The Genetics of Renal Disease

The Genetics of Renal Disease

Frances Flinter Eamonn Maher Anand Saggar-Malik Editors

OXFORD UNIVERSITY PRESS

OXFORD MEDICAL PUBLICATIONS

The Genetics of Renal Disease

OXFORD MONOGRAPHS ON MEDICAL GENETICS General Editors ARNO G. MOTULSKY MARTIN BOBROW PETER S. HARPER CHARLES SCRIVER CHARLES J. EPSTEIN JUDITH G. HALL

16. 18. 21. 22. 24. 25. 26. 27. 30. 31. 32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43. 44. 45. 46. 47. 48. 49.

C. R. Scriver and B. Child: Garrod’s inborn factors in disease M. Baraitser: The genetics of neurological disorders D. Warburton, J. Byrne, and N. Canki: Chromosome anomalies and prenatal development: an atlas J. J. Nora, K. Berg, and A. H. Nora: Cardiovascular diseases: genetics, epidemiology, and prevention A. E. H. Emery: Duchenne muscular dystrophy, second edition E. G. D. Tuddenham and D. N. Cooper: The molecular genetics of haemostasis and its inherited disorders A. Boué: Foetalmedicine R. E. Stevenson, J. G. Hall, and R. M. Goodman: Human malformations A. S. Teebi and T. I. Farag: Genetic disorders among Arab populations M. M. Cohen, Jr.: The child with multiple birth defects W. W. Weber: Pharmacogenetics V. P. Sybert: Genetic skin disorders M. Baraitser: Genetics of neurological disorders, third edition H. Ostrer: Non-mendelian genetics in humans E. Traboulsi: Genetic diseases of the eye G. L. Semenza: Transcription factors and human disease L. Pinsky, R. P. Erickson, and R. N. Schimke: Genetic disorders of human sexual development R. E. Stevenson, C. E. Schwartz, and R. J. Schroer: X-linked mental retardation M. J. Khoury, W. Burke, and E. Thomson: Genetics and public health in the 21st century J. Weil: Psychosocial genetic counseling R. J. Gorlin, M. M. Cohen, Jr., and R. C. M. Hennekam: Syndromes of the head and neck, fourth edition M. M. Cohen, Jr., G. Neri, and R. Weksberg: Overgrowth syndromes R. A. King, J. I. Rotter, and A. G. Motulsky: The genetic basis of common diseases, second edition G. P. Bates, P. S. Harper, and L. Jones: Huntington’s Disease, third edition R. J. M. Gardner and G. R. Sutherland: Chromosome abnormalities and genetic counselling, third edition I. J. Holt: Genetics of mitochondrial disease F. Flinter, E. Maher, and A. K. Saggar-Malik: The genetics of renal disease C. J. Epstein, R. P. Erickson, and A. Wynshaw-Boris: Inborn errors of development: the molecular basis of clinical disorders of morphogenesis 50. H. V. Toriello, W. Reardon, and R. J. Gorlin: Hereditary hearing loss and its syndromes, second edition

Oxford University Press makes no representation, express or implied, that the drug dosages in this book are correct. Readers must therefore always check the product information and clinical procedures with the most up to date published product information and data sheets provided by the manufacturers and the most recent codes of conduct and safety regulations. The authors and the publishers do not accept responsibility or legal liability for any errors in the text or for the misuse or misapplication of material in this work.

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The Genetics of Renal Disease Edited by

FRANCES FLINTER Clinical Genetics Department, Guy’s Hospital St Thomas Street, London SE1 9RT, UK EAMONN MAHER Clinical Genetics Unit, Birmingham Maternity Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham B15 2ST, UK ANAND SAGGAR-MALIK Clinical Genetics Department, St George’s Hospital Medical School, Cranmer Terrace, London SW17 0RE, UK

