic diseases, in a general way, learn how to live with their illnesses. Though, in the chronic renal patient's case, the discovery of the disease happens in a relatively fast way, because many times, when the patient discovers that he has renal failure he already needs to submit to the dialysis treatment. Besides being a chronic disease, it brings a type of inevitable treatment, undelayable and with direct consequences in his whole life. Starting from the beginning of the treatment, everything will be organized in function of this object-machine-kidney (place of living, work schedule, transport, vacations), what represents the medical order in its technical and metaphorical version; the possibility of continuing alive for the chronic renal patient depends on this object and its foundation, while the transplant is not accomplished. The more changes the patient has to do, more he will live the illness and vice-versa. During the hemodialysis process, man and machine interact forming a new system that is at the same time organic and artificial, the man depends on the machine that accomplishes the function of his kidneys. However, in spite of going towards a more and more symbiotic relationship with the machine, a more and more current experience in the medicine, the human being's essence is still the result of the group of meanings built socially, above all the meanings regarding his own body. The perception of the body and the disease is also determined by the cultural values involved. The successful renal transplant allows the recipient to have a "normal" lifestyle again and will liberate him from the dialysis. It is the procedure elected as the best form of treatment, because the dialysis, even each time better and more modern, does not substitute the kidney fully, but the transplanted kidney does. However, a kidney transplant is not a cure; it is the choice treatment for the chronic renal failure. The successful recipients can have a more normal diet and ingest liquids without so many restrictions, and more autonomy in their physical activity. However, even with all the advantages and independently of the type of renal transplant - with cadaveric donors or living donors -, they will need the doctors assistance and the constant use of immunosuppressive medication, because the new kidney is a "stranger" in the recipient’s body.

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There is a consensus that the transplant has a triple objective: in the immediate situation, to preserve the patient's life; after the surgery, to allow the patient to live the most “normal” life possible; and give to the patients, as much as possible, a life expectation, if not equal, at least similar to those not transplanted of the same age [4].

Quality of Life It is thoroughly recognized that the onus of a disease for who has it cannot be entirely evaluated by measures of the clinical state of the disease, of the functional recovery. Psychological and social factors, such as apprehension, difficulty with personal responsibilities, family, financial and other functional losses should also be considered [1,2,7,9]. The research area that was the result of that recognition is "quality of life and health". Besides evaluating the direct manifestations of the diseases, it broadens its interest to study the patient's personal morbidity, i.e., the several effects of the diseases and treatments on the daily life and life satisfaction. Although the evaluation of quality of life addressed to health was almost unknown thirty years ago, it became quickly a relatively recent integral variable in clinical research, generating countless articles on the theme. The individual's quality of life is affected in an intense way on the course of any diseases, however even more if it is a chronic disease, in this case the chronic renal failure. With the progress of medicine, even not considering the possibility of cure, the survival of those patients has been considerably increasing through the dialysis and the transplant, what implicates the necessity of thinking about quality of life during the period of treatment and the whole period of life. The renal transplant, usually, offers better quality of life if compared to the dialysis treatments, in relation to the general improvement of mobility, less dependence on the company of somebody to move from a place to another; smaller restriction in social life, leisure and sexual life; less feelings of sadness, pain, concern, nausea, depression, anxiety, loneliness; decrease of tension before the treatment [1, 11, 12, 13, 14, 15]. Regarding costs, it can be affirmed that the procedure of renal transplant has the lowest, followed by the cost of the peritoneal dialysis and next by the hemodialysis, which is the most expensive procedure [16, 17, 18]. But what kind of life do these kidney recipients have? To which levels of embarrassments are they subjected? And what are their hopes, limits and deceptions? To clarify this situation, it is important to remind that the illness, besides affecting the physical organism, echoes on the individual's general state, in the independence level and in their social relationships. D’Avila and Figueiredo consider that it is possible to establish ideal goals of recovering for a chronic renal failure treatment program, which involve: Taking the individual back to pre-uremic levels of biological, psychological and social adaptation; Causing the minimum adverse effect to the disease related to the survival and adaptation, with the treatment; Producing appropriate psychological and social maturation;

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Maintaining adherence the treatment; Maintaining appropriate family function, with low disturbance and discomfort with the treatment; Causing the smallest problem and the biggest satisfaction level to the health staff, in caring for the patient and his family. [19] Although the importance of quality of life is recognized thoroughly, skepticism and confusion continue regarding how to measure and define quality of life and its usefulness in medical research. Defining quality of life is not really easy. Since the beginning of the renal transplant procedure, people have been asking difficult questions: if after the transplant the individual can return to work, if he can have social life, among others. These questions disturb the researchers, because, can the quality of life of an individual be measured in fact? How to measure life, and, more important, which definition will show that the individual has good or bad health? Many concepts of quality of life were already postulated. The term quality of life has a recent history; it dates from 1970, when it was used in economy and sociology to find indexes of human quality life in different societies [14]. In economy, it emphasizes the social conscience that the goods are finite and the distribution of goods, resources and services should involve the social responsibility; in sociology, it rescues the symbolic dimension of what is shared and built socially, demonstrating the implications of who influences and who is influenced in the several cultural and anthropological contexts; in administration, it is related with the increasing capacity of mobilizing resources, more and more sophisticated and with more impact in technological terms, facing more specific, fast and mutant objectives [20]. The World Health Organization had already established its definition of health, affirming that health is not only the absence of disease or infirmity, but also a state of complete physical, mental and social well-being [21]. This definition shows an initial reference to establish what is health or lack of health; it is a definition sufficiently wide to capture most of the interests. The problem of evaluating quality of life is that the previous definition does not consider the individual's evaluation of his disease or level of inaptness. So, in 1994, looking for an instrument to evaluate quality of life from a genuinely international perspective, the World Health Organization elaborated a collaborative multicentric project, that defined the concept of quality of life as “individuals' perceptions of their position in life in the context of the culture and value systems in which they live and in relation to their goals, expectations, standards and concerns” [22]. It established a differentiated perspective, based on the presupposition that quality of life is a subjective construct (regarding the individual’s perception), multidimensional and composed by positive (mobility) and negative (pain) dimensions. Using that model, several concepts and instruments were created to verify the theme quality of life with a more objective base. Such measures were classified in two general categories: specific disease and general health concepts. Although only some quality of life studies apply the multidimensional definition to renal transplanted patients, research shows that these patients noticed improvements in their global quality of life, especially in the domain of the physical functioning [11, 12, 15, 23, 24]. The

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individual has the opportunity to guide his interests and activities, including a better possibility of having a paid job, once he does not need to interrupt his daily tasks for the dialysis [18, 25, 26, 27, 28, 29, 30]. However, studies show that the renal transplanted patients' quality of life had a general improvement from the level it was before the transplant, but not to the level considered normal in the society [1, 13, 31]. They also showed that the kidney recipient’s quality of life was comparable to the one of chronic hypertension patients. What reinforces the hypothesis that the subjective aspect of being or having been bearer of a chronic disease makes difference in the individual's subjectivity. This data is consistent with all types of transplants and with all measures. But the data that suggests the leveling of improvement of quality of life comparable to the one of the general population raises some researcher’s questions because, for them, this data can be the result of an "adaptation of normal expectations" for transplant recipients [32]. Starting from the facts and considerations described previously, the renal transplant is an answer of the medicine that is consolidated for patients with chronic renal failure. However, for the renal transplant staff the questions about the patients' quality of life are not answered. To understand the impact of the transplant on quality of life, not only should the aspects directly related to the disease be approached, but also the psico-sociocultural factors, such as behavior, family and social relationship, faith, leisure and work opportunities, among others [1, 7, 8, 25]. The transplanted patient is an individual with needs, reasons and desires, that are not always conscious, but that influence his behavior characterizing the reality as a process of permanent construction, that includes the individual's subjectivity, that is why it is necessary to adopt techniques of data collection and analysis to make the analysis of the effects of the unconscious resistances possible. Therefore, it was opted to begin with the quality of life questionnaire, that in general terms would provide an objective and measurable situation, and later to look for a qualitative approach - the not direct interview - to understand episodes lived by those individuals who underwent a transplant starting from their own interpretations.

From the Quality of Life Evaluation to the Subjective Perception Report A study was accomplished with renal transplanted patients at the Renal Transplant Service of Hospital das Clinicas da UFPR. All of them presented good evolution, seric creatinine level = 2,5 mg/dl, without registration of acute renal graft rejection. There was not any previous diagnosis of mental disease or alterations of the mental state because of the medication. They all gave post-informed consent. The instrument used was the World Health Organization multidimensional questionnaire, which evaluates four domains: physical, psychological, social relationships and environment, the WHOQL-Bref and not directive interview. The questionnaire was answered by 100 renal transplanted patients (60 men and 40 women), average age 36 ± 10,4 and median of 35 years, with average post-renal transplant continuation of 87,3 ± 61,6 months and median of 73 months. The married ones were 62%

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and 51% had only fundamental education. To evaluate the domain scores, 12 patients were excluded because their questionnaires were incomplete. The perception of the general quality of life after the renal transplant was considered "Very Good" for 23%, "Good" for 57% and "Neither Good Nor Bad" for 20%. None of the interviewees considered their general quality of life "Bad" or "Very Bad" [33]. The choice of patients' sample for the qualitative investigation (not directing interview) started from the statistical analysis of the anonymous answers for the quality of life questionnaire, just identified by the application order number. The selection of the numbers considered the scores obtained in the questionnaires and, starting from these, selected them by the process of random choice tool of Windows, Excel, a list of thirty numbers from the total questionnaires. So, it can be affirmed that the sample was equally represented by the three answer levels as for quality of life: "Very Good", "Good" and "Neither Good, Nor Bad". After the nominal identification of the patients, a phone call was made to invite and schedule an interview. In the interview, besides the questions about the census data indicated to delineate the group of socioeconomic characteristics of the individual, it was requested that the interviewee reconstituted the history of the disease and of his life, in other words, that he discoursed freely about how his life was before the disease, about the treatment period to the renal transplant and about how his life is after the renal transplant. Twenty-two patients agreed in accomplishing the interview (14 men and 8 women); with average age 39 ± 9,4 and median of 37 years, with average post-renal transplant continuation of 66 ± 70 months and median of 35 months. (The time of transplant varied from less than one year to 18 years.). It was observed that exactly as the result of the patients' profile that answered the quality of life questionnaire, most of them, 72%, is married and the prevalence of low education level was also observed, 68% of them did not study further than the fundamental level. The low education and the low socio-economical level are characteristics of most of the patients that use the service in this public hospital. All patients were treated by hemodialysis. The duration of the hemodialysis treatment varied from less than one year to 5 years, and 41% of the interviewees did not complete one year of hemodialysis before the transplant. The data groups - quantitative and qualitative – are not oppose, they complement each other, and therefore the realities included by them interact dynamically, excluding any dichotomy. As it is known, until some years ago the medicine had as objective to evaluate the level of influence of the disease and its treatment on the patients' global quality of life, what took the World Health Organization to define quality of life as a multidimensional concept, highlighting that it "is not enough to give years to life, but life to years". However, in spite of the clarity of the objective and the definition of quality, many authors share the doubt on the validity of such questionnaires, if they can reach the subjective conception [12, 13, 14]. It was possible to verify that quality of life after the renal transplant is in agreement with the literature, the individuals who underwent transplant got considerably better in comparison with the ones who continue using dialysis methods. The patients recover a good amount of the capacities they had before the chronic renal failure, because the transplant favors a less restrictive diet in some cases, and larger freedom of time and mobility, in other words, except for the daily medication and the routine consultations (usually monthly), the individual has better opportunities to develop his interests and activities.

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In that sense, it is important to consider that quality of life for most of the individuals is seen as the reduction or disappearance of the symptoms caused by the origin disease. Such attitude, probably, is associated to the conception those patients have concerning what is quality of life, not forgetting that the socioeconomic condition of most of them is quite precarious, and that it was already so before the disease. So, quality of life may be limited to the fact of being healthy. In the subjective evaluation, it was confirmed that the perception of welfare does not necessarily have connection with the physical state, a person can have some functional damage or limitation of his capacities and not to feel sick, as well as, have reestablished their functions and physical capacities, but feel limited for life, because it is just not enough that the science advances to "give life" for those patients, but that they can have the opportunity to take advantage of what is offered to them. Some relevant themes had convergences in the interviews, the meaning of the disease, the meaning of the transplant, the "debt" with the donor and the feeling of normality.

Meaning of the Disease The meanings of the disease are built socially by the coexistent routine and typified by the atmosphere at the hospital. The representation is determined by the social interaction in daily life, the group experience makes the establishment of standard behaviors and the apprehension of the other as a model possible. They are individuals that submit their bodies to the medical order because of the renal failure. The construction of the identity of a sick person is the result of the process where the patient starts to represent the name that his condition defines, in other words, the one of a patient. The situation of hemodialysis is par excellence the place of giving meaning to the chronic renal disease, because it is in that space that the disease comes as a deficiency phenomenon and physical-corporal limitation. It is the place where the individual becomes a patient, 16% of the interviewees felt impotent and submissive to the disease. It is also there that the important aspects of the disease are defined, for what it is possible to suffer or have hope, 12% expressed negative or unpleasant feelings in the hemodialysis, while 16% felt discouraged and saw death as the only answer. From the interviewed patients’ point of view, the hemodialysis is a homogenizing practice. It has the characteristic of transforming the disease into a group experience, "where everybody is the same", facing the same fatality, the inevitable, for 28% of the interviewees. The chronic renal disease implicates a series of losses for its bearer. The interviewees referred that they changed their way of thinking when discovered the disease. For 31% the disease altered the body, they started feeling that their body was not the same as before, while for 12% the disease brought the feeling and the concern with the possibility of death, and 14% referred the disease only as one more challenge of life. Besides the health loss, changes in their social relationships and the threat of the unviable continuing their personal and professional projects disturb their usual life performance. The discouragement and the feeling of rupture in their biography with the emergence of the disease are the feelings for 24% of the interviewees. The individual many times is temporarily license from work and is even forced to delegate his household chores to another person; in

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other words, he stops carrying out functions and activities that were previously of his responsibility. This threat can become socially reinforced by losing his job or the functions he had or breaking up a relationship with a partner, 19% of the informers said they feel stigmatized by the fact they are sick. All of this, with no doubt, makes them go through a series of contradictions, because, even if they know about the need of the treatment, they also have to work with the psychosocial losses that result from the treatment.

Meaning of the Transplant The transplant is surrounded by mystery and emotional impact. It is considered a scientific progress and a miracle of the medicine, as liberation of the difficulties of the traditional treatment, but it is also frightening. Because of its imaginary effects, the individual is taken by the innovation and always thinks he is the only one. The transplanted individual feels different from most people of his environment. The feeling of "not knowing himself anymore" after the transplant occurred for 29% of the interviewees and 24% referred the feeling of disability, meaning the low self-esteem. Intimately they are living an experience that the others ignore, they feel alone, and this feeling is an easy way to believe that only people that are living the same situation can understand them. They restricted their net of relationships, besides the contact with other patients, 20% said that they got closer to the family. Some identities are attributed to the individual when he is born: girl or boy; and others are attributed or acquired in a subsequent phase of life: intelligent, brave, etc. The “identity is always assimilated through an interactive process with others. The others identify him in a certain way. Only after the identity is confirmed by the others, it can become real for the individual to whom it belongs” [34]. Most of the time, in search of relief and in the struggle for life, when submitting to the medical discourse, when taking the position of bearer of a disease - "body and soul" - the individual loses his referential and identifies himself with the disease. In other words, from the medical point of view, the transplant, the new kidney, can be a therapeutic solution; from the individual’s point of view it marks his position of depending on the other, another kidney, putting himself in a work of elaboration of his new status. The interviewees' individual perception on how their life changed after the transplant goes from worsened for 2%, except for the medication it returned to normality for 10%, until the meaning of a new life for 4%. The transplant meant retaking of the freedom for 14% and change of physical appearance for 18% of the interviewees. The idea of interaction with the donor through the donated organ is so strong that many believe to have inherited the donor's personal characteristics, such as: “I got strong like him", “I started to like green vegetables”, “I got younger”, etc. In this sense it is important to point the concept of health developed by Canguilhem: "man does not feel in good health - when feeling not only normal, that is to say adapted to one’s environment and its demands, but also normative, capable of pursuing new norms of life" [35]. In other words, many times it is for being too much adapted to a social norm that the individual gets sick or does not let the condition of being sick go. Everything that repeats automatically, by routine and habit, is marked by renouncing the creation, pointing up,

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therefore, to possible situations of diseases. Maybe this justifies that 22% of the interviewees, even with the transplant; do not retake their projects for fearing of harming and/or losing the graft. It is worth to observe the difference between "adaptation" and "adherence1." The patient can creatively get adapted to his disease and the treatment, without necessarily having to choose the extreme directions or live the disease as the only possibility, or he can deny all the medical prescriptions in a frankly attitude of irreverence, putting his life at risk. In the choice of treatment, the information is one of the essential elements for the treatment of a chronic disease, because good part of the result depends on the patient's adhesion. This way, to participate in the choice of the treatment method, when possible, involves the patient in his treatment, and consequently a bigger adhesion is expected, because, as he is the one who chooses, he is responsible by his choice. From the interviewees 46% referred that the family was supportive and got involved in the transplant; 29% referred the transplant as the possibility of not dying and 25% verbalized that they wanted the transplant, but they had in mind they would have an eternal debt with the donor. It is well known that the human beings have a notable capacity of adapting to adverse circumstances: wars, political oppression and also illnesses. People get to find psychological and social mechanisms to go through many distressing situations, living one day after the other. This seems to be the situation of 14% of the interviewees who said they became better people after the transplant, and of 16% who said that the transplant increased their enthusiasm for life. It is possible for some patients to make the problems caused by the chronic renal failure become part of their daily life and be accepted as "something that happens", although it makes them suffer.

The “Debt” with the Donor Technically, the renal transplant process looks simple and calm. The medical science, once again, presents their technological miracles. But, the situation does not involve only the body mechanics, "something" of the "soul" interferes in the lineal course of the events. The relationship donor-recipient involves commitments that go beyond the mere solidarity and praiseworthy altruism in our culture. The subjective position of patient and the real dependence on the hemodialysis machine, make him supposedly dependent, he becomes the total recipient, waiting for a kidney, for caring, for understanding, for all the attention and help that such a needy patient demands. The transplant itself appear as a divine grace, deservedly reached by having been strong to face the disease and by having faced it bravely; it appears as redemption, proportional to the effort made. In spite of receiving all the technical orientations about the transplant, the patients are informed that it is an alternative procedure of treatment and not the cure, nevertheless for many of them the transplant is a "rebirth." The "rebirth" idea makes us suppose the variety of possible subjective significances implicated in the transplant procedure, mainly in living donor transplantation. 1

Adaptation, adjustment of an organism, particularly of the man, to the conditions of the environment; to adapt, to accommodate; to become capable. Adherence, fixation, connection, union; diffused, very connected; abnormal union set among structures of the organism through fibrous tissue [36]

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Only 9% of the interviewees underwent transplants from cadaverous donors, all the others were transplanted with kindred living donors, from these 67% of the donors were the siblings. In a transplant situation, there is the differential that this prolongation opportunity and/or better quality of life is possible because somebody donated part of his body. And, as already seen, for most of them, that makes all the difference. It is observed that there is an internal code among the chronic renal patients: “you shall not covet your neighbor's kidney”. They do not ask for anybody’s kidney, alleging that this type of thing should be offered. And this code is responsible by all the seduction that inflates the passive and sick posture. Touched by the patient's suffering, 46% verbalized that it was the family that motivated the transplant and 32% said that they felt ambivalent, part of the individual waited for the transplant; the other part was worried about submitting a healthy person to a surgery. The idea of receiving a cadaverous organ is less accepted, because, besides the guilty conscience that they may have, in the acceptance of a “dead” person’s organ, once again the idea of constant death in the disease is present. The idea that the transplant only works if it is from body to body, that is, between live persons, is linked to the fact that in the surgery both bodies are side by side. What the patient receives when undergoing the transplant is life - he receives the gift of life, reacquires his health, condensed in the recovery of the renal function. He receives life from a living person. Body-to-body the idea of proximity of bodies is implicit, not only physical but emotional proximity, because the certainty of the donor in donating is an essential issue for the recipient’s success in the post-transplant recovery, in other words, "accepting" what was given to him. The research data shows that 14% of the interviewees would like to have had another donor and attribute the post-transplant problems to the donor. After the transplant a new notion of relationship appears for both donors and recipients. The experience of the transplant recreates consanguinity and alliance bonds and establishes new ones, approximating donor and recipient, making them feel closer, more kindred, and more similar than others with the same level of relationship. For the medicine the transplant is a technical procedure, for the recipient it is a gift with meanings that are attributed according to each individual’s own history. Family relationships are vitalized; the transplant causes a radical transformation in the individual's personal and social life.

Feeling of Normality The normal and the pathological are two pillars of medicine. The normal is symptom less and is not perceived. Only the pathological draws our attention and through disease we appreciate the normal. Nevertheless, the pathological is defined as a deviation from normal. So, the definition of normal and pathological depends upon the circumstances in which they are observed. Canguilhem affirms that "the pathological should be understood as a kind of normal, since the abnormal is not what is not normal, but what is a different normal" [35]. He raises the discussion of the individual's subjective interpretation before these concepts: normal, abnormal and pathological.