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Great Clarendon Street, Oxford OX2 6DP Oxford University Press is a department of the University of Oxford. It furthers the University’s objective of excellence in research, scholarship, and education by publishing worldwide in Oxford New York Auckland Bangkok Buenos Aires Cape Town Chennai Dar es Salaam Delhi Hong Kong Istanbul Karachi Kolkata Kuala Lumpur Madrid Melbourne Mexico City Mumbai Nairobi São Paulo Shanghai Taipei Tokyo Toronto Oxford is a registered trade mark of Oxford University Press in the UK and in certain other countries Published in the United States by Oxford University Press Inc., New York Previously published as The Genetics of Renal Tract Disorders by M. D’A Crawfurd in 1988 © Oxford University Press, 2003 The moral rights of the authors have been asserted Database right Oxford University Press (maker) First published 2003 All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without the prior permission in writing of Oxford University Press, or as expressly permitted by law, or under terms agreed with the appropriate reprographics rights organization. Enquiries concerning reproduction outside the scope of the above should be sent to the Rights Department, Oxford University Press, at the address above You must not circulate this book in any other binding or cover and you must impose this same condition on any acquirer A catalogue record for this title is available from the British Library ISBN 0 19 263146 2 (Hbk) 10 9 8 7 6 5 4 3 2 1 Typeset by Newgen Imaging Systems (P) Ltd., Chennai, India Printed in Great Britain on acid-free paper by Biddles Ltd, Guildford and King’s Lynn

Preface The first part of the twenty-first century is widely predicted to be the ‘age of genomic medicine’. Indeed, the completion of the first draft of the human genome sequence is revolutionizing human genetics research and we can now look forward to a time when the genetic basis of most monogenic disorders is known. There have certainly been huge changes in our knowledge of medical genetics since 1988 when The Genetics of Renal Tract Disorders by M.D’A Crawfurd (Oxford Monographs on Medical Genetics 14) was first published. Genetic factors are important in the pathogenesis of a wide range of renal diseases and we felt that a more contemporary monograph on this subject would be relevant to a wide range of clinicians and other interested groups. Although Dr Crawfurd single-handedly produced an excellent text, given the extent of current knowledge, we decided that a multi-author approach would be the best option. We have been fortunate to recruit a most distinguished group of authors and wish to thank them for their excellent contributions. As with any group of authors, there are bound to be some instances of repetition. We have not attempted to eliminate all of these as different views of the same subject can be most illuminating! Inevitably there will be some topics that have not been covered in as much detail as some readers would wish, and some may have been overlooked. However we believe that our international collection of co-authors have produced a balanced and authoritative book that will address the needs of nephrologists, clinical geneticists, paediatricians, internal physicians, pathologists and urologists and their co-workers. Frances Flinter Eamonn R Maher Anand Saggar Mallick

London, UK Birmingham, UK London, UK

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Contents List of contributors 1 Introduction to clinical genetics Shehla Mohammed 2 Renal function and management of renal disease A. David Makanjuola and John E. Scoble 3 Renal development Paul J. D. Winyard 4 Kidney and lower urinary tract malformations Adrian S. Woolf 5 Urinary tract defects and chromosomal disorders David J. Amor, Lachlan de Crespigny, and R. J. McKinlay Gardner 6 Dysmorphic syndromes with renal involvement Ian D. Young 7 Primary hereditary nephropathies Karl Tryggvason 8 Alport syndrome Frances Flinter 9 Autosomal dominant polycystic kidney disease Anand K. Saggar-Malik and Stefan Somlo 10 Autosomal recessive polycystic kidney disease Lisa M. Guay-Woodford 11 Cystic renal diseases Anand K. Saggar-Malik 12 Primary inherited metabolic diseases of the kidney Margaret Town and William van’t Hoff 13 Genetics of stone forming diseases Pasquale Strazzullo and Pietro Vuotto 14 Disorders of tubular transport David S. Geller, Mark A. J. Devonald, and Fiona E. Karet 15 Tuberous sclerosis complex Astrid P. Weber and Robert F. Mueller 16 Neurofibromatosis Susan M. Huson and Natalie Canham

xi 1 27 57 89 117 147 167 183 203 239 253 261 281 307 337 349

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Contents

17 The Bardet–Biedl and Alström syndromes Philip L. Beales, Patrick S. Parfrey, and Nicholas Katsanis 18 Genetic syndromes with a renal component Richard Sandford and Robin G. Woolfson 19 The genetics of glomerulonephritis and systemic disorders affecting the kidney Stephen H. Powis 20 Wilms tumour and the Wilms tumour predisposition syndromes Richard Grundy 21 Von Hippel–Lindau disease Eamonn R. Maher 22 Inherited predispositions to kidney cancer Berton Zbar, Gladys Glenn, Maria Turner, Gregor Weirich, Peter Choyke, Stephen Hewitt, Michael Nickerson, Naboru Nakaigawa, W. Marston Linehan, and Laura Schmidt 23 Gene therapy in renal disease Anand K. Saggar-Malik 24 Gene therapy for renal cancer Michael J. Gough and Richard G. Vile Index

361 399

417 455 487 497

511 515

557

List of Contributors David J. Amor, Genetic Health Services Victoria, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia Philip L. Beales, Molecular Medicine Unit, Institute of Child Health, University College London, UK Natalie Canham, West Midlands Regional Genetic Service, Birmingham Women’s Hospital, Edgbaston, Birmingham, UK Lachlan de Crespigny, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia Mark A. J. Devonald, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, UK Frances Flinter, Clinical Genetics Department, Guy’s & St Thomas’ NHS Trust, London, UK R. J. McKinlay Gardner, Genetic Health Services Victoria, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia David S. Geller, Yale University School of Medicine, New Haven, Connecticut, USA Michael J. Gough, Molecular Medicine Program, Mayo Clinic, Rochester, Minnesota, USA Richard Grundy, Clinical Genetics Unit, Birmingham Maternity Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK Lisa M. Guay-Woodford, Division of Genetic and Translational Medicine, University of Alabama at Birmingham, USA Susan M. Huson, Clinical Genetics Department, Oxford Radcliffe Hospitals, Oxford, UK Fiona Karet, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, UK Nicholas Katsanis, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, Maryland, USA Eamon R. Maher, Clinical Genetics Unit, Birmingham Maternity Hospital, Queen Elizabeth Medical Centre, Edgbaston, Birmingham, UK A. David Makanjuola, Renal Department, St Helier Hospital, Carshalton, Surrey, UK Shehla Mohammed, Clinical Genetics Department, Guy’s & St Thomas’ NHS Trust, London, UK Robert F. Mueller, Clinical Genetics Department, St James’s Hospital, Leeds, UK Patrick S. Parfrey, Patient Research Centre, Health Sciences Centre, Memorial University, St Johns, Newfoundland, Canada Stephen H. Powis, Centre for Nephrology, Royal Free & University College Medical School, London, UK

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List of Contributors

Anand K. Saggar-Malik, Clinical Genetics Department, St George’s Hospital Medical School, London, UK Richard Sandford, Cambridge Institute for Medical Research, Addenbrooke’s Hospital, Cambridge, UK John E. Scoble, Renal Department, Guy’s & St Thomas’ NHS Trust, London, UK Stefan Somlo, Yale University School of Medicine, New Haven, Connecticut, USA Pasquale Strazzullo, Department of Internal Medicine, Federico II University of Naples Medical School, Italy Margaret Town, Nephro-Urology Unit, Institute of Child Health, University College London, UK Karl Tryggvason, Division of Matrix Biology, Karolinska Institutet, Stockholm, Sweden William van’t Hoff, Nephro-Urology Unit, Institute of Child Health, University College London, UK Richard G. Vile, Molecular Medicine Program, Mayo Clinic, Rochester, Minnesota, USA Pietro Vuotto, Federico II University of Naples Medical School, Naples, Italy Astrid P. Weber, St James’s Hospital, Leeds, UK Paul J. D. Winyard, Institute of Child Health and Urology, Great Ormond Street Hospital NHS Trust, London, UK Adrian S. Woolf, Nephro-Urology Unit, Institute of Child Health, University College London, UK Robin G. Woolfson, University College London Hospitals, London, UK Ian D. Young, Clinical Genetics Department, Leicester Royal Infirmary, Leicester, UK Berton Zbar, Laboratory of Immunology, National Cancer Institute, Frederick Cancer Research & Development Center, Frederick, Maryland, USA