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The subjective effects of the chronic renal failure in the patients that already underwent the renal transplant cannot be expressed by the concepts of normal and pathological, but by how each transplanted patient will symbolically receive this new significant - the transplant as part of his life. However, none of the interviewees consider themselves "normal". For 30% the condition of normality would be the fact of being able to feel the same as other people, healthy; for 15% the condition of normality would be equal to the possibility of feeling happy, feeling calm without constant intercurrent problems; for 40% the condition of normality was linked to the condition of being able to work, being productive in the aid or in the sustenance of the family, and 15% referred that they do not know what is the feeling of normality because they have always had health problems. Starting from that data it can be affirmed that, in agreement with Canguilhem, the normality in abstracto does not exist; therefore it is, to certain extent, a creation in the picture of possibilities that were given to us and the acquisitions we conquered. For Canguilhem the distinction between the normal and the pathological is something very different from a simple quantitative variation. There is a substantial qualitative difference between one state and the other that cannot be reduced to calculations or statistical averages. The pathological implicates a direct and concrete feeling of suffering and impotence, of a contradicted life; and health implicates much more than the possibility of living in accordance with the external environment, it implicates the capacity of instituting new norms. "The man, even in the physical aspect, is not limited to his organism, it is, therefore, beyond the body that is necessary to look, to judge what is normal or pathological for that same body" [35]. Regarding patients bearers of chronic diseases, as the name says, it is about a disease that lasts while life lasts. Differently from an acute disease, that is characterized by other temporality, chronologically shorter, the chronic diseases break the most frequent pattern of medical practice: symptom - diagnosis - treatment - cure. For these, the pattern is always open, it marks a daily chronic existence of the disease, collaborating or defining a restructure in the individual's life. However, in spite of the long duration or permanent characteristics, not all of the chronic illnesses - for instance, heart failure or diabetes - necessarily "dominate" the individual's life. But, those that have chronic renal failure, in function of the disease, need to submit, besides the restrictive alimentary diet, to programs of dialysis that put them before an inevitable institutionalization, they go through significant changes in their lives, completely modifying their routines and social lives. It is not possible to consider that every chronic renal patient that went through dialysis treatment and transplant assume the subjective condition of being sick. Although one can think that the individual has the probability of accepting the "label" that is attributed to him, developing the stigmatized self-image, internalizing the stigma of patient, because the system of expectations, sanctions and social rewards make sure that he does so. It is believed that even having to face adverse answers, these are not necessarily permanent or important. They will become permanent if the self-stigmatization persists. That is due to the continuation of his precarious health condition due to the non recovery of his clinical state with systematic relapses, or the internalization of a new subjective reality given by the identification with the disease. There are two immediate possibilities: one would be the path to alienation (to the diagnosis), and as result the transplanted patient, in other words, the identity with the

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unhealthy, would be noticeable in spite of being clinically capable of doing the activities done before or others that could take him out of the immobility of being sick, the patient would stay in this place. The other possibility would be the case of the individuals that wanted the transplant to continue living, that accept the limitations of the post-transplant treatment as a fact and retake their lives as an individual with transplant, that is not able to do everything (as any human being), but that can venture in life, wish.

Conclusion There is no doubt about the disastrous effects of the chronic disease in the social life of an individual, but in the renal transplant, something new appears. Differently from other chronic diseases, besides the medical treatment, the medicine offers the patient alternatives that substitute the sick kidney: the dialysis and the transplant. Both demand the patient’s submissive acceptance to “something” - machine or grafted organ. In that situation, the individual lives an experience where the intrinsic relationship that is set between his body and all the technological instruments that make his existence possible, affects the way he perceives life. A segment of his body is affected and this affects his life now and forever. However, in spite of the importance of the evaluation of quality of life in the post-renal transplant, the patient's experience with the disease and treatment should be recognized as a central component of medical care and researches. In the subjective evaluation with the data collected through the interviews, it was confirmed that the subjective perception of well-being does not necessarily have connection with the physical state, in other words, an individual can have some functional damage or limitation of his capacities and do not feel sick, as well as he can have his functions and physical capacities reestablished, but feel limited for life [2, 3, 7]. Getting sick! A hole that opens up under the individual’s feet to make him fall? Or does the illness appear in a determined moment of his existence? The interviews showed that before the diagnosis of chronic renal failure, these and other possibilities appeared. The receiving of the diagnosis appeared as essential for how the individual behaves in life, in other words, for some whose life had no longer the same flavor, even before the disease, the routine of the medical treatment had reached the value of a rite, from which he could not abstract. The role of the patient determines a complemental function of the doctor-patient system, disability retirement, and weak family structure. It is also essential to consider in those cases the disease as secondary gain. When the daily professional routine is very boring, when the family everyday life of is not more than a disappointing repetition, a love disillusion, when everything goes against the dreams of a wonderful future, the human being needs something to change the reality. The disease is a welcome refuge. Considering this, it is not possible to limit the concept of health to a "scientific" concept. As it is not possible to relate neither normality with health nor anomaly with pathology, the resource of the statistical averages, the frequent values, the calculations, tell us little about that concept [26]. In this chapter, analyzing the quality of life questionnaire as a complemental instrument of analysis, it was verified that, although it has as purpose the reach of the subjectivity by its multidimensional aspect (physical, psychological, social and

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environment), it propitiates limited information when trying to describe the experience of bearing the disease and its influence in other areas of the patient's emotional and social life. The theory that perfect health does not exist tells us about the legitimacy of considering that the disease is an individual perception, if it is linked to an individual suffering, to a “feeling of impotence and contradicted life”, then, and only then, it can be considered pathology. But those facts, those feelings, are not registered in the statistics that establish frequency and normality. The term "medical order" implicates obligations on the part of the patient; it establishes the routines and determines conducts. It does not exclude the legitimacy of the medicine, it is obvious that, when someone is sick, it is necessary to look for medical treatment, because even if it cannot cure, it is very possible that it can offer alternatives to provide better quality of life - the renal transplant is an exemplary procedure in this case. It was verified that the subjectivity is a component that interferes in the way of accepting and getting adapted to a life condition, of instituting new norms. In this sense, two situations were evident: the one of those transplanted, the ones that were anchored in the situation of being sick, that even not feeling physical limitations restricted their range of possibilities. And those with transplant, for whom the transplant appears as one more of the life difficulties, but it was not enough for giving up on investing in relationships, new jobs, studies, among other situations. In compensation, even if few, we found individuals that rebelled in limiting their conquests starting from the fact of having a transplanted kidney. For these, the "struggle" for life continues every moment. We are referring to those that welcomed the disease with its limitations and creatively instituted new ways to experience life situations. They readapted their projects, but they did not shelve them. They create new norms for themselves. Finally, a wider subject appears: can the submission to the “medical order”, in the case of the transplant, which imposes the permanent compulsory nature of medication and care, be the harbor for an indisposition in life, even with a certain quality? If the differential for the institution of normality depends on subjective components, what measures could be proposed to stimulate those individuals to continue thinking about the future as a project and not just as time of survival? That subject seems to reflect the possibilities of the different actors' performance in the process of construction of the normality so that the individuals who are bearers of renal transplants do not limit their existence only to the care with the disease.

References [1]

[2]

Reimer J; Franke GH; Lütkes P; Kohnle M; Gerken G; et al. Quality of life in patients before and after kidney transplantation. Psychother Psychosom Med Psychol 2002; 52(1):16-23. Hricik DE; Halbert RJ; Barr ML; Helderman JH; Matas AJ; et al. Life satisfaction in renal transplant recipients: preliminary results from the transplant learning center. Am J Kidney Dis 2001; 38(3):580-7.

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Claire Terezinha Lazzaretti and José Miguel Rasia Bakewell AB; Higgins RM; Edmunds ME. Does ethnicity influence perceived quality of life of patients on dialysis and following renal transplant? Nephrol Dial Transplant 2001; 16(7):1395-401. Küss R; Bourget P. An Illustrated History of Organ Transplantation: The Great Adventure of the Century. France: Sandoz. 1992. Pinson CW; Feurer ID; Payne JL; Wise PE; Shockley S; et al. Health-related quality of life after different types of solid organ transplantation. Ann Surg 2000; 232(4):597-607. Ekeberg O; Loge JH. Patients' quality of life after transplantation--what do we know? Tidsskr Nor Laegeforen 1999; 119(26):3920-3. Blake C; Codd MB; Cassidy A; O'Meara YM. Physical function, employment and quality of life in end-stage renal disease. J Nephrol 2000; 13(2):142-9. Keown P. Improving quality of life--the new target for transplantation. Transplantation 2001; 72(12 Suppl):S67-74. Helman CG. Cultura, Saúde e Doença. Porto Alegre: Artes Médicas, 1994. Lindqvist R; Carlsson M; Sjüdén PO. Coping strategies and health-related quality of life among spouses of continuous ambulatory peritoneal dialysis, haemodialysis, and transplant patients. J Adv Nurs 2000; 31(6):1398-408. Fujisawa M; Ichikawa Y; Yoshiya K; Isotani S; Higuchi A; et al. Assessment of healthrelated quality of life in renal transplant and hemodialysis patients using the SF-36 health survey. Urology 2000; 56(2):201-6. Babe RS. Quality of life improvement after transplantation. Curr Opin in Organ Transplant 1999; 4: 173-6. Rebollo P; González MP; Bobes J; Sáiz P; Ortega F. Interpretación de los resultados de la calidad de vida relacionada con la salud de pacientes en terapia sustitutiva de la insuficiencia renal terminal. Nefrologia. 2000; 20:431-9. Ortuzar MG. Ethics and Quality of Life of Kidney Transplant Patient. Transplant Proc. 2001; 33(1-2):1913-6. Andrews PA. Renal Transplantation. BMJ 2002; 321: 530-4. Gokal R. Quality of life in patients undergoing renal replacement therapy. Kidney Int 1996; 43 (Supl. 40): S23-S27. Coelho VSP Interesses e Instituições na Política de Saúde. RBCS 1998; 13:115-28. Arredondo A; Rangel R; Icaza E. Costo-efectividad de intervenciones para insuficiência renal crônica terminal. Rev Saúde Pública 1998; 32 (6): 556-65. D’Ávila DO; Figueiredo AE. Métodos de depuração extra renal: hemodiálise, diálise peritonial e novas técnicas. In: Riella MC. Princípios de nefrologia e distúrbios

hidroeletrolíticos. Rio de Janeiro: Guanabara Koogan; 1996; p. 607-45. [20] França ACL. Qualidade de vida no trabalho: conceitos, abordagens, inovações e desafios nas empresas brasileiras. Rev Bras Medicina Psicossomática 1997; 1 (2): 79-3. [21] WHO –World Health Organization. Constitution of the World Health Organization Chronicle of the World Health Organization 1. Geneva; 1947. [22] WHO –World Health Organization. Development of the World Health Organization WHOQOL – BREF Quality of Life Assessment. Psychol Méd 1998; 28: 551-8. [23] Dew MA; Switzer GE; Goycoolea JM; Allen AS; DiMartini A; Kormos RL; et al. Does transplantation produce quality of life benefits? A quantitative analysis of the literature. Transplantation. 1997; 64:1261-73.

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[24] Jofre R; Lopez-Gomez JM; Moreno F; Sanz-Guajardo D; Valderrabano F. Changes in quality of life after renal transplantation. Am J Kidney Dis. 1998; 32:93-100. [25] Nielens H; Lejeune TM; Lalaoui A; Squifflet JP; Pirson Y; et al. Increase of physical activity level after successful renal transplantation: a 5 year follow-up study. Nephrol Dial Transplant 2001; 16(1):134-40. [26] Harris LE; Luft FC; Rudy DW; Tierney WM. Clinical correlates of functional status in patients with chronic renal insufficiency. Am. J. Kidney Dis. 1993; 21:161-6. [27] DeOreo PB. Hemodialysis patient-assessed functional health status predicts continued survival, hospitalization, and dialysis-attendance compliance. Am. J. Kidney Dis. 1997; 30: 204-12. [28] Fiebiger W; Mitterbauer C; Oberbauer R. Health-related quality of life outcomes after kidney transplantation. Health Qual Life Outcomes. 2004; 2(1):2. [29] Vergoulas VG. Quality of Life in patients with kidney transplantation. Hippokratia 2002; 6(Suppl 1): 91-8 [30] Kennedy SE; Shen Y; Charlesworth JA; Mackie JD; Mahony JD; Kelly JJ; et al. Outcome of overseas commercial kidney transplantation: an Australian perspective. Med J Aust 2005; 182(5): 224-7. [31] Matas AJ; McHugh L; Payne WD; Wrenshall LE; Dunn DL; Gruessner RW; et al. Long-term quality of life after kidney and simultaneous pancreas-kidney transplantation. Clin Transplant 1998; 12:233-42. [32] Limbos MM; Chan CK; Kesten S. Quality of life in female lung transplant candidates and recipients. Chest 1997; 112: 1165-74. [33] Lazzaretti CT; Carvalho JGR; Mulinari RA; Rasia JM. Kidney Improves the Muldimensional Quality of Life. Transplant Proc. 2004; 36:872-3. [34] Berger PL; Berger B. Sociologia: como ser um membro da sociedade In: Farachin MM, Martins JS. Sociologia e Sociedade. Rio de Janeiro: Livros Técnicos e Científicos Editora; 1978. p.200-14. [35] Canguilhem G. O Normal e o Patológico. Rio de Janeiro: Forense; 2000. [36] Ferreira ABH. Novo Dicionário Aurélio da Língua Portuguesa. Rio de Janeiro: Nova Fronteira; 1986.

In: Progress in Kidney Transplantation Editor: Dominick W. Mancuso, pp. 123-135

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter VII

The Cost and Cost Effectiveness of Kidney Transplantation: A Systematic Review and Economic Evaluation Cyril F. Chang* and Stephanie C. Steinberg The Methodist LeBonheur Center for Healthcare Economics, Fogelman College of Business and Economics, University of Memphis, Memphis, TN

Abstract The purpose of this article is to summarize the current knowledge on the economics of kidney transplantation. It systematically reviews and evaluates articles in the peerreviewed literature on topics relating to the cost and cost effectiveness aspects of kidney transplantation. The literature review is conducted through the identification and classification of articles by topic and by the economic evaluation methodology applied by the authors. It includes only articles with publication dates from January 1, 2000 to June 2005 to meet the criteria for "new" research findings in transplantation. Studies in nonEnglish language journals, abstracts, conference papers, and posters are excluded. The main data source of research literature is PubMed, a service of the National Library of Medicine which includes over 15 million citations for biomedical and health services research articles back to the 1950's.

I. Introduction Kidney transplantation is a surgical procedure in which a healthy donated kidney is transplanted into a patient whose own kidneys have failed. It is the treatment of choice in both the United States and most other countries for end-stage renal disease (ESRD). The purpose *

The Methodist LeBonheur Center for Healthcare Economics; Fogelman College of Business and Economics; The University of Memphis; Memphis, Tennessee 38152; E-Mail [email protected]

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of this review article is to summarize the current knowledge on the economics of kidney transplantation. It systematically reviews and evaluates articles in the peer-reviewed literature on topics relating to the cost and cost effectiveness aspects of kidney transplantation. The literature review is conducted through the identification and classification of articles by topic and by the economic evaluation methodology applied by the authors. It includes only articles with publication dates from January 1, 2000 to June 2005 to meet the criteria for "new" research findings in kidney transplantation. Studies in non-English language journals and informal papers such as abstracts, conference papers, and posters are excluded. The main data source of literature search is PubMed, a service of the National Library of Medicine which includes over 15 million citations for biomedical and health services research articles back to the 1950's. These citations are from MEDLINE and additional life science journals that are reputable and peer-reviewed. In the past thirty years, the Medicare end-stage renal disease (ESRD) program has been among the most frequently assessed medical treatment programs. The program's implementation by Medicare in 1973 (Social Security Amendments of 1972 - Public Law 92603 Section 299I) was the first, and thus far the only time, that a Medicare coverage decision was based solely on a single diagnosis (De Lew, 2000). Today, Medicare pays over 80 percent of the total costs of ESRD treatment in the U.S. (USRDS 2004). For ESRD, two major treatment options exist: dialysis and kidney transplantation. Available evidence has shown kidney transplantation as both an efficacious (effective) and a cost-effective treatment option. This paper focuses on the economic aspects of kidney transplantation in comparison with dialysis, with an emphasis upon the identification and comparison of treatment costs and cost-effectiveness analyses for each modality. The structure of the review is as follows. A. Kidney Transplantation vs. Dialysis A.1. Cost Analysis A.2. Cost-Effectiveness Analysis B. Quality of Life and Kidney Transplantation B.1. Cost Analysis B.2. Cost-Effectiveness Analysis C. Drug Therapy Following Kidney Transplantation C.1. Cost Analysis C.2. Cost-Effectiveness Analysis.

II. Relevant Economic Concepts in Clinical Economic Evaluation Economic evaluation of clinical interventions combines information on the health outcomes of an intervention with information on its cost to assist decision making in clinical, health system, and public policy settings (Eisenberg 1989; Drummond et al. 1997). The purpose of this type of analysis, which has drawn increased attention in recent years, is to ensure that the benefits of a clinical intervention are sufficiently large to justify the associated economic costs. This section introduces fundamental and significant clinical economic

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concepts and analytical approaches with widespread use in medical science as well as in health services research, including those that address the outcomes and economic costs of kidney transplantation. In the health care literature, economic evaluation can use one of the following four approaches or methodologies (Muennig 2002): Cost-minimization analysis, Cost-effectiveness analysis, Cost-utility analysis, and Cost-benefit analysis. These methodologies, though similar in some ways, are distinct from each other in terms of the research questions they are designed to address, and how the consequences of the studied interventions are measured and valued.

Cost or Cost-Minimization Analysis A cost analysis or cost-minimization analysis seeks to identify the economic costs of alternative interventions that produce the same or similar outcomes but vary in both the type and quantity of inputs or resources used. The methodology of a cost analysis is twofold: 1) costs of a treatment must be accurately measured and accounted for; and 2) the outcomes can be evaluated against alternative treatments per the premise that if the outcomes are similar or the same, then the least costly intervention is preferred.

Types of Costs Included Most clinical economic analyses include three major categories of costs: 1) direct costs of intervention, 2) indirect costs, and 3) intangible costs. Direct costs include the actual expenses for the use of inputs or resources associated with the medical intervention being examined. These costs reflect the value of resources used to prevent and diagnose a health impairment and to treat the patients. There are two sub-categories of direct costs. Direct medical costs, such as costs of hospitalization and physician services, are those incurred within the health sector. Direct non-medical costs, on the other hand, are those that are directly associated with the diagnosis and treatment but fall outside of the health sector, and are incurred by the patient or family. Some examples of direct non-medical costs include expenses for transportation, lodging, goods, childcare, out-of-pocket purchases of medicine and medical supplies, and special clothing. A second major component of costs in economic evaluation analysis (i.e. indirect or productivity costs of medical intervention) consists of those related to the consequences of morbidity and mortality. Indirect of productivity costs of medical intervention include the costs of job absenteeism to employers, decreased earning ability suffered by the patient and family, and changes in occupation due to, for example, long-term disability. Costs of premature death also fall within the classification of indirect costs.

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Finally, a third type of costs, intangible costs, are those associated with pain, grief, and other personal suffering such as changes in social functioning and activities of daily living. Various approaches have been used to convert these intangible costs into dollars for ease of combining them with the other types of costs in clinical economic evaluation.

Cost Effective Analysis Cost-effectiveness analysis (CEA) is a technique for selecting among alternative and competing interventions that produce similar or the same outcomes in an environment of limited resources. This technique, since its introduction to medical research in the 1960s, has become a common feature in medical literature and clinical guidelines (Gold, 1996). CEA addresses the central question of which among the competing strategies offers the best value by calculating the cost effectiveness ratio:

The CE ratio might be interpreted as the "price" of an additional or extra unit of outcome purchased by switching from current practice to the new strategy (e.g., $10,000 per life year or LY). If the price is low enough when compared to other worthy strategies, the new strategy is considered "cost-effective."

Cost-Utility Analysis Cost-utility analysis (CUA) is a special case of CEA in which intervention strategies produce outcomes that are different in terms of both quantity (e.g., number of LY’s saved or extended) and subjective levels of well-being or quality of life. The best known “utility” measure for evaluating the well-being or quality of life is the “quality-adjusted life years” or QALY’s. In this case, alternative strategies are compared on the basis of cost per extra or incremental unit of QALY.

Cost-Benefit Analysis Cost-benefit analysis (CBA) is a technique used most commonly for evaluating public work projects such as the construction of highways, hydroelectric facilities, and school improvements. Public works projects tend to serve different populations and produce different outcomes, which necessitates the utilization of an analysis technique to effectively capture and compare the relevant costs and benefits associated with the project. CBA identifies, quantifies, and adds all the positive factors and measures these benefits in dollars. Then it identifies, quantifies, and subtracts all the costs associated with the action or program. The difference between the two indicates whether the planned action is advisable. However, the

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accurate identification, inclusion and quantification of the costs and benefits presents a substantial challenge in performing an accurate cost benefit analysis. Due to the unwillingness in the health care field to measure and compare life values, CBA has experienced limited use in medical and health services research.

Discounting The proper quantification and measurement of the costs and benefits of an action or intervention strategy are complicated by the reality that both costs and benefits may not all occur in a single year. In most cases, costs and benefits will emerge over a long period of time. Discounting is a technique that allows the calculation of present values of costs and benefits which accrue in the future. Consistent with its emphasis on time preference, discounting assumes that individuals prefer to forego a part of the benefits if they accrue it now, rather than fully in the uncertain future. By the same reasoning, individuals prefer to delay costs rather than incur them in the present. The strength of this preference is expressed by the discount rate which is inserted in economic evaluations.

Sensitivity Analysis The results and conclusions of economic evaluation can change according to the assumptions that underlie the study. The results and conclusions are also subject to change due to the reality of uncertainty involved in the statistical estimation of the numerical values of the clinical outcomes as well as the dollar amounts of costs and benefits. Sensitivity analysis is a technique that repeats the evaluation analysis by varying the assumptions underlying the estimates. In so doing, sensitivity analysis tests the robustness of the conclusions by varying the items around which there is uncertainty. Given that there will be a degree of uncertainty about some elements of any economic evaluation, sensitivity analysis assists in judging how robust the conclusions will be.

III. Results III.A. Kidney Transplantation Cost Analysis A convenient way to examine the costs of kidney transplantation is to analyze Medicare expenditures for ESRD program in general and kidney transplantations in particular. Since Medicare covers the treatment costs of ESRD patients in the U.S., total and average payments per patient per year based on Medicare claims data provide a reliable account of the costs of dialysis treatment and kidney transplantations. It should be noted that the analysis of Medicare claims data implicitly takes the narrow perspective of the insurer and not that of the society at large.

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Each year, the United States Renal Data System (USRDS) analyzes the finances of Medicare’s portion of the ESRD program and reports the results in an annual publication called the Annual Data Report or ADR of the United States Renal Data System (USRDS 2004). According to the 2004 edition of the ADR, Medicare’s portion of the ESRD program grew from $5.8 billion in 1991 to $17 billion (or 6.7 percent of the Medicare budget) by 2002. On a per-member-per-year basis, the costs for dialysis were approximately $53,000 in 2002. Deductibles and co-payments increased the total dialysis costs to approximately $63,000 per-member-per-year. In comparison, the costs of transplantations were much lower, at about $18,400 per-member-per-year for Medicare. However, the costs of transplantations rise rapidly with age. Transplantation costs have also been reported in peer-reviewed journals. For example, Shireman, Martin and Whiting (2001) analyzed 12 months of retrospective claims data for a population of Medicaid patients who underwent a solid-organ transplantation. Total health care costs, including the initial hospitalization and subsequent outpatient hospital services, physician visits, other ambulatory visits, and prescription drug costs, were compared across transplant types and all costs were captured in 1998 dollars. The authors found that costs were significantly different between the kidney group and the unspecified group, and between the liver group and the unspecified group. Heart transplant recipients had the highest costs that averaged nearly $20,000 per year more than kidney recipients, although this difference was not statistically significant. Liver organ recipients’ costs were closer to those of the kidney recipients, and averaged around $40,000 per transplant. Finally, drug costs were consistent across the four types of transplants. Researchers from many foreign countries have also analyzed and described the costs in U.S. dollars of their kidney transplant programs. Lopez-Neblina, Alvarez and Finkelstein (2000) reported that the cost of a traditional kidney transplant procedure in the private practice in Mexico ranged from US $35,000 to US $40,000 per patient. However, the cost in the first two years of the public-sector High-Efficiency Kidney Transplantation (HEKI) Program was much lower with a range between US $10,000 and US $18,000 range, and experienced a cost variance by geographic region. Nakajima et al. (2001) reported that dialysis patients in Japan numbered over 170,000 in 1998, but only 658 patients received a renal transplant that year. The average cost of all recipients at a single institution in Japan during the first year after renal transplantation was recently reported to be about US $50,000. The average cost dropped to about US $19,000 during the second year. In contrast, the average cost of dialysis treatment during the first year in Japan was estimated to be about $46,000. This relatively high cost of dialysis treatment in Japan suggests that transplantation can become less expensive than dialysis in just two years while providing superior outcomes. Using U.S. Medicare claims data for 42,868 cadaveric (CAD) donor kidney transplants and 13,754 living donor (LD) transplants, Smith et al. (2000) compared the differences in financial costs between these two types of procedures. The average total Medicare payments were $39,534 for CAD and $24,652 for LD transplants during the first posttransplant year. The most significant cost differential between the two procedures was from the inpatient hospitalization. Living donor transplants, the authors concluded, cost remarkably less than cadaveric transplants, and the single largest cost saving emanated from inpatient hospital services.

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It is important to note that most of the cost studies reviewed typically include such direct medical costs as the costs of initial transplantation, subsequent inpatient and outpatient visits and the necessary pharmaceutical therapy to maintain quality of life and prevent acute rejection. Few studies consider the other two categories of costs for which data are more difficult to collect – the indirect (productivity) costs and intangible costs. Another commonality identified among the reviewed cost studies was the measurement of costs in current dollars rather than constant dollars adjusted for medical inflation. Lastly, few of the cost studies applied the concept and technique of discounting to report the net present value of the costs of transplantation as advised by experts in the field of economic evaluation of clinical interventions (Gold et al., 1996). Cost-Effectiveness of Kidney Transplantation Kidney transplantation was first successfully performed in 1954 (Merrill, Hurray, Harrison et al 1956). The development in the 1960s and 1970s of immunosuppressive drugs that prevented the human immune system from rejecting the “foreign” organ dramatically improved the efficacy and popularity of the procedure. Today, the kidney transplantation has reached a high level of maturity and the primary barrier to this life-saving procedure is now the limited availability of kidneys from either living or cardaveric donors (USRDS 2004). The literature research for studies published between 1968 and 2000 that assessed the economic costs and benefits of kidney transplantation was thoroughly reviewed by a metastudy by Winkelmayer, Weinstein, Mittleman et al. (2002). The inclusion criteria of this extensive meta-study specified that the appropriate articles for review must include in the title of the article both an appropriate economic evaluation term such as “cost-effectiveness analysis,” “health care costs,” and/or “economics” and a key renal replacement therapy term such as “renal replacement therapy,” “end-stage renal disease,” or “kidney dialysis.” A total of thirteen studies from seven countries (4 articles from U.S. 2 each from Canada, U.K., and Sweden, and 1 each from Brazil, New Zealand, and the Netherlands) met the inclusion criteria. To compare the relative cost effectiveness of kidney transplantation against its major alternative treatments of center-based hemodialysis and home hemodialysis, costeffectiveness ratios were translated into 2000 U.S. dollars per life-year (LY) saved using the standard meta-study methodology to combine the results based on different research methods and analytical techniques. In summary, the cost-effectiveness of center hemodialysis remained within a narrow range of $55,000 to $80,000 per LY saved in most studies reviewed. In comparison, the cost-effectiveness of home hemodialysis was found to be between $33,000 and $50,000 per LY saved. Kidney transplantation, in contrast, became increasingly more cost-effective overtime, approaching about $10,000 per LY saved in the late 1990s. Several recent studies have analyzed and compared the cost-effectiveness of kidney transplantation versus hemodialysis. Analyzing cost data from eight transplantation centers in New York City in 1998, Loubeau, Loubeau and Jantzen (2001) found that the initial higher costs of transplantation were fully recouped by Medicare 2 years and 10 months after the initial surgery. For patients eligible for Medicare solely due to their end-stage renal disease status (thus they tend to be younger patients), transplantation would save on average $3,800 over dialysis for the two years after the break-even point. For regular Medicare patients, the average savings would be $2,400.

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Similar patterns of cost savings of kidney transplantation over hemodialysis have also been confirmed by foreign studies such as Salonen, Reina, Oksa et al. (2003) for Finland, Kalo (2003) for Spain, and Kaminota (2001) for Japan. However, none of the above costeffectiveness studies took into consideration quality differences in treatment alternatives for end-stage renal disease, thus ignoring an important aspect of clinical economic evaluation that can potentially affect the results of a cost-effectiveness analysis.

III.B. Quality of Life Assessment In the renal transplantation literature, quality of life has long been a focus of research. Klarman et al. (1968) described in a classic cost-effectiveness analysis that renal transplantation not only increased patient survival but also improved quality of life by as much as 25 percent compared with dialysis. This finding has since been confirmed by numerous other studies including Evans, Manninen, Garrison et al. (1985), and Russell, Beecroft, Ludwom et al. (1992) using a wide range of assessment scales. Recently, Parsons and Harris (1997) demonstrated that patients with a successful renal transplant consistently showed superior levels of functional ability when assessed on the basis of their “perceived health status,” “total life satisfaction,” “social well-being,” and “profile of sickness impact.” Similarly, an Canadian study (Laupacis, Keown, Pus et al. 1996) demonstrated convincingly that quality of life based on a utility scale from 0 (death) to 1 (perfect health) increased 25 percent from 0.57 before transplantation while studied patients were on the waiting list to 0.74 at the 12-month endpoint. The same Canadian study also found that the employment rate of transplant patients went up, from 30 percent before transplantation to 45 percent at the 24-month endpoint. However, the presence of diabetes in the ESRD presents a special problem. For example, patients with diabetes mellitus tend to have higher morbidity when compared with patients with renal disease. Studies have also shown that ESRD patients with diabetes mellitus tend to experience inferior quality of life than other ESRD patients (Parsons and Harris 1997; Evans, Manninen, Garrison et al. 1985). To investigate the impact of quality-of-life adjustment on cost-effectiveness analyses, Chapman, Berger, Weinstein et al. (2004) conducted a meta-study of 228 original cost-utility studies on a wide range of clinical interventions published before 1998. Out of these, sixtythree (173 ratio pairs) reported both cost per life year (LY) and cost per quality-adjusted life year (QALY) for the same intervention. The main conclusion of this large scale meta-study was that the mean ratios were $69,100/LY and $103,100/QALY, with corresponding medians of $24,000/LY and $20,400/QALY. The mean value of quality-adjusted cost-effectiveness ratios thus exceeds that of the non-adjusted ratios by $34,300, with a median difference of $1,300. However, 60 percent of the ratio pairs differ by $10,000 per year or less. When the reciprocals of the ratio pairs were compared, the mean difference was 59 life years or quality-adjusted life years per $1 million (median: $2.1 million). Taken together, the results of the meta-analysis suggest that quality adjustment did not substantially alter the estimated cost-effectiveness of an intervention. This conclusion was further confirmed by the calculation of the Spearman rankorder correlation between ratio types. Specifically, quality adjustment led to a ratio moving either above or below $50,000/LY (or QALY) in only 8 percent of the ratio pairs.

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Unlike most other cost-effectiveness analyses that primarily focus on the impact of kidney transplantation on patients’ quality of life, Chang et al. (2004) analyzed the costs and outcomes of an innovative, nurse-led intervention program designed to improve patients’ quality of life during the posttransplantation period. The quality of life intervention program integrated a three-pronged interdisciplinary approach that emphasized: 1) proactive, patientinitiated care to prevent posttransplantation morbidities, 2) employment counseling, and 3) enhancement of social support network. A cost-effectiveness analysis was performed comparing quality of life and costs of two groups of sequential patients, a treatment group (n = 150) and a retrospective control group (n = 30), who received a kidney transplant during December 1998 and August 2001. Cost and quality-of-life data were collected at 6 and 12month endpoints, and the number of quality-adjusted “treatment-free days” was used as the primary outcome. Patients in the treatment group exhibited better outcomes as measured by the average number of treatment-free days (289 vs. 272) at 12 months. Further, the superior outcome was delivered at an incremental cost of only $29 per quality-adjusted treatment-free day. A oneway sensitivity analysis confirmed the robustness of the results.

III.C. Cost and Cost Effectiveness of Immunosuppressive Regimens Cost Analysis Despite impressive improvement in surgical procedures and techniques in renal transplantation, acute rejection remains one of the major complications in kidney transplantation (Vincenti, Larsen, Durrbach, et al. 2005). Sollinger (1995) reported that rejection occurred in about 20 – 30 percent of the cases within the first 12 weeks. The search for better and safer immunosuppressive therapies and the optimization of existing regimens to prevent and treat acute rejection have been a priority among practitioners and researchers. Treatment of Acute Rejection Corticosteroids or glucocorticoids, often just called "steroids," were once thought to be almost miraculous in the 1940’s and 1950’s. However, as the use expanded over the years, side effects emerged and it was realized that high doses given over long periods of time could produce unacceptable risks. Corticosteroids were first introduced as a treatment for acute rejection in 1954 during the first kidney transplant (Nghiem et al. 2005). However, chronic use of this pharmaceutical therapy produced long-term complications such as massive weight gains, diabetes, hypertension, osteoporosis, dyslipidemia, cataract, and increased malignancy. In the1980s and 1990s, many immunosuppressive drugs have been introduced to work alone or in combination with other drugs to supplement conventional steroid withdrawal regimens in order to lower acute rejection rates and to prevent graft loss. These include mycophenolate mofetil (MMF), everolimus (Certican®), cyclosporine (CSA), basiliximab (Simulect®), and anti-thymocyte globulin (Thymoglobulin®). Cost Studies In a retrospective study involving 147 consecutive patients who received a kidney transplant at the Allegheny General Hospital at Pittsburgh, Pennsylvania, Nghiem et al.

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(2005) tracked and compared the costs of four protocols of immunosuppression. The study was motivated by the belief that the concomitant use of T-cell antibody such as IL2, receptor antagonist, and basiliximab with cyclosporine (CSA) and MMF would significantly improve graft survival and decrease the costs of cadaveric kidney transplantations. The first group of study patients (n = 52) were classically treated with a triple therapy of CSA plus MMF plus Prednisone (cortisone taken internally) and served as a historical control. The second group (n = 31), called the “Simulect® induced group,” received addition induction with basiliximab (Simulect®). The third group (n = 30) received basiliximab plus cyclosporine (CSA) plus MMF and was referred to as the “Simulect® and steroid-free group.” The fourth group (n = 34) was the “Thymoglobulin and steroid-free” group which received Thymoglobulin plus CSA plus MMF in conjunction with only four days of steroid. The four groups were similar in donor and recipient characteristics such as gender, race, and cold ischemic time. Costs data for hospital charges for primary hospitalization, readmission for rejection, and complications were collected from computer records of the treating hospitals. All hospital charges were converted into 2004 dollars using the hospital services stratum of the Consumer Price Index (CPI). The results showed similar clinical outcomes among the four groups as measured by their readmission rates and readmission length of stay. Most important, total readmission charges including the charges for immunosuppressive therapies were similar in all four groups. Thus, the three steroid minimization protocols did not reduce either the primary or the readmission lengths of stay, the times to first and second admission, the incidence of delayed graft function, the rejection rate, the graft survival, nor (and) the graft function. The classical triple therapy of CSA + MMF + the internally taken cortisone Prednisone emerged to be cost effective because it was less expensive and equally effective in comparison to the other three alternative protocols. Several additional studies have become available that examine the safety, efficacy, and costs of basiliximab (Simulect®) in both renal transplant induction and in preventing acute rejection. Lilliu and colleagues (2004) in France conducted a cost-minimization study comparing the costs of Simulect® and Thymoglobulin® that had been shown to have equivalent efficacy in renal transplant induction. Outcomes of the study indicated the decrease in the costs of care for the Simulect® patients, as analyzed from the hospital perspective, resulted from a significant reduction in the initial hospital length of stay and the number of infectious episodes. Consequently, direct cost savings of 1,159 Euros per patient in the Simulect® group more than compensated for the higher price of this immunosuppressive drug. Cost-Effectiveness Analysis Separately in the U.K., an economic evaluation was undertaken by Chilcott and colleagues (2002) alongside a multicenter international trial of basiliximab (Simulect®) in preventing acute rejection in renal transplantation. Clinical outcomes and resource utilization in a double-blind trial were monitored and analyzed in the 12 months following transplantation. The results showed a statistically significantly lower rejection rate in the basiliximab group than in the placebo group (37.9% vs. 54.8%) and no statistically significant differences in the mean cost of treatment per patient. The mean cost of treating basiliximab patients was US $47,940 and US $46,280 for placebo patients. Basiliximab was found to be a

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cost-effective therapy with a reduced rate of rejection and a consistent average cost of treatment for participants in both the treatment and placebo groups.

IV. Conclusion Management of ESRD is a special challenge for the American health care system because the costs of treatment of this disease are primarily paid for by Medicare, a federal health insurance program that has been under constant financial stress. The optimal allocation of available resources to satisfy the competing demands of the ESRD population is essential in ensuring best possible outcomes under the ever-tightening financial and regulatory constraints. The paper summarizes the current knowledge on the economics of ESRD with a focus on kidney transplantation. It systematically reviews topics relating to the cost and cost effectiveness aspects of kidney transplantation in the peer-reviewed literature and reports their key findings. The evidence summarized in this review suggests the following conclusions. First, kidney transplantation is both effective and cost-effective treatment option when compared with dialysis, the main alternative when an appropriate organ is unavailable or when the patient is not suited for this high-risk and more invasive surgical alternative. The major challenge today is primarily the limited supply of suitable kidneys from either cardaveric or living donors. The front-end costs of kidney transplantation are high, with patients having a transplant event during the year consuming about $100,000 in services and pharmaceuticals in 20012002 (USRDS 2004). However, the $100,000 investment in a patient who received a transplant yielded an average of $50,000 per year of savings compared to dialysis. In addition, the quality of life of transplant treatment has also been shown to be superior. Thus when compared to the willingness to pay benchmark of $55,000 per life year established by Winkelmayer, Weinstein, Mittleman et al. (2002), renal replacement transplantation appears to be reasonably well justified from the societal perspective. Another factor that has contributed to the success of kidney transplantation has been the introduction of newer and more effective immunosuppressive drugs such as mycophenolate mofetil (MMF), everolimus (Certican®), cyclosporine (CSA), basiliximab (Simulect®), and anti-thymocyte globulin (Thymoglobulin®). When used alone or in combination with other classic anti-inflammation drugs, these newer drugs have been shown to be effective as a group contributing greatly to the increased attractiveness of kidney transplantations compared to dialysis. This and other data have led researchers such as Yen and colleagues at Washington University Medical School to urge the extension of Medicare coverage of immunosuppressive medications throughout the life of the transplant recipient (Yen et al. 2004).

References Chang CF, Winsett RP, Gaber AO, et al. Cost-Effectiveness of Post-Transplantation Quality of Life Intervention Among Kidney Recipients. Clin. Transplant 2004 Aug;18(4): 40714.

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Chapman RH, Berger M, Weinstein MC, et al. When Does Quality-Adjusting Life-Years Matter in Cost-Effectiveness Analysis? Health Econ 2004 May;13(5):429-36. Chilcott JB, Holmes MW, Walters S, et al. The Economics of Basiliximab (Simulect) in Preventing Acute Rejection in Renal Transplantation. Transpl Int 2002 Oct;15(9-10): 486-93. De Lew N. Medicare: 35 years of service. Health Care Financing Review 2000 ;22(1) :75103. De Vit GA, Ramsteijn PG, De Charro FTA. Economic evaluation of end stage renal disease treatment. Health Policy. 1998;44:215-32. Drummond MF, O’Brien B, Stoddart G, et al. Methods for the economic evaluation of health care programmes. New York: Oxford University Press, 1997. Eisenberg JM. Clinical economics. A guide to the economic analysis of clinical practices. JAMA. Nov 24, 1989;262(20):2879-86. Evans RW, Manninen Dl, Garrison LP, et al. The Quality of Life of Patients with End-Stage Renal Disease. The New England Journal of Medicine, 1985, 312, 553 – 559. Gold MR, Siegel JE, Russell LB, et al. Cost-Effectiveness in Health and Medicine. New York: Oxford University Press, 1996 Kalo Z, Jaray J, Nagy J. Economic Evaluation of Kidney Transplantation Versus Hemodialysis in Patients With End-Stage Renal Disease in Hungary. Prog. Transplant. 2001. Sept; 11(3):188-193. Kaminota M. Cost-Effectiveness Analysis of Dialysis and Kidney Transplants in Japan. J. Med. 2001 Jun;50(2):100-8. Klarman HE, Francis JO, Rosenthal JD. Cost-effectiveness analysis applied to the treatment of chronic renal disease. Medical Care 1968;6:48 -54. Laupacis A, Keown P, Pus N, et al. A study of quality of life and cost-utility of renal transplantation. Kidney Int. 1996;50:235-42. Lilliu H, Brun-Strang C, Le Pen C, et al. Cost-Minimization Study Comparing Simulect vs. Thymoglobulin in Renal Transplant Induction. Clin Transplant. 2004 Jun;18(3):247-53. Lopez-Neblina F, Alvarez JH, Finkelstein LI. High-Efficiency Kidney Transplantation: Concept, Technique, Results, and Cost Analysis. Transplant Proc 2000 Feb;32(1):141-2. Loubeau PR, Loubeau JM, Jantzen R. The Economics of Kidney Transplantation Versus Hemodialysis. Prog. Transplant 2001 Dec;11(4):291-7. Merrill JP, Murray JE, Harrison JH, et al. Successful homotransplantation of the human kidney between identical twins. JAMA 1956;160:277-82. Muennig P. Designing and Conducting Cost-Effectiveness Analyses in Medicine and Health Care. San Francisco: Jossey-Bass, 2002. Nakajima I, Akamatsu M, Tojimbara T, et al. Economic Study of Renal Transplantation: A Single-Center Analysis in Japan. Transplant Proc 2001 Feb-Mar;33(1-2):1891-2. Nghiem DD, Carpenter BJ, Schlosser JD, et al. The Cost of Steroid-Free Protocols in Primary Cadaveric Kidney Transplantation (In Process Citation). Transplant Proc 2005 May;37(4):1797-9. Parsons DS, Harris DC. A review of quality of life in chronic renal failure. Pharmacoeconomics 1997;12:140-60. Russell JD, Beecroft ML, Ludwom D, et al. The quality of life in renal transplantation - a prospective study. Transplantation 1992; 54 (4): 656-60.