1 Introduction to clinical genetics Shehla Mohammed

Human chromosome constitution Introduction This introductory chapter aims to provide some basic information about human genetics, the nature of genetic counselling and to highlight some of the issues and dilemmas that may arise following the diagnosis of a genetic disorder in a family. The double stranded DNA (deoxyribonucleic acid) helix is partitioned into small units called chromosomes, located in the nucleus of each cell: the word chromosome being derived from the Greek, chroma (colour) and soma (body). The chromosomes exist in 23 pairs. Of these 46 chromosomes, the autosome pairs are numbered sequentially from 1 to 22 in descending order of size. The remaining pair, the sex chromosomes, comprises two X chromosomes in females and an X and a Y chromosome in males. The presence of paired chromosomes implies that all somatic cells contain two copies of every gene, also referred to as the diploid genome. In the gametes, each mature egg and sperm contains a single haploid set of genes, thereby ensuring that each individual inherits one chromosome of each pair from each parent. Chromosome structure Each chromosome is comprised of a densely compacted coil of DNA, bonded with protein molecules called histones (Fig. 1.1). This nucleic acid–histone complex is folded in an orderly manner, allowing large stretches of DNA to be accommodated in the finite space of the cell nucleus. Furthermore, the folding of a particular section of the DNA molecule can influence the activity of genes encoded in that region. The length of the DNA loop can vary in size from about 20,000 to 80,000 nucleotide pairs, resulting in an average human chromosome being about 100 m long. Human chromosomes are readily visualized by halting them at a specific stage of the cell cycle and using special stains, which are selectively taken up by the DNA in each chromosome. This is normally done during metaphase, a stage of cell division when the chromosomes are maximally contracted and can therefore be visualized easily by light microscopy (Fig. 1.2(A)). A specific banding pattern gives each chromosome a characteristic appearance (Fig. 1.2(B)). Euchromatin contains actively expressed genes and stains as light bands, while the darker bands comprise heterochromatin, which is believed to contain largely inactive or silent DNA. Each chromosome is comprised of two halves, called chromatids, which are separated from each other along the length of the chromosome, except at a narrowing called the centromere. The size, banding pattern, and position of the centromere identify a particular chromosome.

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Shehla Mohammed Histone “bead” DNA duplex

DNA helix

100 Å Chromatin fibre

Nucleosomes

Loop

Chromosome

Fig. 1.1 Coiling of DNA and association with histone. (Reproduced with kind permission of The Open University from Science: a Second level course in genetics—Units 1 and 2, 1990.)

A

B

1

6

13

2

3

7

8

14

15

9

10

16

4

5

11

12

17

18

19 20 21 22 X Three chromosomes 21 instead of two

Y

Fig. 1.2 A: Chromosomes under light microscopy. B: Ideogram and notation. (See Plate 1 of the Colour Plate Section at the centre of this book.)

The centromere divides each chromosome into short and long arms, the standard notation being ‘p’ for petit, and ‘q’ (the next letter in the alphabet), respectively (Fig. 1.3). The ends of chromosomes are referred to as telomeres, which have an important role in maintaining the integrity of chromosomes by effectively ‘sealing’ their ends. Chromosome notation There is an agreed nomenclature for describing the chromosomal constitution (Paris Conference 1971). The total number of chromosomes per cell is described using an Arabic number, followed by the sex chromosome constitution. If there is an extra chromosome, its number is preceded by ‘’ plus symbol. A missing chromosome is denoted by a minus ‘–’ symbol. Therefore, Down syndrome (trisomy 21 in a female) is notated as 47,XX21. A reciprocal translocation is denoted by ‘t’ followed by parentheses containing the number and arm of the chromosome involved. For example, t (13p; 18q) indicates the exchange of chromosomal material between the short arm of chromosome 13 and the long arm of chromosome 18. More complex structural rearrangements use the centromere as the point of reference and designate the terminal ends as ‘ter’; ‘pter’ being the end of the short and ‘qter’ the end of the long arm.

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22 21 15.2 15.3 15.1 p 14 13 11.2 12 11.1 11.1 11.22 11.21 11.23

q

21.1 21.2 21.3 22 31.1 31.2 31.3 32 33 34 35 36 7

Fig. 1.3 Chromosome 7 ideogram with division into dark and light bands. (See Plate 2 of the Colour Plate Section at the centre of this book.)