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Sakamaki H, et al. Cost effectiveness analysis of mycophenolate mofetil treatment for intractable acute rejection in renal transplantation recipients [abstract]. Value in Health 1999;2:204-5. Salonen T, Reina T, Oksa H, et al. Cost Analysis of Renal Replacement Therapies in Finland. Am J Kidney Dis 2003 Dec;42(6):1228-38. Shireman TI, Martin JE, Whiting JF. The Cost of Transplant Graft Maintenance Following Solid Organ Transplantation. Transplant Proc 2001 Feb-Mar;33(1-2):1920-1. Smith CR, Woodward RS, Cohen DS, et al. Cadaveric Versus Living Donor Kidney Transplantation: a Medicare Payment Analysis. Transplantation. 2000 Jan 27; 69(2):3114. Sollinger HW. US Renal Transplant-Mycophenolate Mofetil Study Group. Mycophenolate mofetil for the prevention of acute rejection in primary cadaveric renal allograft recipients. Transplantation 1995;60:225-32. Vincenti F, Larsen C, Durrbach A, et al. Costimulation blockade with belatacept in renal transplantation. N Engl J Med. 2005;353(8):770-81. U.S. Renal Data System, USRDS 2004 Annual Data Report: Atlas of End-Stage Renal Disease in the United States,National Institutes of Health, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, 2004. Winkelmayer WC, Weinstein MC, Mittleman MA, et al. Health economic evaluations: The special case of end-stage renal disease treatment. Medical Decision Making. 2002;22(5):417-30. Yen EF, Hardinger K, Brennan DC, et al. Cost-effectiveness of extending Medicare coverage of immunosuppressive medications to the life of a kidney transplant. American Journal of Transplantation 2004;4:1703-1708.

In: Progress in Kidney Transplantation Editor: Dominick W. Mancuso, pp. 137-142

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter VIII

Ethical Aspects of Kidney Transplantation Paolo Bruzzone* Divisione Trapianti d’Organo, Dipartimento “Paride Stefanini”, Università di Roma “La Sapienza”, Rome, Italy

Abstract Kidney transplantation from living donors is widely performed all over the world. Living nephrectomy for transplantation has no direct advantages for the donor other than an increased self-esteem, but at least remains an extremely safe procedure, with a worldwide overall mortality of 0.03%. This theoretical risk for the donor seems to be justified by the socioeconomic advantages and increased quality of life of the recipient, especially in selected cases, such as paediatric patients, when living donor kidney transplantation can be performed in a preuremic phase, avoiding the psychological and physical stress of dialysis, which in children is not well tolerated and cannot prevent the retarded growth. According to the Ethical Council of the Transplantation Society, commercialism must be effectively prevented, not only for ethical but also medical reasons. The risks are too high not only for the donors, but also for the recipients, as a consequence of poor donor screening and evaluation with consequent transmission of HIV or other infective agents, as well also of inappropriate medical and surgical management of donors and also of recipients, who are often discharged too early. Most public or private insurance companies are considering kidney donation a safe procedure without long-term impairment and therefore do not increase the premium, while recipients’ insurances of course should cover hospital fees for the donors. "Rewarded gifting" or other financial incentives to compensate for the inconvenience and loss of income related to the donation are not advisable, at least in our opinion. Our Centre does not perform anonymous living organ donation or “cross-over” transplantation. *

Corresponding Author: Paolo Bruzzone MD. Home address: Via Santa Maria Goretti 38/10 00199 Rome Italy. Phone: 39-6-86209440 Fax: 39-6-4463667 E-mail [email protected]

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Paolo Bruzzone In the last few years kidney, liver, pancreas, heart and lungs transplantation have been increasingly performed with declining morbidity and mortality. However the inadequate availability of cadaver human donors significantly reduces the number of patients who can undergo this kind of treatment. An alternative procedure is xenotransplantation, introduced in Europe in 1966 by our Institution using primates as donors. More recently other animal species, such as pigs, who are not under risk of extinction, have been preferred; this "discordant" model of xenotransplantation is associated with unreversible hyperacute rejection on a humoral basis, involving complement activation. This phenomenon could be prevented by creating transgenic pigs expressing on their cell membranes complement inhibitors such as Decay Accelerating Factor (DAF), Membrane Cofactor Protein(MCP) and CD59.

Key words: Kidney transplantation, living related kidney donors, living unrelated kidney donors, Cyclosporine, xenotransplantation.

Introduction Solid organ transplantation has been occasionally as well as unsuccessfully performed since 1906 [1]. Progressive improvements in surgical technique and immunosuppressive therapy made possible in the sixties to obtain prolonged survival after kidney transplantation from chimpanzee to human both in U.S.A. and Europe[2]. The increasing availability of dialysis therapy and kidney transplantation from cadaver or living related and unrelated kidney donors reduced the interest for xenotransplantation, which however has recently become again an attractive option, at least for heart and liver temporary replacement. However chimpanzee and baboons are expensive, have reduced sizes[3] and can cause zoonoses[4]. A better choice for xenotransplantation may be offered by pigs, that have anatomical and metabolic close similarities to humans[5]. Such a "discordant" xenotransplantation, such as from swine to human, are associated with humoral hyperacute rejection, due to preformed antibodies, together with complement system activation (both classical and alternative ways) and cellular necrosis[6]. Complement activation inhibitors may be soluble (H or C4bp) or membrane bound: CR1(CD35), Decay Accelerating Factor (DAF or CD55)[7], Membrane Cofactor Protein (MCP or CD46) and CD59. The organs harvested from pigs transgenics for DAF, MCP and CD59, would be protected by complement attack when grafted into human recipients[8, 9, 10]. After having obtained an adequate number of transgenic pigs, their organs have been harvested and perfused with oxigenated, heparinized human blood[11,12 13]; histology and electron microscopy studies have shown protection from human complement attack. With the same aim pancreatic islets[14] and solid organ transplantation[15, 16] will be done from transgenic pigs to adult baboons. Finally, after adequate approval from competent authorities, islets transplantation from transgenic pigs into diabetic human recipients could be performed, as well as heterotopic liver or heart transplantation in fulminant hepatitis[17] or in case of intractable heart failure, as a bridge to homotransplantation.

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These initial clinical trials will assess the possibility of permanent replacement of human organs with heart, livers and kidneys harvested from transgenic pigs. Kidney transplantation from living donor, accomplished for the first time in 1954 by Murray, who confirmed the possibility of successfully performing this kind of procedure with a long-term recipient survival, is now considered a good clinical solution, complementary to cadaver (CD) kidney transplantation, for increasing the donors’ pool. With the increasing availability of dialysis, many centres subsequently discouraged transplantation from living donors: however the introduction of Cyclosporine A with a significant improvement of the results in cadaver kidney transplantation together with a dramatic growth of the number patients in the waiting list, due to inadequate supply of cadaver kidneys, prompted again the expansion of the criteria for acceptable living donors. The validity of this kind of procedure is based upon many ethical and clinical consideration, including the results which in most reports are better than with cadaver donor kidney transplantation, the free willingness of the donor and the limited amount of risks for his health: both conventional and laparoscopic living donor nephrectomy is a safe procedure, with a worldwide overall mortality of 0.03%[18]. However, at least 4 kidney donors all over the world developed end stage renal failure and underwent kidney transplantation[19]. Our Center activated the Living Related Donor (LRD) kidney transplantation program in 1967 and the Living Unrelated Donor (LURD) kidney transplantation program in 1968, performing 62 kidney transplants (of which 6 LURD) under conventional therapy. In the Cyclosporine era, our group did the first living kidney transplant in 1982 and realized the first LURD transplant in Europe in 1983.

Materials and Methods The study population consisted of 398 LRD- of which 35 pairs shared 2 haplotypes, 338 shared 1 haplotype(Group A) and 25 had no haplotypes in common - and of 206 LURD(Group B): 171 between spouse pairs(Group C) – 123 from wife to husband (Group C1) and 33 from husband to wife with pre-transplant pregnancies (Group C2) as well as 33 between relatives in law or emotionally related patients(Group D). 194 pairs showed 3-6 HLA A B Dr mismatches(MM) with the donor and in 10 cases 0-2 MM. Donor and recipient mean age was 48±87 and 34.8±8.2 in Group A and respectively 46±11.2 and 47±11.2 in Group B. The post-transplant immunosuppression therapy was based on Cyclosporine A(CAS). χ2 test was used to assess statistical significance.

Results Donor mortality was 0%; perioperative morbidity was 15.2%. Graft function immediately started after surgery. The actuarial 1yr,5yrs,10yrs and 15yrs graft survival was in Group A: 92%, 87%, 78%, 64% vs. Group B 89%, 78%, 71%, 70%(NS), Group C1: 90%, 75%, 67%, 69% vs. Group C2: 81%, 74%, 72%, 62%(NS) and Group C: 88%, 78%, 66%, 60% vs. Group D: 91%, 80%, 71%, 61%(NS).

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Discussion and Conclusions There was no statistically significant difference between LURD and LRD as far as graft survival. Immunological factors, such as HLA compatibility, pre-transplant blood transfusions and previous pregnancies seem not to influence graft survival. While, according to various authors, morbidity ranged from 10% to 20%, major complications such as severe haemorrhages, pulmonary embolism and pneumothorax can be prevented by an accurate surgical technique and a prompt donor mobilization after surgery. According to published data[20] as well as to our personal experience[21, 22, 23], presently there could be no clinical but only ethical objections to LURD kidney transplantation. Many donors report increased self-esteem, as a consequence of having helped to restore the health and improve the quality of life of a significant one. Some people in fact could argue that the theoretical risk for the donor, which as previously discussed is extremely low, does not compensate for the evident socioeconomic advantages and increased quality of life of the recipient, who avoids a long waiting list time and is usually able to return to social activity and work earlier. In selected cases, such as younger recipients, kidney transplantation can be performed in a pre-uremic phase, avoiding to the patients the psychological and physical stress of dialysis, which in paediatric cases is not well tolerated and cannot prevent the retarded growth. The consent has to be free and without any form of coercion, that could be very subtle and difficult to detect, as some forms of psychological conditioning are difficult to be recognised in a family setting, while economic dealings may be on purpose concealed even by the donor. According to the Consensus Statement of the Amsterdam Forum on the Care of the Live Kidney Donor, organized on April 1-4, 2004 by the Ethics Committee of the Transplantation Society, “Minors less than 18 years of age should not be used as living kidney donors”[24]. In agreement with the guidelines suggested by the Ethical council of the Transplantation Society, commercialism must be effectively prevented, may be by using as living unrelated donors only spouses or relatives in law with a great interest in recipient's health conditions. One must remind that results of truly commercial kidney transplants are very discouraging, providing a further issue against transplant from paid donors, as a consequence of poor donor screening with consequent transmission of HIV and other infective agents, as well also of inappropriate medical and surgical management of recipients, who present an unacceptable morbidity rate and, often after having been discharged too early, seek medical attention in their native countries. "Rewarded gifting" or other financial incentives to compensate for the inconvenience and loss of income related to the donation are not advisable, at least in our opinion. All over the world most public or private insurance companies are considering living kidney donation a safe procedure without long-term harm or impairment and therefore do not increase the premium for these donors, while recipients’ insurances of course should cover the donors hospital fees. Due to obvious ethical reasons, transplant physicians and surgeons cannot take part in kidney donation from prisoners, even such a procedure has been proposed to death-row inmates as an alternative to execution.

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In order to increase the possibility of LURD kidney transplantation between blood group incompatible pairs, Rapaport proposed in 1986 a national network to exchange kidneys harvested from living donors with a blood group not compatible with their emotionally related recipients. This suggestion was proposed again in 1997 by Ross as a local program, by Park[25] and by other authors. This so called cross-over procedure could allow to perform living kidney transplantation also in case of couples with direct positive cross-match and, if applied on a larger scale, could allow also the HLA matching, which however is probably very useful in this kind of transplant. In our experience only 5% of couples could benefit from cross-over, which could theoretically have negative psychological and ethical effects, such as a decreased willingness to donate to a stranger or a higher risk of donor coercion and organ commercialism. Therefore, our Centre does not perform “cross-over” transplantation. In conclusion, we certainly agree with the guidelines issued by the International Congress on Ethics in Organ Transplantation (Munich, December 10-13, 2002): kidney transplantation from living donors is a safe and effective procedure and should not be discouraged.

References [1] [2] [3] [4]

[5] [6] [7]

[8]

[9]

Jaboulay M.: Greffe de reins au pli du coude par soutures arterielles et veineuses. Lyon Med 107, 575-577,1906. Bailey L., Nehlsen-Cannarella S.L., Concepcion W. et al.: Baboon-to-human cardiac xenotransplantation in a neonate. J Am Med Ass 254,3321-3329,1985. Niekrász M., Ye Y.,Rolf L.L. et al.: The pig as organ donor for man. Transplant Proc 24(2):625-626,1992. Kalter S.S.: The baboon. Microbiology, clinical, chemistry and some hematological aspects. In Primates in Medicine,Vol.8, Series Editors: Goldsmith E.I. and MortJankowski J.,Karger S.Publishing, Basel,1973. Coates M.E., Gustafsson B.E.(eds,): The germ-free animal in biomedical research. Laboratory Animals Ltd.,London,1984. Ross D.N. In Experience with human heart transplantation (edited by H.Shapiro),Butterworths,Durban,1969. Cary N., Moody J., Yannoutsos N. et al.: Tissue expression of human decay accelerating factor, a regulator of complement activation expressed in mice: a potential approach to inhibition of hyperacute xenograft rejection. Transplant Proc 25(1):400401,1993. White D.J.G., Oglesby T., Liszewski M.K. et al.: Expression of human decay accelerating factor or membrane cofactor protein genes on mouse cells inhibits lysis by human complement. Transplant Proc 24(2):474-476,1992. Pascher A., Poehlein Ch. et al.: Expression of human decay accelerating factor (hDAF) in transgenic pigs regulates complement activation during ex vivo liver perfusion - immunopathological findings. Transpl Int (Suppl 1): S 385-387,1996.

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[10] McCurry K.R., Kooyman D.L. et al.: Human complement regulatory proteins protect swine-to-primate cardiac xenografts from humoral injury. Nature Medicine 1:423427,1995. [11] Terajima H., Shirakata Y. et al.: Long duration xenogenic extracorporeal pig liver perfusion with human blood. Transpl Int (Suppl.1):S388-S391,1996. [12] Pohlein Ch., Pascher A. et al.: The function of transgenic human DAF-expressing porcine livers during hemoperfusion with human blood. Transpl Int (Suppl 1): S392396, 1996. [13] Schon MR, Lemmens HP et al.: Improved xenogenic extracorporeal liver perfusion. Transplant Proc 26(3):1293-97,1994. [14] Mirenda V., Charreau J., Sigalla J. et al.: Xenoreactivity in the pig islet to human combination: feasibility of adenovirus-mediated gene transfer into pig islets. Transplant Proc 28(2):808-810,1996 [15] Waterworth P.D., Cozzi E, Tolan M.J et al.: Pig-to-primate cardiac xenotransplantation and cyclophosphamide therapy.Transplant Proc, in press. [16] Tolan M.J., Friend P.J., Cozzi E. et al.: Life-supporting transgenic kidney transplants in a pig-to-primate model. Transplant Proc, in press. [17] Makowka L., Cramer D.V., Hoffman A. et al.: Pig liver xenografts as a temporary bridge for human allografting. Xeno, 1(2):27-29, 1993. [18] M.Ciszek,L.Paczek and W.Rowinski. Clinical outcome of living kidney donation. Transplantation Proceedings 35,1179-1181,2003. [19] C.Gracida,R.Espinoza and J.Cancino. Can a living kindey donor become a kidney recipient?. Transplant Proc 36,1630-1631,2004. [20] D’Alessandro A.M.,Pirsch J.D., Knechtle S.J. et al. Living unrelated renal donation: The University of Wisconsin experience. Surgery 124:604-11,1998. [21] P.Berloco,D.Alfani,P.Bruzzone et al. Is unrelated living donor a valid organ source in renal transplantation under CyA therapy? Transplantation Proceedings,23,912913,1991. [22] D.Alfani,R.,Pretagostini,P.Bruzzone,et al. Kidney transplantation from living unrelated donors. Clinical Transplants 1998. Eds. J. Michael Cecka and Paul I.Terasaki, UCLA Tissue Typing Laboratory,Los Angeles, 1999.Pagg. 205-212. [23] Cortesini,R.,Pretagostini,P.Bruzzone,D.Alfani. Living unrelated kidney transplantation. World Journal of Surgery 26,238-242,2002. [24] The Ethics Committee of the Transplantation Society. The Consensus Statement of the Amsterdam Forum on the Care of the Live Kidney Donor. Transplantation 78(4):491492,2004. [25] Park L,Moon JI,Kim SI,Kim YS, Exchange donor program in kidney transplantation. Transplantation 27;67(2):336-8,1999.

In: Progress in Kidney Transplantation Editor: Dominick W. Mancuso, pp. 143-162

ISBN 1-60021-312-X © 2006 Nova Science Publishers, Inc.

Chapter IX

State of the Art in Kidney Xenotransplantation Cristina Costa* and Rafael Manez Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain

Abstract Research in kidney xenotransplantation aims to solve the great shortage of kidneys for transplantation. In fact, more than half of the patients in the waiting list remain without transplant. The potential benefits of xenotransplantation raised great expectations in the early 90s, but the combination of several hurdles has precluded its clinical application. The main impediment is the strength of the immune response triggered by the xenograft. Porcine kidneys were chosen for its availability and human-like physiology as the best candidates for clinical xenotransplantation. However, transplantation of such kidneys in nonhuman primate models results in xenograft rejection in a period of weeks to months in spite of all available immunosuppression. This process is called acute humoral xenograft rejection (AHXR) because it comprises a very strong humoral immune response and thrombosis. The presence of an innate cellular component (NK cells and macrophages) has also been described. The approaches developed to date, including genetic engineering of the donor pig to express human complement regulatory proteins or removal of the Gal α1,3-Gal antigen, have averted xenograft hyperacute rejection (HAR). Moreover, the prolonged survival of kidneys from pigs deficient in the Gal α1,3-Gal antigen (up to 83 days for the thymokidney) suggests that this carbohydrate also contributes to AHXR. Nevertheless, further modifications of the donor pig are needed that target other key pathways of AHXR, such as coagulation incompatibilities, to allow progress toward the clinic. Finally, potential xenozoonotic infections and ethical factors are not to be left aside. In this regard, all studies to date suggest these issues can be addressed with careful monitoring and regulation. In summary, great advances have been made in the *

Correspondence should be addressed to Cristina Costa: Institut d’Investigació Biomèdica de Bellvitge (IDIBELL), Hospital Duran i Reynals, Gran Via km. 2,7, 08907 L’Hospitalet de Llobregat, Barcelona, SPAIN; TEL: 34932607775 ext. 3068, FAX: 34-932607426, E-mail: [email protected].

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Cristina Costa and Rafael Manez xenotransplantation field that encourage to actively continue this research. Further genetic engineering of the donor organ may lead to successful kidney xenotransplantation.

Introduction Kidney transplantation benefits many patients who suffer renal failure. Advances in immunosuppression and the use of both cadaveric and living donors allows progress in the field. However, the availability of human kidneys is limited and the demand of such transplants continues on the rise. In Spain, world leader in organ donation, more than half of the patients in the waiting list remain without transplant [1]. Aiming to solve the great shortage of solid organs for transplantation, research in xenotransplantation became the focus of attention in the early 1990s [2-4]. At this time point, the pig was chosen as the most appropriate source of xenogeneic organs and tissues after the first transgenic pigs were generated [2-4]. Prior clinical work using nonhuman-primate kidneys resulted in poor outcome (graft survivals of days to a few months) and confirmed the difficulty of obtaining organs from this source (Table 1). Moreover, the risk of transmitting infection diseases to humans in this setting was too high (think of AIDS for instance). On the contrary, the pig was domesticated, reproduced with large litters and was already in use for the food industry as well as for some medical applications (insulin, heart valves). The potential to genetically modify the donor pig together with its primate-like physiology and anatomy led to consider the possibility of “humanizing” the porcine organs to the point of successful clinical transplantation. Significant advances toward this end have been made, but progress in the field has been slow mainly due to technological difficulties. A decade later, we are still facing various barriers that have precluded its clinical application. In this work we are going to thoroughly review these hurdles and describe the approaches developed to date to overcome them. Table 1. Experience in clinical renal xenotransplantation using nonhuman primate donors Year 1910 1913 1964 1964 1964 1964 1964 1964 1965 1966

Surgeon Unger Schonstadt Reemtsma Reemtsma Hitchcock Stazl Hume Traeger Goldsmith Cortesini

Donor Species Monkey Monkey Chimpanzee Monkey Baboon Baboon Chimpanzee Chimpanzee Chimpanzee Chimpanzee

Number of cases (n) 1 1 12 1 1 6 1 3 2 1

Graft survival < 3 days ? ≤ 9 months 10 days 5 days ≤ 2 months 1 day < 49 days 4 months 1 month

Modified from Taniguchi S. and Cooper D. K. C. (1997) Clinical Xenotransplantation-A brief review of the world experience. In: D. K. C Cooper, E. Kemp, J. L. Platt and D. J. G. White (Eds): Xenotransplantation (2ond edition, pg 776-784). Heidelberg: Springer-Verlag.