Chromosome abnormalities The frequency of chromosome abnormalities at birth is approximately 9.2 per 1000 (Jacobs 1992). Approximately 50–60 per cent of all spontaneous miscarriages and 8 per cent of all clinically recognized pregnancies have an underlying chromosomal anomaly. These may be numerical or structural abnormalities (Table 1.1). The gain or loss of chromosomal material is referred to as aneuploidy. Aneuploidy of the autosomes tends to be associated with significant learning and/or physical disabilities, whereas an extra sex chromosome (e.g. 47,XXX) tends to have less effect on the phenotype. Structural abnormalities may be ‘balanced’ or ‘unbalanced’. Balanced translocation carriers are, themselves, healthy, but are at risk of having a fetus with an unbalanced chromosome constitution, which may miscarry spontaneously, or be viable, with a significant risk of physical and learning disability. Table 1.1 Numerical and structural abnormalities Numerical abnormalities Structural abnormalities Trisomy Monosomy Polyploidy

Deletions Duplications Inversions Ring chromosomes Translocations Fragile sites

Mendelian inheritance Disorders caused by a defect in a single gene, the so-called ‘Mendelian’ disorders, follow a pattern of inheritance as described by Gregor Mendel. Traits or disorders determined by

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a gene on an autosome show autosomal inheritance. Those determined by a gene on one of the sex chromosomes show sex-linked inheritance. These single, or monogenic, disorders can be subdivided into the following. Autosomal dominant (AD) inheritance In autosomal dominant (AD) inheritance, the disorder manifests in the heterozygote state; that is, in a person who has both an abnormal copy of a gene and a ‘matched’ normal homologous copy. If an individual has an AD condition, each of their offspring has a 50 per cent chance of inheriting the abnormal gene, irrespective of the sex of the parent or the child (Fig. 1.4). Examples of AD disorders include autosomal dominant polycystic kidney disease, tuberous sclerosis, and myotonic dystrophy (Table 1.2). Variable expression In AD disorders, the clinical features can show striking variation from person to person, even within the same family. In conditions that are variably expressive (e.g. neurofibromatosis),

Affected father

Mother

D d

d d

D d

d d

D d

d d

Affected son (25%)

Normal daughter (25%)

Affected daughter (25%)

Normal son (25%)

Fig. 1.4 Segregation of autosomal dominant trait. Reprinted with kind permission of the publisher from Greenwood Genetics Centre (1995). Counseling aids for geneticists, Lippincott, Williams & Wilkins, Greenwood, USA. Table 1.2 Features of autosomal dominant inheritance Multiple generations affected Both males and females are affected with equal frequency and severity Transmission of the disorder from one generation to the next Male-to-male transmission observed Each offspring of an affected parent has a 50% chance of being affected and a 50% chance of being unaffected

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it can be difficult to establish the gene carrier status in apparently unaffected individuals if no direct gene test is available. Variable penetrance Most AD disorders are completely penetrant, with all gene carriers showing some clinical features; however, some conditions show incomplete or reduced penetrance, i.e. a proportion of heterozygotes for a dominant gene may not express the trait. This explains the presence of an apparently unaffected individual who has both a parent and a child with the same AD disorder leading to the impression that a disorder can apparently ‘skip’ a generation (Fig. 1.5). Variable penetrance may also result from the now well-recognized phenomenon of germinal (or gonadal) mosaicism, where an individual’s gonadal cells alone carry a mutation, whilst the somatic cells are normal. This explains the recurrence of a genetically dominant disorder in other children of unaffected parents (e.g. tuberous sclerosis). Penetrance is usually quoted as a percentage. For example, a figure of 90 per cent means that 9 out of 10 of all heterozygotes will exhibit some features of the condition.

I:1

I:2

II:1

II:2

II:3

III:1

III:2

Fig. 1.5 Autosomal dominant pedigree, showing a non-penetrant carrier.

Autosomal recessive (AR) inheritance Autosomal recessive (AR) disorders manifest in the homozygote state, that is, in an individual who has two copies of the abnormal gene (and no normal copy). If an individual has an AR condition, both of his or her parents will be heterozygotes, that is, usually healthy carriers of a single copy of the abnormal gene. If a couple are both carriers of an autosomal recessive gene, each of their offspring has a 25 per cent chance of inheriting a ‘double dose’ of the gene and, hence, being affected with the disorder. Each of their offspring also has a 50 per cent chance of being a healthy carrier and a 25 per cent chance of not inheriting the abnormal gene at all (Fig. 1.6). AR disorders are generally severe and are found in many metabolic conditions and complex malformation syndromes. Examples of autosomal recessive disorders include autosomal recessive polycystic kidney disease, Bardet–Biedl syndrome and cystic fibrosis (Table 1.3).

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Carrier father

Carrier mother

R r

R R

R r

R r

R r

r r

Normal

Carrier

Affected

(25%)

(50%)

(25%)

Fig. 1.6 Segregation of autosomal recessive trait. Reprinted with kind permission of the publisher from Greenwood Genetics Center (1995). Counseling aids for geneticists, Lippincott, Williams & Wilkins, Greenwood, USA. Table 1.3 Features of autosomal recessive inheritance Usually affects only one generation in a single sibship with unaffected parents Males and females affected with equal frequency and severity The recurrence risk for each offspring of carrier parents is 25% Higher incidence of consanguinity Certain recessive disorders are more common in specific ethnic groups, e.g. sickle cell disease and congenital Finnish nephrotic syndrome

X-linked recessive inheritance An X-linked recessive trait is one determined by a gene on the X chromosome and usually manifests itself in males only (Table 1.4). A disorder is transmitted by healthy female heterozygotes to affected males (Fig. 1.7). In each pregnancy, a woman carrying an X-linked recessive disorder has a ● ● ● ●

25 per cent chance of having an affected son 25 per cent chance of having an unaffected son 25 per cent chance of having a carrier daughter 25 per cent chance of having a non-carrier daughter

Rarely, a woman can exhibit signs of an X-linked recessive disorder, because of ‘skewed X-inactivation’. This X-inactivation, or Lyonization, occurs during early embryonic life and

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is random, but only one X chromosome is inactivated. If this inactivation is ‘non-random’ or ‘skewed’, and the X chromosome that remains active in the cells is the one that contains the abnormal gene, carriers of X-linked recessive traits may occasionally develop signs and symptoms of the disorder. Another cause of a manifesting female carrier is the presence of a structural abnormality involving the X chromosome, for example, an X-autosome translocation (the X chromosome which remains active in most of the cells is the one which contains the abnormal gene).

Father

Carrier mother

X Xr

X Y

X X

X Y

X Xr

Normal daughter

Normal son

Carrier daughter

(25%)

(25%)

(25%)

Xr Y

Affected son (25%)

Fig. 1.7 Segregation of X-linked recessive trait. Reprinted with the kind permission of the publisher from Greenwood Genetics Center (1995). Counseling aids for geneticists, Lippincott, Williams & Wilkins, Greenwood, USA.

Table 1.4 Features of X-linked recessive disorders Usually only males affected Maternally related affected males in more than one generation* No male-to-male transmission All daughters of an affected male are obligate gene carriers (and all his sons are unaffected) * A family history may not always be positive, as X-linked disorders have a high incidence of new mutations. This pattern of inheritance, therefore, needs to be considered in the event of an isolated affected male, as female relatives are at risk of being carriers and having affected males irrespective of whom they marry (Kingston 1998).

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Many X-linked recessive disorders are severe or usually lethal, so that affected males may not survive to reproductive age. The recognition of gonadal mosaicism in X-linked recessive disorders (e.g. Duchenne muscular dystrophy, DMD), means there is an appreciable risk of recurrence (10 per cent) in a situation of an apparently isolated case of DMD. X-linked dominant inheritance These rare disorders are determined by genes on the X chromosome and are manifest in the heterozygote female as well as in the hemizygous male. In some disorders, the effects on males are often so profound that the disorder is usually lethal. Examples include vitamin-Dresistant rickets and Incontinentia Pigmenti (Table 1.5; Fig. 1.8).

Table 1.5 Features of X-linked dominant inheritance Transmission through many generations Both males and females affected but with an excess of affected females Females usually less severely affected than males No male-to-male transmission An affected male transmits the disorder to all his daughters An affected female transmits the disorder to half her daughters and sons

I:1

II:1

male III:1

male III:2

II:2

male III:3

I:2

II:3

III:4

II:4

II:5

III:5

Fig. 1.8 Segregation of X-linked dominant trait. Reprinted with the kind permission of the publisher from Greenwood Genetics Center (1995). Counseling aids for geneticists, Lippincott, Williams & Wilkins, Greenwood, USA.