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Challenges in Renal Xenotransplantation Immunological Barriers The main impediment to the clinical application of xenotransplantation is the strength of the immune response triggered by the xenograft. Transplantation of porcine kidneys in nonhuman primate models results in xenograft rejection by humoral and cellular mechanisms [4]. In solid organ xenotransplantation, three types of rejection have been described that differ in the time of onset and the immune pathways involved [2-4]. Hyperacute rejection is the fastest rejection process and takes place within minutes to hours after xenotransplantation. HAR is initiated by a humoral immune response in which xenoreactive natural antibodies (XNA) (preexisting in the host) are deposited on the donor endothelium resulting in complement activation and edema [2-4,5]. This process also triggers the coagulation cascade leading to thrombosis, ischemia and necrosis. The major xenoepitope recognized by XNA is the carbohydrate epitope Gal α1,3-Gal, which is highly expressed in pig tissues and is synthesized by the α1,3-galactosyltransferase (α1,3-GT) [6,7]. Humans and Old World primates lack a functional α1,3-GT and produce anti-Gal α1,3-Gal antibodies in high titers [6-8]. Several strategies that prevent XNA reactivity and/or complement activation have successfully overcome HAR (described bellow). Acute humoral xenograft rejection (AHXR), also named acute vascular rejection or delayed xenograft rejection, occurs in a period of days to months in spite of available immunosuppression [2-4,9,10]. It shows similarities to HAR, as it comprises a very strong humoral immune response with antibody and complement deposition, as well as thrombosis. However, there are some key differences in AHXR such as the origin of the antibodies, which in this case involves an antibody response elicited by the xenograft. High-affinity anti-Gal α1,3-Gal antibodies are found between these induced antibodies [11]. Moreover, AXHR is characterized by the presence of an innate cellular infiltrate (NK cells and macrophages) and a type II endothelial cell activation that promotes intravascular thrombosis and fibrin deposition [12,13]. Acute cellular rejection occurs within days after transplantation and is predominantly a T cell-mediated response to donor antigens. The activation of T cells during transplant rejection is mediated by a primary signal through the T cell receptor and costimulatory secondary signals that are preserved crossspecies [2,14,15]. At this moment, it is believed that the cellular response can be controlled using standard immunosuppression. However, this cannot be fully evaluated unless the principal triggers of AHXR are averted.

Tools to Combat Hyperacute Rejection Multiple strategies have been developed to overcome xenograft rejection. These can be divided in two main types, those focused on modifying the donor organ and those that use systemic treatments to alter the recipient. The most elegant and with potential for long-term clinical success are those based on genetic engineering of the donor pig because they are the less detrimental to the patient, minimizing the need for immunosuppression and conditioning therapies. However, they are technologically challenging and involve labor-intensive, slow

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and costly procedures. Systemic treatments, on the contrary, may help us to identify key molecules or pathways of the rejection process at a faster pace and accelerate the clinical use of xenotransplantation. The key role of XNA and complement in causing HAR was confirmed by the efficacy of antibody absorption by plasmapheresis and systemic complement inhibition [16-18]. The first approaches developed to counteract HAR by genetic engineering focused on inhibiting complement activation by expressing human complement regulatory proteins in transgenic pigs [19-23]. The function of complement regulatory proteins is highly restricted by the species [24]. It was therefore reasoned that the porcine molecules were ineffective to control human complement activation. Initially, in vitro studies showed that expressing human CD59 (hCD59) or human CD55 (hDAF) significantly protected pig cells from human serummediated cytolysis [25-27]. Ex vivo human blood perfusion experiments also demonstrated that transgenic kidneys and hearts from hCD59-expressing pigs functioned longer than control organs [28]. Subsequently, prolonged survival of transgenic pig organs expressing human complement inhibitors was observed in pig-to-primate transplant models [3,21,26,27,29]. However, these approaches did not accomplish complete protection from humoral xenograft rejection, as they did not address the massive XNA reactivity. In consequence, others and we pursued the generation of transgenic pigs as donors of organs with reduced human serum antibody reactivity. Before homologous recombination and knockout technology was available in the pig, one of the most actively investigated strategies was based on transgenic expression of human α1,2-fucosyltransferase (H transferase, HT) [30-36]. HT generates fucosylated residues (Hantigen, the O blood group antigen) that are universally tolerated. HT was shown to efficiently compete with α1,3-GT for the same acceptor substrate, N-acetyl lactosamine, impeding the transfer of the terminal galactose residue that gives rise to the Gal α1,3-Gal antigen [30]. The reduction in the Gal α1,3-Gal epitope in HT engineered cells resulted in decreased human antibody reactivity and serum-mediated cytolysis [30-35]. Moreover, transgenic mouse hearts expressing human HT also exhibited enhanced survival when challenged with human serum [34] or transplanted into a α1,3-GT knockout mouse model of HAR [36]. As HT or other competitive enzymes were unable to completely remove the Gal α1,3-Gal antigen, this approach was combined with expression of human complement inhibitors. In the mouse model, genetic engineering strategies that comprised a reduction of antibody reactivity and specific complement inhibition showed an additive effect in protecting cells and organs to the challenge with human serum [37,38]. The generation of double transgenic pigs that co-expressed HT and a complement inhibitor allowed the confirmation of these observations in the appropriate setting. Coexpression of HT and hCD59 in peripheral blood mononuclear cells and aortic endothelial cells from the double transgenic pigs led to the highest protection from human serum-mediated lysis when compared to controls and single transgenic cells [39]. Moreover, the double transgenic cells maintained their resistance to XNA and complement after a 24-hour treatment with porcine proinflammatory cytokines [39]. Despite these advances, these pigs were developed for cellular transplantation applications and xenotransplantation of kidneys from these doubletransgenic pigs has not been tested in nonhuman primates. The technology to produce knockout pigs was subsequently developed in order to eliminate the Gal α1,3-Gal antigen from pig tissues. It became feasible after 1996, when the

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team of Ian Wilmut succeeded to carry out somatic cell nuclear transfer in a sheep [40]. Several teams engaged then in a race to produce the first pig deficient in the α1,3-GT gene. There were concerns weather those pigs would be viable, but the first piglets null for α1,3-GT and subsequent litters were all healthy and developed well [41-44]. A recent report has shown survivals of more than 20 days for kidneys from α1,3-GT knockout pigs transplanted to baboons proving these are protected from HAR [45]. However, further work is needed to understand the effect of Gal α1,3-Gal antigen removal in the absence of other modifications, as these studies included a CVF treatment and T cell depletion [45]. Another carbohydrate antigen of interest when addressing HAR is the blood-group A. As in humans, this antigen is also present in some pigs and can be recognized by human antigroup A antibodies [46]. Human serum reactivity of the IgM isotype was detected toward this epitope in an A-positive pig kidney (in distal tubules, tubules of Henle and collecting ducts), which was extracorporeally connected to a volunteer dialysis patient [46]. However, the group A antigen does not pose any threat to xenotransplantation progress because it can be monitored for matching as in allotransplantation. Nevertheless, the most practical solution to this issue is to eliminate this antigen from the donor pig colony by selection.

Tools to Combat Acute Humoral and Cellular Xenograft Rejection The approaches described above, alone or in combination, have successfully prevented HAR. However, it has proven far more difficult to address AHXR. In this section, we are going to describe the efforts made so far to control both AHXR and cellular rejection. It is difficult to separate these two processes because they overlap in the time of progression and probably share immunological pathways. The studies conducted to date suggest there is no single pathway that triggers AHXR, but a series of them. The main goal at this point is to find the combination of modifications and/or treatments that leads to long-term xenograft survival without compromising the life of the recipient. Most of the information we can find today on preclinical kidney xenotransplantation comes from studies that utilized hDAF-transgenic pigs as donors and cynomolgus monkeys or baboons as recipients (tables 2 and 3). This genetic modification, alone or combined with hCD59, not only protects from HAR, but confers some advantage in front of AHXR [47,48]. We will therefore focus our attention on those works that involve genetically modified donors, which are summarized in tables 2 and 3. These documented primate models are all life supporting with bilateral nephrectomy of the recipient and most commonly heterotopic transplantation of a single porcine kidney. The graft renal vein and artery are thus anastomosed to the inferior vena cava and aorta and the ureter is anastomosed to the recipient bladder. The species of the primate recipient affects the outcome, as mean survival times are longer in the cynomolgus than in the baboon. Although the baboon is expected to be a closer and better model to the human patient (Table 3), the cynomolgus monkey may be very useful as well for identifying pathways involved and treatments (Table 2). Without immunosuppression, AHXR occurs in 4 to 5 days after transplantation of hDAF kidneys in baboons [49-51]. We found no reports of such experiment in cynomolgus monkeys to compare with. Addition of a basic immunosupression regime including cyclosporine A (CsA), cyclophosphamide (CyP) and corticosteroids (CS) led to a short improvement, 7.5 days mean survival in the baboon and about 12 days in cynomolgus monkeys [3,29]. Further

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prolongation required strong immunosuppression including splenectomy or addition of newer drugs such as rapamycin-based medication, methotrexate, etc [47,48,52-61]. However, survival was only prolonged for a few more days attaining survival means of 25-35 days in the cynomolgus monkey and 9-20 days in the baboon. Interesting results were also obtained when adding systemic complement inhibition using C1-inhibitor (C1-inh) or soluble complement receptor 1 (sCR1, TP10) [52,55]. Studies in the cynomolgus monkey indicate that a 3-day treatment with C1-inh can reverse a diagnosed AHXR [52,55]. However, longterm treatment with such complement inhibitors that target the C3 complement component is not advised due to the resulting susceptibility to infections [52,55]. As the anti-Gal α1,3-Gal antibodies were thought to play a role in AHXR, a series of polymers were developed containing multiple Gal α1,3-Gal epitopes. The most broadly tested was GAS914, a Gal α1,3-Gal trisaccharide with a molecular weight of 500 kDa [62]. Intravenous infusion of GAS914 successfully depleted the anti-Gal α1,3-Gal antibodies [62] and reduced the degree of kidney xenograft rejection [57]. Unexpectedly, administration of GAS914 did not improve survival [57], suggesting that anti-nonGal α1,3-Gal antibodies are also very important. Nevertheless, the generation of the α1,3-GT knockout pigs has overcome the problem of the anti-Gal α1,3-Gal antibodies [45]. The most impressive results yet have been obtained when using kidneys from pigs deficient in the Gal α1,3-Gal antigen into baboons, especially when combining those kidneys with protocols promoting tolerance through chimerism [45]. A mean survival time of 34.1 days was attained when cotransplanting the kidney with a vascularized thymic lobe after a T-cell-depletion conditioning regime and a mean survival of 44.8 days with a survivor of 83 days, a record to date, when using the thymokidney protocol. The thymokidney is formed after a donor thymus is grafted under the donor kidney capsule prior to xenotransplantation with the aim to promote tolerance to the xenograft. These results are thus not readily comparable to those previously mentioned based on pharmacological immunosuppression. On the contrary, they should be compared to previous work of the same group that reported that hDAF-expressing thymokidneys survived for 24.4 days as a mean (up to 30 days) [58]. Thus, the Gal α1,3-Gal null phenotype seemed advantageous, suggesting that this carbohydrate also contributes to AHXR. A role of the Gal α1,3-Gal antigen in amplifying the rejection process has been observed in a small animal model [63]. Nevertheless, it remains to be determined whether and how much the α1,3-GT knockout extends survival when kidneys are transplanted under standard immunosuppression. In fact, the community is now waiting for results to come from using the combination of α1,3-GT knockout and human complement inhibitors, as further protection from AHXR would be expected. One main issue that strikes out from all these studies is that treatments that fight the strong humoral response such as cyclophosphamide in high doses or the systemic complement inhibitors described, even GAS914, may actually diminish or avert AHXR in some cases [45,47,52,55,57]. However, they are too harsh and lead to an excessive immunosuppression that is accompanied by serious side effects (gastrointestinal injury, anemia, infections, etc.) that compromise the life of the recipient. The solution must come therefore from genetic engineering of the donor tissue to make it more compatible and at the same time target the pathways that trigger the uncontrolled antibody response. Lessons can be learnt from the experience in heart xenotransplantation, in particular from the grafting of α1,3-GT knockout hearts into baboons carried out by the team of David Cooper [64]. In this case, an effort has been made to diminish the immunosuppression to levels acceptable for the

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clinical practice. Although, most of the α1,3-GT null hearts were rejected under these circumstances with signs of AHXR or thrombotic microangiopathy alone, these modified hearts attained the longest survivals reported to date [64]. Most experiments point out that the thrombosis problem will probably substantially ameliorate when the antibody response is controlled. In fact, the long-term survivors show healthy kidneys in biopsy specimens collected prior to rejection or at necropsy when died due to non-rejection causes [45,47,52,56,59]. Nevertheless, further modifications of the donor pig are recommended to address the coagulation incompatibilities between pig and man. These will be discussed in the next section covering the physiological barriers. Table 2. Experience in preclinical renal xenotransplantation using genetically-modified porcine donors and cynomolgus monkey recipients First author / year

[Ref.]

Treatment

Zaidi / 1998 Cozzi / 2000

[29] [47]

Vangerow / 2001

[52]

Cozzi / 2003

[53]

Cozzi / 2003

[54]

Lam / 2005

[55]

hDAF-tg + CsA + CyP + CS hDAF-tg + CsA + CyP + CS + Spx + EPO hDAF-tg + CsA + CyP + CS hDAF-tg + CsA + CyP + CS + C1INH hDAF-tg + CsA + CyP + CS + Spx + EPO + MPS hDAF-tg + CsA + CS + Spx + MPS+ MTX hDAF-tg + CsA + CyP + CS + MPS + GAS914 hDAF-tg + CsA + CyP + CS + MPS + GAS914 + sCR1 hDAF-tg + CsA + CyP + CS + MPS + GAS914 prior Tx + sCR1

Number of cases 7 9

Mean survival (range) 13 days (6-35) 35.2 days (5-78)

4 4

11.5 days (9-15) 33.7 days (18-68)

10

24.1 days (2-51)

4

26.7 days (16-41)

4

21.5 days (6-37)

2

12.5 days (10,15)

4

24.7 days (10-37)

Notes: hDAF-tg, kidney from transgenic pig expressing hDAF; CsA, cyclosporine A; CyP, cyclophosphamide; CS, corticosteroids; Spx, splenectomy; EPO, erythropoietin; C1-INH, C1inhibitor; MPS, mycophenolate sodium; MTX, methotrexate; sCR1, soluble complement receptor 1; Tx, transplantation.

It cannot be denied that one of the main advantages of xenotransplantation is the possibility to engineer the donor organ. I suggest that, apart from targeting the coagulation incompatibilities, the next modifications should involve reducing the immunogenicity of the donor organs. The next step would be to diminish the activation of the immunological pathways engaged by the porcine cell. An example of such targets is the CD80-CD86/CD28 pathway, which is preserved in the pig-to-human xenogeneic combination. Experiments with porcine aortic endothelial cells (PAEC) show that these cells provide strong costimulation to human T cells [2,14,15] and trigger human NK cell-mediated cytotoxicity [65] through porcine CD86. Porcine CD86, as opposed to the human counterpart, is expressed in a wide variety of cells and tissues. Moreover, the CD28 pathway is resistant to immunosuppression by calcineurin inhibitors [66]. Another potential point of interest for therapeutic intervention are the powerful TNF cytokines, α and β. Incorporation of strategies that block TNF may prove useful in the development of xenografts resistant to AHXR, as the contribution of TNF

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has been shown in small-animal transplant models [67-69]. In general, such approaches would be expected to reduce the need for immunosuppression of the patient, increase the chances for tolerance induction and improve overall the quality of life. Table 3. Experience in preclinical renal xenotransplantation using genetically-modified porcine donors and baboon recipients First author / year Lawson / 1997 Diamond / 1997 Cowan / 2000

[Ref.]

Treatment

[3] [3]

Buhler / 2001

[48]

Cowan / 2002

[50]

Ghanekar / 2002

[56]

hDAF and hCD59-tg + CsA + CyP + CS hDAF and hCD59-tg + CsA + CyP + CS + MMF hDAF and hCD59 and HTlow-tg + LMWH hDAF-tg + IA + CVF + CyP + CS + MMF + PGE + ATG + aCD154 + Spx + TmI + Heparin hDAF and hCD59 and HTlow-tg + ATIII ± LMWH hDAF-tg + CsA + CyP + CS + GAS914 + RAD hDAF-tg + CsA + CS + GAS914 + RAD + RATS hDAF-tg + CsA + CS + GAS914 + RATS hDAF-tg + CsA + CyP + CS + RAD low hDAF-tg + CsA + CyP + CS + RAD low + GAS914 low hDAF-tg + CsA + CyP + CS + RAD high + GAS914 high hDAF-tg + CsA + CyP low + CS low + RAD low + GAS914 high hDAF-tg + CVF + IA + CyP ± CS + MMF + PGE + Tmx or ATG + Spx + aCD2 + aCD154 + TmKid hDAF-tg + CsA + CyP + CS + MMF+ IA hDAF-tg + CsA + CyP high + CS + MMF hDAF-tg + CsA + CyP high + CS + MMF + IA hDAF-tg + CsA + CyP + CS + MMF

Zhong / 2003

[49]

[57]

Barth / 2003

[58]

Ashton-Chess/ 2003

[59]

Ashton-Chess/ 2004

González / 2004

[60]

[61]

hDAF-tg + CsA + CyP high + CS + MMF + Mx hDAF-tg + CsA + CyP + CS + MPS + GAS914 hDAF-tg + CsA + CyP + CS + GAS914 + FTY720 hDAF-tg + CsA + CS + GAS914 + FTY720 + Bas

Number of cases 6 7

Mean survival (range) 7.5 days (<10) 7.6 days (<15)

6

4 days (3-5)

3

28.7 days (28-29)

4

5 days (4-6)

2

28 days (20-36)

4

23 days (20-26)

3

20 days (18-22)

4 4

18.5 days (4-40) 19.2 days (10-37)

4

14.5 days (9-25)

4

18.7 days (7-36)

5

24.4 days (18-30)

4

6 days (5-9)

4

9.2 days (7-12)

4

7 days (5-10)

2

9 days (9)

4

8 days (6-10)

10

7 days (1-31)

3

8 days (4-28)

3

8 days (3-13)

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Table 3 Continued First author / year González / 2004 (Cont’d) Ménoret / 2004 Yamada / 2005

[Ref.]

[51] [45]

First author / year Lawson / 1997

[3]

Diamond / 1997

[3]

Cowan / 2000

[49]

Buhler / 2001

[48]

Cowan / 2002

[50]

Ghanekar / 2002

[56]

Zhong / 2003

[Ref.]

[57]

Treatment hDAF-tg + CS + GAS914 + FTY720 + Bas + RAD hDAF and hCD59-tg Gal KO + CVF + MMF + CS + ATG + aCD2 + aCD154 + Spx + Tmx ± WBI Gal KO ± CVF + MMF + CS + ATG + aCD154 + Spx + Tmx ± WBI + VTL Gal KO ± CVF + MMF + CS + ATG + aCD154 + Spx + Tmx ± WBI + TmKid Treatment hDAF and hCD59-tg + CsA + CyP + CS hDAF and hCD59-tg + CsA + CyP + CS + MMF hDAF and hCD59 and HTlow-tg + LMWH hDAF-tg + IA + CVF + CyP + CS + MMF + PGE + ATG + aCD154 + Spx + TmI + Heparin hDAF and hCD59 and HTlow-tg + ATIII ± LMWH hDAF-tg + CsA + CyP + CS + GAS914 + RAD hDAF-tg + CsA + CS + GAS914 + RAD + RATS hDAF-tg + CsA + CS + GAS914 + RATS hDAF-tg + CsA + CyP + CS + RAD low hDAF-tg + CsA + CyP + CS + RAD low + GAS914 low hDAF-tg + CsA + CyP + CS + RAD high + GAS914 high hDAF-tg + CsA + CyP low + CS low + RAD low + GAS914 high

Number of cases 4

Mean survival (range) 9 days (1-20)

2 3

5.5 days (5-6) 29 days (20-34)

6

34.1 days (4-68)

5

44.8 days (16-83)

Number of cases 6

Mean survival (range) 7.5 days (<10)

7

7.6 days (<15)

6

4 days (3-5)

3

28.7 days (28-29)

4

5 days (4-6)

2

28 days (20-36)

4

23 days (20-26)

3

20 days (18-22)

4

18.5 days (4-40)

4

19.2 days (10-37)

4

14.5 days (9-25)

4

18.7 days (7-36)

hDAF-tg, kidney from transgenic pig expressing hDAF with or without other indicated transgenes; CsA, cyclosporine A; CyP, cyclophosphamide; CS, corticosteroids; MMF, mycophenolate mofetil; MPS, mycophenolate sodium; CVF, cobra venom factor; PGE, prostacyclin PGE2; ATG, antithymocyte globulin; aCD154, anti-CD154 mAb; Spx, splenectomy; TmI, thymus irradiation; LMWH, low molecular weight heparin; ATIII, antithrombin III; RAD, a rapamycin derivative; RATS, rabbit anti-thymocyte serum; IA, immunoglobulin adsorptions; Mx, mitoxantrone; Bas, basiliximab or anti-IL2R mAb; Gal KO, α1,3-GT knockout; Tmx, thymectomy; WBI, whole body irradiation; VTL, vascularized thymic lobes; TmKd, thymokidney.