Y-linked inheritance Traits caused by genes on the Y chromosome (also referred to as holandric inheritance) are only transmitted from father to son. Hairy ears show this pattern of inheritance. In addition, the H–Y histocompatibility antigen and genes involved in spermatogenesis are carried on the Y chromosome. No pathological traits have been described with this mode of inheritance.

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Mapping disease genes Introduction Rapid advances in molecular genetic techniques for the detection of specific DNA sequences have led to an exponential increase in the discovery of human disease genes. This is reflected in the increasing number of genes being identified: 200 in 1986, with more than 9800 currently, with the numbers increasing rapidly and a total of 30,000 genes predicted to be identified on completion of the Human Genome Mapping Project (Cooper et al. 1998, HGMD April 2000 htpp://uwcm.ac.uk/uwcm/mg/hgmdo.html). This section deals briefly with some of the methods used for identifying genes. In lowresolution mapping, a gene is assigned to a particular chromosome or a specific region on a chromosome. More detailed analysis using high-resolution techniques can then be used to isolate the gene of interest. Restriction enzymes: site-specific DNA cleavage Restriction enzymes are endonucleases produced by bacteria. These enzymes, by recognizing specific short ‘palindromic’ DNA sequences, cleave double stranded DNA in a highly specific manner. They act like ‘molecular scissors’, with each particular restriction enzyme yielding the same reproducible double stranded DNA fragments. As change of a single base pair within a recognition site will abolish cleavage, these enzymes can also be used to detect variation in DNA sequence at a specific site. Some endonuclease rare cutter sites are often associated with regions of actively transcribed genes. Recognition of such sites can, therefore, be used as pointers to the sites of these genes. Linkage analysis The first step in identifying a disease gene is usually a linkage study, whereby the chromosomal location of the gene is established. Utilizing this approach, a gene is mapped by localizing its proximity to another nearby locus on the same chromosome. Such loci are said to be ‘linked’. Using polymorphic DNA markers, multiple affected family members are typed and the inheritance of the marker traced through the family until a marker is found which co-segregates with the disease (Fig. 1.9). For conditions with an autosomal pattern of inheritance,

2 3 3 1

1 3 2 2

3 5 1 3

3 5 2 3

4 5 3 3

3 5 1 3

Fig. 1.9 Pedigree showing linkage.

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this will include markers from chromosomes 1–22 and for X-linked diseases, from the X chromosome. A DNA marker of known chromosomal localization, segregating with the disease in multiple families, will identify the location of the gene; however, sometimes it can be difficult to identify a sufficient number of affected families to provide statistical power for the analysis. Linkage analysis relies on estimating the frequency of crossing over (or recombination) that occurs between homologous chromosomes at meiosis. A DNA marker that is not close to a gene on a particular chromosome (or is on a different chromosome) will be inherited independently of the gene. The closer a marker is to the gene, the less likely it is that the two will be separated during meiosis. In practical terms, markers that show less than 5 per cent recombination with a disease gene are useful in carrier detection and prenatal diagnosis. The statistical likelihood that two markers are linked is measured as the logarithm of the odds (LOD). A LOD score of 3 is usually taken as the threshold of significance, while a LOD score below 2 suggests that the two loci are not linked. LODs between 2 and 3 are not significant. Cytogenetic abnormality Some disease genes may be localized if an affected individual has a chromosomal aberration that points to a specific gene location. The astute clinical observation of such rare situations, where an individual has been recognized to have a monogenic disorder and also found to have a constitutional chromosomal abnormality has allowed the isolation of genes for a number of single gene disorders. Although individually rare, this is true for the positional cloning of several important genes in humans, including Duchenne muscular dystrophy (DMD). The DMD gene was localized to the short arm of the X-chromosome, following identification of several females with DMD, who also had an X-autosome translocation (a chromosomal rearrangement between the short arm of the X-chromosome and an autosome). Isolation of DNA clones spanning the region of the X-chromosome involved in the rearrangement provided detailed gene mapping information and the eventual cloning of the DMD gene (the genes for neurofibromatosis and familial polyposis coli were also identified in this manner (Ledbetter et al. 1989, Herrera et al. 1986)). Similarly, the occurrence of individuals with multiple abnormalities may be reflective of several closely occurring genes being involved. It was such a contiguous gene syndrome in an individual with autosomal dominant polycystic kidney disease and tuberous sclerosis that led to the identification of one of the polycystic kidney disease genes (PKD1) on chromosome 16 (Sampson et al. 1997, the European PKD Consortium 1994). Although individuals with a chromosomal abnormality and a single gene disorder are rare, their recognition is important, as this can often provide the first clue for gene mapping studies. Low-resolution mapping Somatic cell hybridization The technique of fusing cells from two different species has long been used as a gene mapping technique. A somatic cell is any cell of an organism that is not a gamete. Fusion of human with mouse or hamster cells is facilitated in the presence of a virus or chemical which causes membrane fusion. The resulting somatic cell hybrids, when grown in continued culture, show preferential loss of human chromosomes from the cells. This loss of chromosomes is random, with no selectivity for individual chromosomes. A panel of hybrid cells can, therefore,