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Non Immunological Barriers Xenograft Physiology It is well established that xenogeneic proteins such as porcine insulin can work properly in humans. However, it is unclear whether and for how long xenografts will perform their functions in a different environment for which they have not been genetically programmed. Pig kidneys have sustained the life of nonhuman primates for several months [45,47,52]. Xenograft function has failed apparently as a result of rejection and not because of existing physiological incompatibilities between the species. Recipients of kidneys from the α1,3-GT knockout pigs required continuous treatment with human albumin to maintain proteins in the normal range [45]. This was due to the proteinuria that emerged after transplantation and persisted during the three-month xenograft survival. Clinical symptoms (edema) caused by proteinuria were different according to the therapeutic protocol, suggesting that proteinuria resulted from the rejection process and not from a physiological mutual incompatibility. Moreover, nonhuman primates transplanted with α1,3-GT null kidneys did not show the anemia previously described with cyclophosphamide immunosuppression, confirming that this was a consequence of treatment toxicity instead of an incapacity of pig erythropoietin to sustain erythropoiesis in the recipient [70]. Porcine hearts and kidneys have thus confirmed their human-like physiology and are candidates for the first solid-organ clinical xenotransplantation. On the contrary, other organs such as pulmonary and hepatic xenografts have not survived beyond a few days (they have demonstrated their capacity to support the recipient’s live during this short time, though). Some physiological differences between donor and recipient regarding these organs are expected, especially in the case of the liver with its complex metabolic system. However, even for the heart and kidney, some minor incompatibilities cannot be ruled out in the long term. An example could be the clotting abnormalities described in AHXR. For the moment, it is unclear whether they are reliant or independent of the deposition of xenoantibodies in the xenograft. Porcine cells in vitro have an inherent tendency to spontaneously clot human plasma [71], an effect that appears dependent on certain molecular incompatibilities between human and porcine regulators of coagulation [72]. In particular, porcine tissue factor pathway inhibitor (TFPI) does not effectively block human factor Xa and porcine thrombomodulin (a key anticoagulant expressed by endothelial cells) hardly activates human protein C relative to its human counterpart [72]. Thus, if clotting abnormalities occur independently of antibody, it could explain why AHXR has been so difficult to prevent or treat in pig-to-nonhuman primate xenotransplantation. Nevertheless, the accumulated experience suggests that physiology should not be an unsolvable obstacle for the clinical application of pig organ xenotransplantation. Several approaches, including genetic engineering of the porcine organs, are currently being developed to address the clotting incompatibilities [73]. Other issues that may arise after solving AHXR could be treated as well by incorporating the appropriate human gene/protein in the donor pig.

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Risk for Transmission of Infections from the Xenograft The success of organ allotransplantation requires a balance between the immunosupressive treatment necessary to prevent graft rejection and the risk of opportunistic infections or cancer that can appear as a result of this treatment. Experimental data generated so far in xenotransplantation indicate that the immunosuppression required to prevent AXHR must be initially more potent than that employed in allotransplantion. Therefore, theoretically, the risk of opportunistic infections is higher in xenotransplantation than in allotransplantation. In addition, xenotransplantation of cells, tissues or organs of nonhuman origin will increase the range of opportunistic infections, since it will also have to include those diseases that affect the animal species used as a graft source. However, it will probably reduce the impact of infections specific for human organs as porcine cells are resistant to many human pathogens. Nevertheless, the possibility that by means of a xenograft a new pathogenic agent is introduced in the recipient, and from this extend to the general population (as it happened with AIDS), concerns scientists and those responsible for the public health. The terms "xenosis" and "xenozoonosis" have been proposed to describe those infections produced by infectious agents from other animal species which do not cause infection in humans under natural conditions, but may be transmitted from a xenograft [74,75]. The probability that certain microorganisms from other animal species develop a disease in humans is unknown. Theoretically, it could be high in the case of microorganisms zoonotic in normal conditions (e.g. Toxoplasma gondii), also for microorganisms similar to those causing infections in allotrasplantation (e.g. cytomegalovirus CMV), microorganisms with capacity to infect a broad range of species (e.g. Pneumocystis carinii), and those microorganisms capable of establishing replicative infection in human cells in vitro. Nevertheless, it is unlikely that bacterial, fungal and parasitic xenosis or xenozoonosis, caused either by common as by species-specific pathogens, will imply a special risk for a xenograft recipient, and less for the public health. The reason is that this type of infections can be prevented in the animal source of organs. Production of animals in closed and isolated areas, screened for likely pathogens will minimize the risk of graft-derived infections with known pathogens. Rigorous protocols of clinical and microbiological evaluation using both immunocompetent and immunosuppressed animals can detect animals with infection signs, those carrying latent microorganisms similar to those causing infection in allotransplantation or those carrying pathogens for different species. Removal of these animals would lead to the development of “designated pathogen free” (DPF) pig herds liberated of several microorganisms [76]. In addition to these causes of exclusion for any animal, specific criteria can be included according to the organ that will be transplanted. Thus, the presence of Mycoplasma sp would discard animals as possible source for lung xenografts, or the Coxsackie virus for cardiac xenografts. The risk of transmission of infection could still be further reduced with the use of pigs totally free of germs (gnotobiotic). Although at the moment there are no facilities that allow the production of mammals in this state, its construction is totally feasible, being the high cost the most important disadvantage. Gnotobiotic animals are less robust than those with normal microbiological colonization and, at this moment, they do not offer any advantage to DPF animals to diminish the risk of xenosis or xenozoonosis transmission. Therefore, the production of gnotobiotic pigs has been relegated until the clinical experimentation demonstrates its necessity.

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All animal species have viruses that persist within the cells in a latent state. These include the herpesvirus and retrovirus, but also hepatitis virus, adenovirus, rabies and pseudorabies virus, reovirus, papovavirus and others. At the moment, it is not known whether nonhuman latent viruses represent a risk of infection and disease for humans. Nevertheless, it is hard to believe that an animal such as the pig, that has been in contact with humans for thousands of years, can transmit an infection to immunocompetent individuals that has not become apparent yet. The infection and disease of pig abattoir workers from Asia caused by Nipah virus, a paramyxovirus that infects pigs [77], or the spread of the severe acute respiratory syndrome (SARS) caused by a coronavirus after consumption of infected exotic animals in China, are good examples of how easy is the infection and propagation of those zoonosis. A special remark deserves the potential reactivation of latent herpesvirus infection after pig organ xenotransplantation. Three herpesviruses have been identified in swine: porcine CMV (PCMV), and porcine lymphotropic virus-1 and -2 (PLHV-1, -2), which are associated with a syndrome of lymphoide proliferation in pigs undergoing bone marrow transplantation similar to the post-transplantation lymphoproliferative disease (PTLD) described in allotransplantation [78]. Herpesviruses are species-specific and therefore, if infection occurs after xenotransplantation, this should be limited to the xenograf. Replication of PCMV has been demonstrated in pig xenografts transplanted in nonhuman primates, causing tissueinvasive infection and contributing to endothelial cell injury and consumptive coagulopathy [79]. Exclusion of PCMV from swine herds has been possible by early weaning of newborns. The lack of PCMV was associated with a reduction in coagulopathy and improved xenograft survival in pig-to-nonhuman primate xenotransplantation. In contrast, no activation of PLHV1 has been demonstrated to date in solid organ xenotransplantation [80]. However, unlike PCMV, this virus cannot be removed from the donor animals by early weaning of newborns [81], remaining a potential pathogen in pig organ xenotransplantation. DPF pigs lack all the exogenous microorganisms that these animals can acquire, limiting their infections to viruses that are vertically transmitted or latent viruses that cannot be removed from the animal source by early weaning of newborns or by harvest prior to birth. Those transmitted vertically from the sow to the piglets include endogenous retroviruses, which are viruses that have been integrated permanently in the genome of the host during the evolution of mammals. Although they are not pathogenic in the host, these retroviruses can be xenotropic or able to infect other species. Two porcine endogenous retroviruses (PERV) have been described with capacity to infect human cells in vitro [82], which lead to consider the possibility of recombination or complementation of xenograft endogenous retroviruses with viruses present in human tissues. Search for crossspecies transmission of PERV in patients treated with living pig tissues [83], in abattoir pig workers and transplant recipients in contact with pigs [84], and in nonhuman primates transplanted with pig organs and treated with an intense immunosupression [85], failed to demonstrate PERV replication in both humans and nonhuman primates. On the other hand, it has been identified an inbred miniature swine that failed to produce replication of PERV in human cells [86], indicating that this infection could also be prevented by selecting the animals used as source of organs. In summary, with the information available to date, the risk for xenozoonosis should be no reason to give up on pig organ xenotransplantation. In contrast to the apparent situation found a few years ago, the risks have now been identified, investigated and tests have been developed that allow to conduct controls in preclinical and clinical studies. Although we

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cannot completely rule out the possibility that infection emerging from the xenograft could affect the public health, the risk appears negligible and preventable by means of an intensive monitoring of xenograft donors and recipients.

Conclusion Kidney xenotransplantation has great potential to solve the pressing shortage of human organs for transplantation and represents a future alternative to allotransplantation. Advances in the field have been slow at some point due to technological problems such as the impossibility of knocking out genes in pigs and the difficulty in assessing the xenozoonotic risks. However, now the tools are in place and can be further developed. The major barrier that remains to be addressed is immune rejection, especially the humoral response triggered by the xenograft. The identification of the key pathways and molecules involved in this rejection process and the development of strategies to overcome them is just a matter of time. The use of organs deficient in the Gal α1,3-Gal antigen has already shown some improvement relative to prior studies in nonhuman primate models. Further genetic engineering of the donor organ may lead to successful kidney xenotransplantation.

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Cristina Costa and Rafael Manez receptor type 1 on acute humoral xenograft rejection in hDAF-transgenic pig-to-primate life-supporting kidney xenografts. Xenotransplantation 12: 20-9. Ghanekar, A.; Lajoie, G.; Luo, Y.; Yang, H.; Choi, J.; Garcia, B.; Cole, E. H.; Greig, P. D.; Cattral, M. S.; Phillips, M. J.; Cardella, C. J.; Levy, G. A.; Zhong, R. and Grant, D. R. (2002) Improvement in rejection of human decay accelerating factor transgenic pigto-primate renal xenografts with administration of rabbit antithymocyte serum. Transplantation 74: 28-35. Zhong, R.; Luo, Y.; Yang, H.; Garcia, B.; Ghanekar, A.; Luke, P.; Chakrabarti, S.; Lajoie, G.; Phillips, M. J.; Katopodis, A. G.; Duthaler, R. O.; Cattral, M.; Wall, W.; Jevnikar, A.; Bailey, M.; Levy, G. A. and Grant, D. R. (2003) Improvement in human decay accelerating factor transgenic porcine kidney xenograft rejection with intravenous administration of GAS914, a polymeric form of alphaGAL. Transplantation 75: 10-9. Barth, R. N.; Yamamoto, S.; LaMattina, J. C.; Kumagai, N.; Kitamura, H.; Vagefi, P. A.; Awwad, M.; Colvin, R. B.; Cooper, D. K.; Sykes, M.; Sachs, D. H. and Yamada, K. (2003) Xenogeneic thymokidney and thymic tissue transplantation in a pig-to-baboon model: I. Evidence for pig-specific T-cell unresponsiveness. Transplantation 75: 161524. Ashton-Chess, J.; Roussel, J. C.; Bernard, P.; Barreau, N.; Karam, G.; Dantal, J.; Moreau, A.; Letessier, E.; Nagasaka, T.; Emanuele, C.; Minault, D.; Soulillou, J. P. and Blancho, G. (2003) The effect of immunoglobulin immunadsorptions on delayed xenograft rejection of human CD55 transgenic pig kidneys in baboons. Xenotransplantation 10: 552-561. Ashton-Chess, J.; Meurette, G.; Karam, G.; Petzold, T.; Minault, D.; Naulet, J.; Tesson, L.; Plat, M.; Anegon, I.; Soulillou, J. P. and Blancho, G. (2004) The study of mitoxantrone as a potential immunosuppressor in transgenic pig renal xenotransplantation in baboons: comparison with cyclophosphamide. Xenotransplantation 11: 112-22. Gonzalez Martin, M.; Garcia Buitron, J.; Alonso Hernandez, A.; Centeno Cortes, A.; Lopez Pelaez, E.; Vazquez Martul, E.; Mosquera Reboredo, J.; Requejo Isidro, I. and Manez Mendiluce, R. (2004) [Renal xenotransplantation from hDAF pig to baboon. Experience and review] Actas Urol. Esp. 28: 161-74. Katopodis, A. G., Warner, R. G., Duthaler, R. O.; Streiff, M. B.; Bruelisauer, A.; Kretz, O.; Dorobek, B.; Persohn, E.; Andres, H.; Schweitzer, A.; Thoma, G.; Kinzy, W.; Quesniaux, V. F.; Cozzi, E.; Davies, H. F. Manez, R. and White, D. (2002) Removal of anti-Galalpha1,3Gal xenoantibodies with an injectable polymer. J. Clin. Invest. 110: 1869-77. Costa, C.; Brokaw, J. L.; Wang, Y. and Fodor, W. L. (2003) Delayed rejection of porcine cartilage is averted by transgenic expression of alpha1,2-fucosyltransferase. FASEB J. 17: 109-11. Kuwaki, K.; Tseng, Y. L.; Dor, F. J.; Shimizu, A.; Houser, S. L.; Sanderson, T. M.; Lancos, C. J.; Prabharasuth, D. D.; Cheng, J.; Moran, K.; Hisashi, Y.; Mueller, N.; Yamada, K.; Greenstein, J. L.; Hawley, R. J. Patience, C.; Awwad, M.; Fishman, J. A.; Robson, S. C.; Schuurman, H. J.; Sachs, D. H. and Cooper, D. K. (2005) Heart transplantation in baboons using alpha1,3-galactosyltransferase gene-knockout pigs as donors: initial experience. Nat Med. 11: 29-31.

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[65] Costa, C.; Barber, D. F. and Fodor, W. L. (2002) Human NK cell-mediated cytotoxicity triggered by CD86 and Galα1,3-Gal is inhibited in genetically modified porcine cells. J Immunol. 168: 3808-3816. [66] June, C. H.; Ledbetter, J. A.; Gillespie, M. M.; Lindsten, T. and Thompson, C. B. (1987) T cell proliferation involving the CD28 pathway is associated with cyclosporine resistant interleukin-2 gene expression. Mol. Cell. Biol. 7: 4472-4481. [67] Lin, Y.; Vandeputte, M. and Waer, M. (1997) Contribution of activated macrophages to the process of delayed xenograft rejection. Transplantation 64: 1677-1683. [68] Lin, H.; Gastman, B. R.; Wei, R. Q.; Kunkel, S. L.; Gordon, D. and Bolling, S. F. (1997) Phase-directed therapy and cardiac xenograft survival. J. Surg. Res. 72: 84-88. [69] Costa, C.; Bell, N. K.; Stabel, T. J. and Fodor, W.L. (2004) Use of porcine tumor necrosis factor receptor 1-Ig fusion protein to prolong xenograft survival. Xenotransplantation 11: 491-502. [70] Lam, T. T.; Hausen, B.; Squiers, E.; Cozzi, E. and Morris, R. E. (2002) Cyclophosphamide-induced postoperative anemia in cynomolgus monkey recipients of hDAF-transgenic pig organ xenografts. Transplant Proc. 34: 1451-1452. [71] Siegel, J. B.; Grey, S. T.; Lesnikoski, B. A.; Kopp, C. W.; Soares, M.; Schulte am Esch, J. 2nd.; Bach, F. H. and Robson, S. C. (1997) Xenogeneic endothelial cells activate human prothrombrin. Transplantation 64: 888-896. [72] Robson, S. C.; Cooper, D. K. and d’Apice, A. J. (2000) Disordered regulation of coagulation and platelet activation in xenotransplantation. Xenotransplantation 7: 166176. [73] Chen, D.; Weber, M.; McVey, J. H.; Kemball-Cook, G.; Tuddenham, E. G.; Lechler, R. I. and Dorling, A. (2004) Complete inhibition of acute humoral rejection using regulated expression of membrane-tethered anticoagulants on xenograft endothelium. Am. J. Transplant. 4: 1958-1963. [74] Fishman, J. A. (1994) Minituare swine as organ donors for man: Strategies for prevention of xenotransplant-associated infections. Xenotransplantation 1: 47-57. [75] Chapman, L. E. ; Folks, T. M.; Salomon, D. R.; Patterson, A.; Eggerman, T. E. and Noguchi, P. D. (1995) Xenotransplantation and xenogeneic infections. N. Engl. J. Med. 333: 1498-1501. [76] Fishman, J. A. (1995) Preventing infections in xenotransplantation: xenosis from minuature swine. Xeno 3: 72-77. [77] Chang, K. P.; Rollin, P. E.; Ksiazek, T. G.; Leo, Y. S.; Goh, K. T.; Paton, N. I.; Sng, E. H. and Ling, A. E. (2002) A Survey of Nipah virus infection among various risk groups in Singapore. Epidemiol. Infect. 128: 93-98. [78] Huang, C. A.; Fuchimoto, Y.; Gleit, Z.; Ericsson, T.; Griesemer, A.; Scheier-Dolberg, R.; Melendy, E.; Kitamura, H.; Fishman, J. A.; Ferry, J. A.; Harris, N. L.; Patience, C. and Sachs, D. H. (2001) Posttransplantation lymphoproliferative disease in miniature swine after allogeneic hematopoietic cell transplantation: similarity to human PTLD and association with porcine gammaherpesvirus. Blood 97: 1467-1473. [79] Gollackner, B.; Mueller, N. J.; Houser, S.; Qawi, I.; Soizic, D.; Knosalla, C.; Buhler, L.; Dor, F. J.; Awwad, M.; Sachs, D. H.; Cooper, D. K.; Robson, S. C. and Fishman, J. A. (2003) Porcine cytomegalovirus and coagulopathy in pig-to-primate xenotransplantation. Transplantation 75: 1841-1847.

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[80] Mueller, N. J.; Livingston, C.; Knosalla, C.; Barth, R. N.; Yamamoto, S.; Gollackner, B.; Dor, F. J.; Buhler, L.; Sachs, D. H.; Yamada, K.; Cooper, D. K. and Fishman, J. A. (2004) Activation of porcine cytomegalovirus, but not porcine lymphotropic herpesvirus, in pig-to-baboon xenotransplantation. J. Infect. Dis. 189: 1628-1633. [81] Mueller, N. J.; Kuwaki, K.; Knosalla, C.; Dor, F. J.; Gollackner, B.; Wilkinson, R. A.; Arn, S.; Sachs, D. H.; Cooper, D. K. and Fishman, J. A. (2005) Early weaning of piglets fails to exclude porcine lymphotropic herpesvirus. Xenotransplantation 12: 59-62. [82] Patience, C.; Takeuchi, Y. and Weiss, R. A. (1997) Infection of human cells by an endogenous retrovirus of pigs. Nature Med. 3: 282-286. [83] Paradis, K.; Langford, G.; Long, Z.; Heneine, W.; Sandstrom, P.; Switzer, W. M.; Chapman, L. E.; Lockey, C.; Onions, D. and Otto, E. (1999) Search for cross-species transmission of porcine endogenous retrovirus in patients treated with living pig tissue. Science 285: 1236-1241. [84] Hermida, M.; Domenech, N.; Moscoso, I.; Diaz, T.; Ishii, I.; Salomon, D. and Mañez, R. (2003) Lack of porcine endogenous retrovirus (PERV) transmission to pig abattoir workers. Xenotransplantation 5: 521. [85] Moscoso, I.; Hermida, M.; Máñez, M.; López-Peláez, E.; Centeno, A.; Díaz, T. M. and Doménech, N. (2005) Lack of cross-species transmission of porcine endogenous retrovirus (PERV) in pig-to-baboon xenotransplantation with sustained depletion of anti-α Gal antibodies. Transplantation 79: 777-782. [86] Oldmixon, B. A.; Wood, J. C.; Ericsson, T. A.; Wilson, C. A.; White-Scharf, M. E.; Andersson, G.; Greenstein, J. L.; Schuurman, H. J.; Patience, C. (2002) Porcine endogenous retrovirus transmission characteristics of an inbred herd of miniature swine. J. Virol. 76: 3045-3048.

Index A acceptance, 107, 116, 118 access, 39, 47 accumulation, vii, 1, 2, 3, 4, 6, 12, 18, 38, 39, 40, 46, 65, 72, 82, 86 accuracy, 27, 28, 29, 30, 33, 35 acid, 38, 39, 42, 46, 48, 52, 60, 65, 66, 73, 78, 79, 87, 94 acidosis, 5 ACL, 120 acquisitions, 117 activation, x, 16, 39, 40, 47, 49, 52, 65, 94, 100, 101, 102, 103, 104, 138, 141, 145, 146, 149, 154, 155, 156, 157, 161 active transport, 64 activity level, 87 acute leukemia, 81 acute lymphoblastic leukemia, 65, 80, 81 adaptation, 106, 107, 108, 109, 111, 115 adenosine, 57, 62 adenosine triphosphate, 62 adenovirus, 142, 154 adhesion, 40, 46, 47, 52, 90, 94, 100, 115 adipocyte, 94 adjustment, 12, 115, 130 adolescence, 3 adolescents, 42, 50 adults, 34, 38, 44, 50, 58, 61 adverse event, 48 aerobic exercise, 42 affect, viii, ix, 32, 45, 47, 48, 53, 58, 65, 89, 99, 105, 130, 153, 155 African Americans, 68, 71, 87 age, ix, 5, 7, 14, 25, 26, 30, 31, 35, 43, 44, 76, 105, 109, 111, 112, 128, 139, 140 agent, 33, 153

AIDS, 144, 153 albumin, 39, 152 alienation, 117 allele, 58, 59, 60, 61, 62, 64, 65, 66, 67, 68, 69, 70, 75, 81, 83, 85 allograft survival, 33, 45, 48 alternative, x, 9, 10, 24, 25, 32, 33, 42, 106, 115, 125, 126, 129, 132, 133, 138, 140, 155 alternatives, viii, 23, 118, 119, 130 altruism, 115 ambivalent, 116 American Heart Association, 40, 42, 50 amino acids, 63, 64 analgesic, 3 anatomy, 144 anemia, 38, 49, 148, 152, 161 aneurysm, 4 anhydrase, 95 animals, ix, 71, 73, 89, 91, 99, 153, 154 ANOVA, 43 antibody, 82, 132, 145, 146, 148, 152, 155, 156, 157 anticoagulant, 4, 9, 152 antigen, xi, 47, 53, 96, 103, 143, 146, 147, 148, 155, 159 antihypertensive drugs, 3, 7 antioxidant, 41 anxiety, 109 aorta, 147 APC, 4, 9 apoptosis, 47 arrest, 11, 95 artery, 9, 45, 147 Asia, 19, 154 assessment, 25, 32, 33, 44, 77, 130 association, 44, 45, 46, 56, 58, 64, 67, 68, 69, 70, 71, 72, 73, 74, 81, 85, 86, 161 assumptions, 127 asymptomatic, 2, 10

164

Index

atherogenesis, 50 atherosclerosis, viii, 37, 44, 47, 48, 49, 50, 53, 77 atherosclerotic plaque, 41 ATP, 57, 62, 63, 64, 82, 83 atrial fibrillation, 12 atrophy, 6, 7 attachment, 46 attacks, 4 attention, viii, 9, 23, 115, 116, 124, 140, 144, 147 attitudes, 106 attractiveness, 133 autoantibodies, 6, 40 autonomy, 106, 108 availability, x, 4, 72, 129, 138, 139, 143, 144 avoidance, 52 Azathioprine, 65, 66, 84

B barriers, 144, 149 BBB, 63 behavior, 103, 106, 111 beneficial effect, viii, 30, 37, 47 bias, 27, 35, 92 bile, 38, 39, 64, 75 bilirubin, 79 binding, 40, 47, 57, 62, 63, 82, 83, 93, 94, 95, 96, 97, 101, 157 bioavailability, 63, 67, 68, 71, 74, 82, 86 biomarkers, 49 biopsy, 5, 6, 10, 14, 19, 20, 45, 64, 68, 149 biosynthesis, 42 bladder, 24, 64 blood, 4, 9, 33, 35, 39, 40, 45, 50, 56, 57, 63, 66, 67, 68, 69, 70, 72, 74, 81, 82, 85, 91, 138, 140, 141, 142, 146, 147, 159 blood flow, 4 blood group, 141, 146, 159 blood pressure, 45 blood stream, 39, 40 blood transfusions, 140 blood-brain barrier, 57 BMI, 42 body, 26, 32, 35, 38, 40, 72, 108, 113, 114, 115, 116, 117, 118, 151 body mass index, 35 body weight, 32 bonds, 116 bone marrow, 73, 154 brain, vii, viii, 1, 9, 52, 60, 63, 82, 83, 84, 89, 90, 91, 94, 96, 97, 99, 100, 101, 102, 103, 104 Brazil, 105, 129 break-even, 129

bronchitis, 39

C cadaver, x, 138, 139 calcification, 45 calcium, 94 calibration, 32, 35 Canada, 129 cancer, 52, 62, 104, 153 candidates, x, 24, 25, 76, 107, 121, 143, 152 capillary, 63 capsule, 148 carbohydrate, xi, 143, 145, 147, 148, 157, 158 carbon, 45 cardiac involvement, 3, 4, 11, 12, 20 cardiomyopathy, 3, 4, 8, 13, 14, 15 cardiovascular disease, viii, 41, 47, 48, 55, 56 cardiovascular morbidity, viii, 37, 48 carrier, 10, 16, 39 cartilage, 160 catabolism, 44 catalytic activity, 62, 78, 81 cataract, 4 catheter, 91 Caucasian population, 61, 62, 64 Caucasians, 59, 61, 66, 70, 71, 75 C-C, 94 cDNA, 60, 73, 79, 80, 82, 92, 155 cell, x, 7, 10, 11, 19, 39, 40, 46, 47, 53, 63, 81, 82, 83, 86, 90, 94, 95, 101, 132, 138, 145, 148, 149, 154, 157, 158, 160, 161 cell line, 63, 83, 86, 158 cell membranes, x, 39, 138 cell signaling, 47 cell surface, 40, 82, 158 central nervous system, 2, 3 cerebral blood flow, 4 cerebrovascular disease, 4 chemical properties, 39 chemokines, 100 chemotherapy, 62 childcare, 125 childhood, 3, 5, 80, 84 children, x, 38, 41, 42, 43, 44, 48, 51, 61, 65, 81, 137 chimpanzee, 138 China, 154 cholesterol, 39, 41, 44, 47, 50, 51, 52, 53 chromosome, 2, 6, 58, 59, 60, 64, 79 chronic illness, 117 chronic renal failure, vii, 1, 26, 51, 107, 109, 115, 117 cimetidine, 33, 35

Index circulation, 64, 73 classes, 48, 93 classification, ix, 29, 34, 40, 90, 123, 124, 125 clinical presentation, 78 clinical trials, vii, viii, 1, 11, 23, 24, 25, 27, 28, 32, 33, 35, 45, 139 cloning, 60, 79, 80, 82, 158 clusters, ix, 89, 93, 94 CMC, 89 CNS, 18, 82 coagulation, xi, 94, 99, 143, 145, 149, 152, 161 coagulopathy, 154, 159, 161 coding, 58 codon, 60 coercion, 141 cohort, 2, 4, 5, 7, 8, 9, 15, 16, 26, 27, 28, 29, 30, 31, 32, 43, 67 collagen, 40 colon, 59, 63, 73, 78, 79 colonization, 153 common presenting symptoms, 2 communication, 75 community, 56, 148 compatibility, 140 compensation, 119 complement, x, xi, 6, 39, 47, 112, 138, 141, 142, 143, 145, 146, 148, 149, 155, 156, 157, 159 complementary DNA, 81 complexity, vii, 96, 99, 106, 108 compliance, 42, 91, 121 complications, vii, 1, 4, 8, 9, 56, 77, 131, 132, 140 components, 100, 106, 119 composition, 39 compounds, 45, 57, 63, 64 concentration, 2, 9, 25, 26, 32, 33, 34, 51, 52, 56, 63, 66, 67, 68, 69, 70, 72, 73, 74, 75, 78, 84, 92 conception, 112, 113 concrete, 117 conditioning, 140, 145, 148 conduct, 154 conduction, 5 confusion, 56, 110 conjugation, 57, 87 connective tissue, vii, 1, 2 consanguinity, 116 consensus, 58, 109 consent, 76, 140 construction, 113, 119, 126, 153 consumption, 154 contamination, 39, 91 context, ix, 28, 33, 89, 105, 106, 107, 110 control, 5, 9, 13, 43, 44, 45, 63, 91, 93, 99, 131, 132, 146, 147

165

control group, 44, 45, 131 controlled trials, 48 conversion, 42, 59 cornea, 2, 4 corneal opacities, 2, 4 coronary arteries, 41 coronary heart disease, 51 coronavirus, 154 correlation, 2, 7, 18, 26, 27, 63, 68, 82, 83, 84, 130 correlation analysis, 82 correlation coefficient, 26, 27 cortex, 63 corticosteroids, 85, 86, 147 cost benefit analysis, 127 cost effectiveness, ix, 123, 124, 126, 129, 133 cost saving, 128, 130, 132 costs, 109, 124, 125, 126, 127, 128, 129, 131, 132, 133 couples, 141 coverage, 124, 133, 135 covering, 4, 149 creatine, 48 creatinine, 11, 12, 25, 26, 32, 33, 34, 35, 111 crescentic glomerulonephritis, 6 CRP, 39, 41, 47 cultural values, 108 culture, ix, 105, 107, 110, 115 CXC, 104 cyclophosphamide, 142, 147, 148, 149, 151, 152, 160 cyclosporine, 48, 50, 51, 53, 56, 69, 70, 72, 73, 75, 77, 84, 85, 86, 131, 132, 133, 147, 149, 151, 161 cytochrome, 48, 57, 58, 77, 78, 84, 85 cytochrome p450, 84 cytokines, 39, 90, 99, 100, 146 cytomegalovirus, 153, 161, 162 cytoplasm, 100, 101 cytotoxicity, 46, 52, 149, 157, 161

D daily living, 126 damage, 7, 9, 10, 12, 15, 29, 39, 41, 90, 94, 95, 113, 118, 157 data collection, 111 database, 93 death, viii, 8, 9, 38, 41, 47, 89, 90, 91, 96, 97, 99, 100, 101, 102, 103, 104, 113, 116, 130, 140 debt, 113, 115 decay, 141, 156, 157, 160 decision making, 124 defects, 40 defense, 82, 94

166

Index

deficiency, 15, 16, 19, 60, 61, 65, 80, 81, 113 definition, 49, 56, 110, 112, 116 degradation, 60, 81, 101 delivery, 41 demand, 107, 118, 144 demographic characteristics, 43 demographics, 32 density, 40, 49, 50, 51, 76 deposition, 3, 4, 5, 9, 10, 12, 40, 145, 152 deposits, 6, 7, 9, 10, 11, 71 depression, 109 derivatives, 53 detection, 5, 6, 10, 16, 48, 63 deviation, 27, 116 diabetes, 35, 117, 130 diabetic nephropathy, 7 dialysis, vii, 1, 7, 9, 14, 15, 16, 19, 32, 38, 39, 44, 108, 109, 111, 112, 117, 118, 120, 124, 127, 128, 129, 138, 147 diaphragm, 6 diet, 26, 34, 42, 108, 112, 117 differentiation, 46, 47, 95 direct costs, 125 direct measure, 26 disability, 114, 118, 125 discomfort, 110 discounting, 127, 129 discourse, 114 disequilibrium, 62 disintegrin, 95 disorder, vii, 1, 2 dispersion, 27 disposition, 56, 57, 64, 75, 76, 77, 83, 85, 86 disseminated intravascular coagulation, 159 distribution, 11, 57, 60, 63, 81, 110 diversity, 2, 78, 108 DNA, viii, 66, 76, 77, 89, 90, 91, 92, 95, 99 doctors, 108 domain, 63, 95, 96, 110, 112 donors, viii, ix, x, 10, 55, 56, 72, 75, 89, 90, 101, 102, 103, 108, 116, 129, 133, 137, 138, 139, 140, 141, 142, 144, 146, 147, 149, 150, 155, 157, 159, 160, 161 dosage, 65 dosing, 84 double blind study, 45 double-blind trial, 132 Drosophila, 95 drug metabolism, 48, 57, 58, 59, 68 drug targets, 77 drug therapy, 69, 80 drugs, 4, 7, 9, 42, 47, 56, 57, 58, 59, 63, 64, 65, 73, 76, 82, 131, 133, 148

Dubin-Johnson syndrome, 64 duodenum, 64 duration, 12, 14, 103, 112, 117, 142 dyslipidemia, viii, 37, 38, 39, 41, 42, 43, 45, 48, 49, 51, 131

E economics, ix, 123, 124, 129, 133, 134 edema, 145, 152 elaboration, 114 elderly, 47, 49, 53 electron microscopy, 6, 10, 138 electrophoresis, 91 emergence, 113 employment, 9, 120, 130, 131 encoding, ix, 56, 72, 80, 89, 94 endothelial cells, 10, 40, 46, 47, 52, 53, 63, 64, 146, 149, 152, 156, 157, 161 endothelium, vii, 1, 2, 3, 4, 6, 7, 10, 40, 41, 47, 145, 156, 161 end-stage renal disease, 49, 50, 120, 123, 124, 129, 130, 135 energy, 57, 62, 63 England, 50, 101, 134 enthusiasm, 115 environment, 38, 62, 111, 114, 115, 117, 119, 126, 152 environmental factors, 76 enzymatic activity, 10, 58, 66, 74, 76, 82 enzymes, 38, 48, 56, 57, 58, 70, 71, 74, 77, 86, 146 epidemic, 108 epithelia, 64, 83 epithelial cells, 6, 10, 47, 63 epithelium, 6, 10, 79 ERA, 8, 16 erythrocyte, 80, 84 erythropoietin, 152 esophagus, 59 ester, 51, 52, 73 estimating, 26, 27, 34, 35 estriol, 79 estrogen, 96 ethics, 91 ethnic diversity, 59 ethnic groups, 76, 85 ethnicity, 61, 120 etiology, 38 Europe, vii, x, 1, 138, 139 everyday life, 118 evidence, 15, 35, 45, 48, 57, 63, 65, 78, 81, 82, 87, 124, 133 evolution, 7, 65, 78, 111, 154

Index exclusion, 153 excretion, 25, 47, 57, 63, 64, 73, 75 execution, 6 exercise, 3 exons, 58, 59, 60, 64, 71, 78 expenditures, 127 experimental condition, 91 experts, 129 exposure, 43, 48, 68, 69, 70, 71, 72, 74, 75, 87 expression, 2, 3, 4, 7, 40, 42, 46, 47, 52, 53, 57, 58, 59, 60, 62, 63, 64, 67, 68, 69, 71, 74, 75, 77, 78, 79, 80, 82, 83, 84, 85, 87, 90, 91, 93, 97, 99, 141, 146, 156, 157, 158, 159, 160, 161 extinction, x, 138

F Fabry disease, vii, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 21, 22 failure, vii, viii, 34, 38, 43, 48, 51, 55, 62, 64, 106, 107, 108, 109, 111, 112, 113, 117, 118, 134, 144 faith, 111 false positive, 93 familial hypercholesterolemia, 42, 51 family, 46, 57, 59, 62, 63, 64, 79, 82, 95, 96, 106, 107, 109, 110, 111, 114, 115, 116, 117, 118, 125, 140 fasting, 43 fat, 42 fatigue, 3 feelings, 109, 113, 119 feet, 3, 118 females, 2, 6, 7, 8, 10, 14, 16 fetus, 19, 63 fever, 2, 3 fibrin, 145 fibrinogen, 41 fibroblasts, 49, 158 fibrosis, 6, 7, 47, 53 fibrous tissue, 115 filtration, viii, 6, 19, 23, 24, 34, 35, 39 Finland, 130, 135 fixation, 115 flavor, 118 fluid, 82 focusing, 76 food, 76, 144 food industry, 144 forgetting, 113 formamide, 92 fractures, 47 France, 23, 55, 120, 132 free will, 139

167

freedom, ix, 105, 112, 114 friendship, 107 fuel, 41 functional changes, 57

G gallbladder, 83 ganglion, 4 gastrointestinal tract, 59, 63, 74 gel, 91 gender, 35, 132 gene, ix, 2, 15, 46, 51, 57, 58, 59, 60, 62, 63, 64, 66, 68, 69, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 87, 89, 90, 92, 93, 94, 95, 96, 100, 101, 103, 142, 147, 152, 157, 159, 160, 161 gene expression, 51, 62, 94, 100, 101, 103, 157, 161 gene mapping, 78 gene promoter, 74, 81, 87 gene therapy, 15 gene transfer, 142 generation, 74, 146, 148 genes, ix, 40, 56, 58, 59, 61, 63, 73, 76, 77, 79, 83, 84, 86, 89, 90, 91, 92, 93, 94, 96, 97, 99, 100, 101, 103, 141, 155 genetic factors, 56, 64, 69 genetic mutations, 16 genetics, 56 genome, 56, 76, 154 genotype, 2, 5, 7, 11, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 74, 75, 76, 78, 81, 82, 84, 85, 86 Germany, 92 gift, 116 gland, 60 glomerulonephritis, 10 glucose, 38 glucoside, 87 glutathione, 39, 64, 77 glycosylation, 11, 63 goals, 109 gold, 24, 27, 35 graph, 96 grief, 126 groups, 14, 28, 30, 31, 43, 45, 62, 67, 68, 90, 91, 99, 108, 112, 131, 132, 133, 161 growth, x, 95, 96, 104, 137, 139, 140 growth factor, 96 guidelines, 27, 29, 34, 91, 126, 140, 141 guilty, 116 gut, 68, 73, 75

168

Index

H haemopoiesis, 73 half-life, 68, 70, 90 hands, 3, 5 haplotypes, 69, 86, 139 harm, 140 harmful effects, 41 HE, 134 health, viii, ix, x, 34, 37, 41, 105, 107, 108, 109, 110, 113, 114, 116, 117, 118, 119, 120, 121, 123, 124, 125, 127, 128, 129, 130, 133, 134, 139, 140 health care, 125, 127, 128, 129, 133, 134 health care costs, 128, 129 health insurance, 133 health problems, ix, 105, 107, 117 health services, x, 123, 124, 125, 127 health status, 121, 130 heart failure, 34, 117, 138 heart transplantation, 138, 141 heart valves, 144 heat, 94, 103 heat shock protein, 103 height, 25 hematocrit, 66 heme, 103 heme oxygenase, 103 hemiplegia, 4 hemodialysis, 16, 19, 49, 108, 112, 113, 115, 120, 129, 130 hemoglobin, 66 hepatitis, 138, 154 hepatocytes, 63 hepatotoxicity, 48 herpesviruses, 154 heterogeneity, 49, 68, 76 heterozygote, 2, 21, 65 high density lipoprotein, 51 highways, 126 hip, 47, 53, 111 hip fractures, 47, 53 histochemistry, 21 histology, 10, 15, 138 HIV, x, 137, 140 HLA, 10, 90, 139, 140, 141 HO-1, ix, 89 homeostasis, 60 homocysteine, 38 hormone, viii, 89, 90, 96 hospitalization, 121, 125, 128, 132 host, 10, 43, 145, 154 human brain, 80 human genome, 76

human xenotransplantation, 159 husband, 139 hybridization, 92 hydrolysis, 57, 62, 83 hypercholesterolemia, 42, 43, 50 hyperlipidemia, 38, 40, 41, 42, 48, 51 hyperparathyroidism, 38 hyperplasia, 46 hypertension, 38, 49, 111, 131 hypertriglyceridemia, 43 hypertrophic cardiomyopathy, 2, 5 hypertrophy, 5, 16 hypotensive, 102 hypothesis, 10, 28, 56, 66, 72, 111

I ICAM, 40, 41, 47 identical twins, 134 identification, ix, 16, 56, 59, 77, 79, 80, 90, 100, 106, 112, 117, 123, 124, 127, 155 identity, ix, 105, 113, 114, 117 IL-6, 39, 47 IL-8, 47 ileum, 63 immediate situation, 109 immune activation, 93, 99 immune response, x, 47, 143, 145 immune system, 129 immunogenetics, 77 immunogenicity, 100, 149 immunoglobulin, 6, 151, 156, 160 immunomodulatory, viii, 37, 45, 46, 48 immunoreactivity, 12 immunosuppression, 73, 84, 139, 144, 145, 148, 149, 153, 159 immunosuppressive agent, 48, 86 immunosuppressive drugs, viii, 9, 14, 39, 55, 56, 57, 71, 75, 76, 77, 129, 131, 133 immunosuppressive therapies, 131, 132 implementation, 124 impotence, 119 in vitro, 46, 52, 53, 58, 62, 64, 73, 74, 146, 152, 153, 154 incentives, x, 137, 140 incidence, 2, 4, 5, 8, 17, 43, 45, 66, 71, 84, 85, 132 inclusion, 6, 127, 129 income, x, 137, 140 incompatibility, 152 independence, 109 indicators, 43 indices, 32, 70 individual differences, 64

Index individual perception, 114 inducer, 100 induction, 52, 66, 91, 96, 100, 101, 102, 103, 132, 150 infection, 144, 153, 154, 155 inferior vena cava, 147 inflammation, viii, 37, 38, 39, 41, 47, 48, 49, 50, 53, 100, 101, 103, 133 inflammatory disease, 41 inflammatory mediators, 90, 100, 103 inflation, 129 influence, 16, 32, 33, 48, 56, 57, 68, 70, 75, 85, 86, 106, 111, 112, 119, 120, 140 informed consent, 111 ingest, 108 ingestion, 106 inheritance, 56, 80 inhibition, 19, 42, 46, 47, 52, 53, 66, 71, 75, 141, 146, 148, 156, 161 inhibitor, 10, 14, 47, 52, 71, 72, 95, 103, 146, 148, 149, 152, 156 injury, ix, 7, 11, 38, 39, 41, 48, 90, 91, 95, 101, 142, 148, 154, 156 inmates, 140 inner ear, 4 innovation, 114 insertion, 58 insight, 90, 100 instability, 99 instruments, 107, 110, 118 insulin, 152 insurance, x, 137, 140 integration, 61 intensity, 92 interaction, 47, 63, 75, 82, 83, 85, 113, 114 interactions, 53, 56, 57, 72, 75, 77, 100 intercellular adhesion molecule, 40 interest, 38, 45, 56, 108, 109, 138, 140, 147, 149 interference, 3 internalization, 47, 53, 117 internalizing, 117 interpretation, ix, 33, 89, 90, 96, 100, 108, 116 interrelationships, 39 interval, 5, 87 intervention, 100, 124, 125, 126, 127, 130, 131, 149 intervention strategies, 126 interview, 111, 112 intestine, 60, 64, 68, 72, 73, 75 intima, 40, 41 intracranial pressure, 91 intravenously, 11 investment, 133 ionizing radiation, 102

169

irradiation, 151 ischemia, ix, 90, 91, 102, 145 isolation, 91 isozymes, 21 Italy, 1, 14, 22, 137

J Japan, 128, 130, 134 jejunum, 63 jobs, 119 Jordan, 21

K kidney(s), vii, viii, ix, x, 1, 2, 3, 6, 9, 10, 11, 13, 14, 17, 18, 19, 20, 21, 22, 23, 24, 27, 29, 32, 33, 34, 35, 37, 38, 44, 48, 51, 53, 58, 60, 66, 72, 73, 83, 84, 85, 87, 89, 90, 91, 93, 94, 96, 97, 99, 100, 101, 102, 103, 105, 106, 108, 109, 111, 119, 121, 123, 124, 125, 127, 128, 129, 130, 131, 133, 134, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 151, 152, 155, 159, 160 knowledge, ix, 15, 76, 77, 123, 124, 133

L labeling, 66, 83 labor, 145 language, x, 123, 124 LDL, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51 lead, viii, ix, xi, 6, 23, 25, 37, 40, 44, 90, 105, 144, 148, 153, 154, 155 learning, 119 lecithin, 44, 51 leisure, 109, 111 lesions, 4, 6, 10, 40, 46, 49 leukemia, 61, 78, 84 leukopenia, 72 LFA, 47, 53, 156 liberation, 114 life quality, ix, 105 life satisfaction, 109, 130 lifestyle, 108 ligands, 156 likelihood, 15 limitation, 113, 118 linkage, 58, 70 links, 93 lipid metabolism, viii, 37, 38 lipid peroxidation, 40

170

Index

lipids, 6, 41, 44, 45, 50 lipoproteins, 38, 39, 40 liquids, 106, 108 liver, 11, 34, 40, 41, 58, 59, 60, 63, 64, 66, 68, 69, 71, 72, 73, 74, 75, 78, 79, 80, 83, 85, 87, 100, 102, 128, 138, 141, 142, 152 liver disease, 83 liver transplant, 68 liver transplantation, 34, 69, 71, 75, 78, 85, 87 localization, 21, 79, 82, 83, 84, 93, 96, 97 location, 96 locus, 58, 59, 60, 62, 77, 78, 79, 84 loneliness, 109 lovastatin, 47 love, 118 low-density lipoprotein, 38, 51 lupus, 18, 35 lymphocytes, 73 Lyon hypothesis, 2 lysis, 141, 146, 157

M mAb, 151 macrophages, xi, 40, 90, 143, 145, 161 magnetic resonance, 4 magnetic resonance imaging, 4 major histocompatibility complex, 53 males, 2, 3, 5, 6, 7, 10, 14, 15, 26 malignancy, 131 management, x, 15, 19, 91, 137, 140 mapping, 83 mass, 5, 12, 32 matrix, 7, 47 maturation, 43, 109 MCP, x, 94, 138, 157 MCP-1, 94 meanings, ix, 105, 107, 108, 113, 116 measurement, 24, 28, 29, 32, 33, 35, 66, 127, 129 measures, 27, 43, 44, 107, 108, 109, 110, 111, 119, 126 median, 61, 92, 93, 111, 112, 130 Medicaid, 128 Medicare, 124, 127, 128, 129, 133, 134, 135 medication, 56, 75, 76, 108, 111, 112, 114, 119, 148 melanoma, 104 membranes, 39, 52 men, 25, 111, 112 mental state, 111 metabolic syndrome, 50 metabolism, 19, 24, 38, 48, 51, 56, 57, 58, 59, 60, 63, 66, 68, 70, 71, 72, 73, 75, 76, 77, 78, 79, 87 metabolites, 57, 59, 73

metabolizing, 56, 57, 58 methodology, ix, 26, 29, 105, 123, 124, 125, 129 methylation, 57, 65 Mexico, 128 MHC, 40, 47, 156 mice, 21, 141, 157, 158 microscopy, 4, 6, 83 microsomes, 74, 87 migration, 46 mitogen, 101, 104 mitral insufficiency, 5 mixing, 92 mobility, 109, 110, 112 mode, 63 modeling, 47 models, viii, xi, 46, 56, 64, 89, 90, 99, 143, 145, 146, 147, 150, 155 mold, 106 molecular biology, 77 molecular mass, 64 molecular weight, 148, 151 molecules, 40, 45, 46, 47, 53, 62, 63, 90, 92, 99, 100, 102, 146, 155 monitoring, xi, 14, 22, 38, 53, 56, 66, 72, 76, 77, 84, 85, 86, 143, 155 monolayer, 40 morbidity, vii, x, 1, 2, 3, 4, 8, 9, 14, 49, 107, 109, 125, 130, 138, 139, 140 mortality, vii, viii, x, 1, 2, 3, 4, 9, 14, 37, 48, 49, 125, 137, 138, 139 mortality rate, 48 MRI, 4 mRNA, 51, 58, 60, 64, 68, 75, 77, 94, 101 mucosa, 58, 63, 80 multidimensional, 107, 108, 110, 111, 112, 118 multiple regression, 75 multiple regression analysis, 75 muscle mass, 32 mutant, 46, 60, 61, 66, 69, 80, 81, 110 mutant proteins, 60 mutation, 58, 60, 69, 80 myocardial infarction, 4, 45 myocardium, 12, 16 myopathy, 47

N National Institutes of Health, 135 natural killer cell, 52 nausea, 4, 109 needs, 9, 38, 64, 76, 101, 108, 118 nephrectomy, x, 137, 139, 147 nephritis, 6

Index nephron, 32 nephropathy, viii, 3, 6, 10, 11, 15, 19, 20, 37, 38, 47, 49, 50, 53, 77 nephrotic syndrome, 6 nerve, 17 Netherlands, 89, 91, 92, 129 network, 93, 96, 97, 100, 101, 131, 141 neuropathic pain, 3, 10, 11, 17 neutrophils, 39, 47, 66 New Zealand, 129 nitric oxide, 40, 46, 53 nitric oxide synthase, 40, 46 nitrogen, 25 NK cells, xi, 143, 145 North America, 48 nucleus, 100, 101 null hypothesis, 27

O obligate, 16 observations, 14, 45, 46, 68, 92, 146 Oceania, 19 oil, 103 oncogenes, 52 oppression, 115 optimization, 70, 131 organ, viii, ix, x, xi, 9, 11, 15, 19, 22, 55, 56, 63, 71, 72, 73, 74, 76, 90, 99, 100, 101, 102, 105, 107, 114, 116, 118, 120, 128, 129, 133, 137, 138, 141, 142, 144, 145, 149, 152, 153, 154, 155, 156, 157, 161 organism, 109, 115, 117 organization, 15 osmolality, 33 overtime, 129 overweight, 42 oxidation, 39, 40, 41, 50, 57 oxidative stress, 38, 39, 49

P pain, 3, 4, 8, 9, 11, 109, 110, 126 palliative, vii, 1, 3, 4 pancreas, x, 121, 138 parathyroid, 49 parathyroid hormone, 49 parenchymal cell, 71 paresthesias, 2, 3 particles, 38, 40, 44, 45, 49 passive, 116 pathogens, 153

171

pathology, 18, 19, 118, 119 pathophysiology, 155 pathways, ix, xi, 39, 57, 76, 89, 90, 100, 143, 145, 146, 147, 148, 149, 155 PCR, 61, 64, 66, 91, 100 perceptions, 110 perfusion, 86, 141, 142, 146, 157 peripheral blood, 146 peripheral blood mononuclear cell, 146 peripheral nervous system, 3, 4 permeability, 82 permit, 5, 76 peroxidation, 39 perspective, 107, 110, 121, 127, 132, 133 PGE, 150, 151 pharmaceuticals, 133 pharmacogenetics, 56, 57, 65, 77, 80, 81, 84, 85 pharmacokinetics, 11, 16, 48, 56, 63, 70, 71, 75, 76, 84, 85, 86 phenol, 79 phenotype, 2, 7, 10, 15, 61, 67, 82, 148, 158 phosphorylation, 94, 101 physical activity, 108, 121 physical interaction, 100 physiology, x, 52, 143, 144, 152 pigs, x, xi, 138, 139, 141, 143, 144, 146, 147, 148, 152, 153, 154, 155, 156, 157, 158, 159, 160, 162 pilot study, 17, 43, 45, 51 placebo, 19, 44, 45, 52, 73, 132 placenta, 57, 63, 64, 83 planned action, 126 plaques, 40 plasma, 11, 13, 21, 22, 26, 28, 29, 33, 35, 39, 48, 50, 51, 52, 64, 73, 87, 103, 152 plasma levels, 39, 48 plasmapheresis, 146 plasminogen, 47 platelets, 41 plexus, 82 PM, 52 pneumothorax, 140 point mutation, 81 polymerase, 61 polymerase chain reaction, 61 polymers, 148 polymorphism, 56, 58, 65, 66, 68, 69, 70, 71, 72, 73, 75, 77, 78, 80, 83, 84, 85, 87 polypeptide, 94 poor, x, 4, 8, 29, 30, 44, 66, 99, 137, 140, 144 population, viii, 2, 6, 8, 14, 26, 29, 30, 32, 37, 38, 39, 44, 45, 47, 56, 58, 61, 63, 65, 67, 68, 70, 71, 76, 81, 85, 111, 128, 133, 139, 153 posture, 116

172

Index

prediction, 26, 27, 28, 29, 32, 33, 34, 35, 66 predictors, 32, 43, 44 prednisone, 43, 51 preference, 127 prejudice, 107 premature death, viii, 55, 56, 125 present value, 127, 129 pressure, 50 prevention, 50, 51, 71, 73, 86, 100, 135, 156, 161 primate, xi, 142, 143, 144, 145, 146, 147, 152, 154, 155, 156, 157, 159, 160, 161 principal component analysis, 92 principle, 47 prisoners, 140 private practice, 128 probability, 7, 117, 153 probe, 92 prodrugs, 57 producers, 108 production, 11, 39, 41, 46, 50, 58, 74, 101, 153 productivity, 125, 129 program, 22, 109, 124, 126, 127, 128, 131, 133, 139, 141, 142 proliferation, 40, 46, 52, 73, 101, 154, 161 promoter, 58, 62, 65, 75, 77, 82, 85, 87, 95, 96 propagation, 154 prophylactic, 4, 9 prostate, 60, 78 protein family, 62, 82, 83 proteins, ix, xi, 40, 41, 46, 52, 56, 57, 59, 60, 62, 72, 73, 83, 89, 94, 96, 100, 142, 143, 146, 152, 156 proteinuria, 3, 5, 6, 7, 10, 11, 12, 47, 53, 152 proteolysis, 60, 80 protocol, 91, 92, 148, 152 pseudogene, 58, 67, 68, 74 psychological problems, 106 psychosocial factors, 107 public health, 153, 155 public policy, 124 pulmonary embolism, 140 pumps, 63 P-value, 30, 31

Q quality of life, x, 3, 4, 8, 11, 19, 107, 108, 109, 110, 111, 112, 113, 116, 118, 119, 120, 121, 126, 129, 130, 131, 133, 134, 137, 140 questioning, 25

R race, 132, 147 radio, 33 radiotherapy, 84 range, vii, 2, 4, 5, 14, 28, 29, 42, 43, 60, 63, 90, 91, 100, 119, 128, 129, 130, 149, 150, 151, 152, 153 reactant, 41 reading, 60 reality, 111, 117, 118, 127 reasoning, 127 receptors, 40, 42, 47, 51, 53, 56, 104 recognition, 4, 5, 7, 10, 107, 109, 156 recombination, 146, 154 reconstruction, ix, 105 recovery, 106, 109, 116, 117 recurrence, 3, 10, 21 red blood cells, 39, 60, 66 reduction, 5, 12, 39, 42, 43, 45, 47, 48, 57, 60, 66, 75, 92, 113, 132, 146, 154 regeneration, ix, 89, 95 Registry, 9, 16, 19 regression, 43, 77, 92 regression analysis, 43 regulation, viii, xi, 44, 51, 52, 57, 73, 78, 80, 89, 90, 93, 94, 96, 97, 100, 102, 103, 143, 157, 161 regulators, 152 rehabilitation, vii, 106 rejection, vii, viii, x, xi, 9, 10, 14, 24, 45, 49, 50, 51, 55, 66, 68, 71, 72, 73, 85, 86, 102, 103, 111, 129, 131, 132, 135, 138, 141, 143, 145, 146, 147, 148, 149, 152, 153, 155, 156, 158, 159, 160, 161 relapses, 117 relationship, 26, 27, 61, 62, 68, 69, 71, 75, 78, 81, 108, 114, 115, 116, 118 relationships, 106, 114, 116, 119 relatives, 139, 140 relevance, 71, 80 reliability, 32 remodelling, 157 renal dysfunction, 7 renal failure, vii, 1, 7, 10, 19, 20, 108, 139 renal replacement therapy, vii, 1, 3, 16, 120, 129 repair, ix, 90, 91, 94, 101 replacement, vii, 1, 3, 8, 13, 15, 16, 17, 18, 19, 20, 21, 22, 63, 129, 133, 138, 139, 155 replication, 154 repression, 96, 100 residues, 64, 146 resistance, 4, 9, 17, 62, 64, 73, 82, 83, 86, 87, 146, 158 resolution, 53, 92 resources, 76, 110, 125, 126, 133

Index responsibility, 110, 114 restriction fragment length polymorphis, 61 reticulum, 59 retirement, 118 retrovirus, 162 retroviruses, 154 returns, 40 reverse transcriptase, 91, 92 rhabdomyolysis, 48 rheumatoid arthritis, 41, 50 ribosomal RNA, 94 risk, x, 4, 8, 10, 19, 24, 38, 41, 43, 44, 47, 48, 49, 50, 51, 53, 66, 72, 86, 90, 103, 115, 133, 137, 138, 140, 141, 144, 153, 154, 161 risk factors, 49, 50, 51 RNA, ix, 89, 90, 91, 92, 99 robustness, 127, 131 room temperature, 92 routines, 117, 119

S sadness, 109 safety, vii, 2, 3, 11, 16, 18, 22, 50, 51, 72, 76, 132 sample, ix, 35, 62, 91, 92, 105, 112 sampling, 70 sanctions, 117 sarcoidosis, 43, 51 SARS, 154 satisfaction, 110, 119 saturated fat, 42 savings, 129, 133 school, 126 scientific progress, 114 sclerosis, 6, 7 scores, 112 search, 107, 114, 124, 131 searches, ix, 76, 89, 93, 94 searching, 93 secretion, 24, 33 sediment, 6, 18 selecting, 126, 154 selenium, 39 self, ix, x, 105, 114, 117, 137, 140 self-esteem, x, 114, 137, 140 self-image, 117 senescence, 49 sensitivity, 127, 131 sepsis, 8, 65 septum, 5 series, 8, 40, 67, 69, 113, 114, 147, 148 serum, 2, 4, 7, 11, 12, 14, 25, 26, 32, 33, 34, 35, 39, 42, 43, 47, 51, 101, 146, 147, 151, 155, 158, 160

173

serum albumin, 25 services, 110, 125, 128, 132, 133 severe acute respiratory syndrome, 154 severity, 4, 18 sheep, 147 shock, 94, 95 shortage, x, 143, 144, 155 siblings, 116 side effects, 72, 74, 131, 148 sign, 14, 40 signalling, 100, 103, 104 signals, 41, 46, 145 similarity, 83, 161 simulation, 30, 31 Singapore, 161 single-nucleotide polymorphism, 87 sites, 63, 83, 96 skeletal muscle, 47, 96 skin, 4, 11, 77, 87 skin cancer, 77, 87 small intestine, 58, 64, 73, 78, 83, 86 smooth muscle, 6, 7, 40, 47, 52 smooth muscle cells, 6, 47, 52 SNP, 58, 63, 65, 67, 68, 69, 70, 71, 72, 74, 75, 84 sociability, 106 social life, 118 social relations, 109, 111, 113 social relationships, 109, 111, 113 social rewards, 117 social support, 131 sodium, 52, 94, 149, 151, 159 software, ix, 89, 90, 92, 100 solidarity, 115 somatic cell, 147 Spain, 130, 143, 144 species, x, 138, 146, 147, 152, 153, 154, 162 specificity, 63, 77, 79, 81 spectrum, 27, 57 speech, 107 spleen, 11 SPSS, 92 squamous cell, 87 squamous cell carcinoma, 87 stability, viii, 89, 90, 103 stabilization, vii, 1 stages, 6, 11, 29, 39 standard deviation, 27, 30, 31 standards, viii, 23, 29, 110 statin, 45, 46, 47, 48, 52, 53 statistics, 119 steroids, 32, 38, 44, 63, 72, 80, 131, 159 stigma, 117 stigmatized, 114, 117

174

Index

stomach, 59 storage, 3, 4, 6, 12, 16 strategies, viii, 23, 24, 33, 61, 76, 120, 126, 145, 146, 149, 155 stratification, 34 strength, x, 27, 127, 143, 145 stress, x, 39, 49, 100, 101, 133, 137, 140 structural changes, 18 subjectivity, 107, 108, 111, 118, 119 substitution, 60, 65 substrates, 46, 57, 58, 59, 63, 64, 72, 78, 79 Sun, 53 superiority, 28, 44 supply, 9, 133, 139 surface area, 26 surgical resection, 64 survival, vii, viii, xi, 1, 2, 3, 8, 10, 14, 15, 17, 23, 24, 45, 48, 55, 77, 89, 90, 99, 101, 102, 103, 106, 107, 109, 119, 121, 130, 132, 138, 139, 140, 143, 144, 146, 147, 148, 149, 150, 151, 152, 154, 156, 157, 158, 159, 161 survival rate, 2, 3, 8, 14, 103 survivors, 49, 149 susceptibility, 41, 72, 87, 148 Sweden, 129 switching, 126 symptom, 10, 116, 117 symptoms, vii, 1, 2, 3, 4, 5, 8, 9, 10, 11, 13, 14, 15, 17, 48, 113, 152 syndrome, 51, 154 synergistic effect, 156 synthesis, 38, 39, 43, 45, 46, 73 systemic change, 99 systems, 39, 110

T T cell, 145, 147, 149, 156, 161 tandem repeats, 82 targets, 56, 101, 103, 149 technology, 77, 146 teenagers, 3 tension, 109 TGF, 77 theory, 119 therapeutics, 56 therapy, vii, 1, 3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 30, 43, 45, 51, 52, 53, 65, 71, 77, 81, 84, 85, 86, 129, 131, 132, 133, 138, 139, 142, 156, 161 thinking, 109, 113, 119 threat, 113, 147 threshold, 28, 32

thrombomodulin, 152 thrombosis, 4, 9, 40, 145, 149 thymus, 148, 151 time, 5, 6, 10, 12, 13, 14, 24, 32, 33, 43, 44, 70, 73, 76, 85, 90, 100, 102, 108, 112, 114, 119, 124, 127, 131, 132, 139, 140, 144, 145, 147, 148, 152, 155 timing, 68 tissue, 5, 9, 11, 12, 16, 45, 46, 52, 59, 71, 77, 79, 91, 94, 103, 148, 152, 154, 159, 160, 162 tissue plasminogen activator, 45 TNF, 149 total costs, 124 toxic effect, 71 toxic products, 65 toxic substances, 63 toxicity, 65, 66, 73, 81, 86, 152 traits, 76 transaminases, 48 transcription, ix, 58, 82, 89, 92, 93, 95, 96, 101, 103 transcription factors, ix, 89, 93, 103 transduction, ix, 89, 94, 95 transformation, 52, 116 transition, 58, 60, 81 transition mutation, 60 translation, 56 translocation, 95 transmission, x, 137, 140, 153, 154, 162 transplant recipients, viii, 9, 14, 17, 32, 35, 37, 38, 42, 43, 45, 49, 50, 51, 52, 56, 66, 67, 69, 70, 74, 77, 84, 85, 86, 87, 111, 119, 128, 154 transplantation, vii, viii, ix, x, 1, 3, 8, 9, 11, 13, 14, 17, 19, 20, 21, 22, 23, 24, 30, 31, 32, 33, 34, 35, 37, 38, 43, 49, 50, 51, 52, 53, 55, 56, 65, 68, 69, 70, 71, 72, 73, 75, 76, 77, 85, 86, 87, 90, 91, 99, 100, 101, 102, 103, 115, 119, 120, 121, 123, 124, 125, 127, 128, 129, 130, 131, 132, 133, 134, 135, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 149, 152, 154, 155, 159, 160, 161 transport, 39, 41, 47, 56, 57, 62, 63, 72, 73, 75, 82, 83, 86, 94, 108 transportation, 125 trauma, 91 trend, 44, 67, 71 trial, 11, 16, 19, 21, 26, 28, 30, 34, 45, 47, 50, 52, 86, 132 triggers, 101, 145, 147 tumor cells, 62 tumor necrosis factor, 161 tumors, 62, 63, 78, 83, 161 two-sided test, 24 type 2 diabetes, 19, 50 tyrosine, 46

Index

U UK, 92 uncertainty, 127 uniform, 6, 12 United States, 16, 77, 102, 123, 128, 135 urea, 25 ureter, 147 urine, 5, 10, 22, 24, 25, 57

V validation, 93 validity, viii, 23, 26, 27, 29, 32, 100, 112, 139 values, 27, 28, 44, 92, 93, 118, 127 variability, 2, 10, 12, 27, 56, 62, 63, 66, 72, 75, 77, 78 variables, 2, 6, 7, 25, 32, 62, 78, 81, 82, 109 variance, 62, 128 variation, 58, 78, 81, 84, 86, 91, 94, 117 vascular cell adhesion molecule, 40 VCAM, 40, 41, 45 vegetables, 114 vein, 4, 52, 147 ventilation, 91 vessels, 4, 6, 10 virus infection, 161 viruses, 154 vitamin C, 39 VLDL, 40, 44, 52 vomiting, 4

weight gain, 131 weight loss, 42, 43 welfare, 113 well-being, 110, 118, 126, 130 wild type, 60, 61, 62, 69 withdrawal, 43, 51, 52, 131 women, 2, 8, 25, 111, 112 words, 30, 32, 106, 107, 108, 112, 113, 114, 116, 117, 118, 138 work, 70, 107, 108, 110, 111, 113, 114, 117, 126, 131, 140, 144, 147, 148, 152 workers, 154, 162 World Health Organization, 110, 111, 112, 120

X xenografts, 142, 149, 152, 153, 154, 156, 159, 160, 161 xenotransplantation, x, 138, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 152, 153, 154, 155, 157, 159, 160, 161, 162 X-inactivation, 2

Y yeast, 60 yield, 46, 76, 90 young adults, 41, 48

Z zinc, 39

W water, ix, 39, 57, 89 weakness, 106

175