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be produced with different combinations of human chromosomes. By karyotypically characterizing the hybrids, it is possible to identify which human chromosomes have been retained. Genomic DNA is prepared from the hybrid panel and hybridized with the human probe to be mapped. By determining which panel shows a hybridization signal, and comparing it with the karyotype of the cell, it is possible to infer which chromosome carries the gene of interest. In situ hybridization In situ hybridization is another means of mapping a gene. In this technique, a DNA probe is labelled with a fluorochrome and hybridized directly with a metaphase chromosome spread on a microscope slide. Using chromosome-specific probes, fluorescent in situ hybridization (FISH) can distinguish each human chromosome. FISH is particularly useful for determining the respective location of two markers on the same chromosome. By using two probes labelled with different fluorochromes, the technique can be used to order probes along a chromosome when they are separated by an order of one to several megabases (Fig. 1.10). Higher resolution, down to a range of 20–30 kb, is achieved by studying interphase nuclei, as in this phase, the DNA is less tightly packed.

Fig. 1.10 Fluorescent in situ hybridization (FISH). William syndrome is associated with a submicroscopic deletion in the long arm of one homologue of chromosome 7 in band 7q11.23. The deletion is not visible on G-banded chromosome preparations, but can be detected using FISH. (See Plate 3 of the Colour Plate Section at the centre of this book.) (The pictures were provided by C. Mackie Ogilvie and P. N. Scriven, Department of Cytogenetics, Guy’s Hospital, London UK.)

High-resolution mapping Once a gene has been mapped to a particular region of a chromosome, even with closely linked markers, the gene of interest may still reside in a region covering several million base pairs. There are a number of techniques that can be used to narrow down the region of interest and include the following.

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Pulse field gel electrophoresis (PFGE) Pulse field gel electrophoresis (PFGE) is a technique that permits separation of large fragments of DNA. The extraction of DNA for PFGE is done in situ in the gel, as conventional DNA extraction techniques can result in random shearing and fragmentation of the DNA. Using infrequently cutting restriction enzymes, which have 6–8 bp nucleotide recognition sequences, results in relatively larger DNA fragments. Following the application of pulsed, perpendicular electrical fields to the gel and electrophoresis, long stretches of DNA (several million base pairs in length) become separated as large molecules change direction less rapidly as they move through the agarose gel. The location of a gene within this long-range physical map can be determined by constructing physical maps of large stretches of DNA. Yeast artificial chromosomes (YACs) Yeast artificial chromosomes (YACs) are a cloning vector, which also allow the cloning of large segments of DNA. By inserting a specific segment of DNA into a yeast vector, milligram amounts of DNA can be prepared that will allow detailed analysis of gene structure. YACs can carry between 50 and 1000 kb of foreign DNA. By producing a series of overlapping YAC clone sequences, distal to the starting point, a probe can be defined. High-resolution FISH A technique known as chromatin fibre-FISH, which artificially stretches the chromatin fibres, can resolve distances down to several kilobases. Positional cloning Once a gene has been localized to a particular chromosomal region, it becomes possible to identify the gene by positional cloning. Even after narrowing the candidate region to a relatively small interval of 1 cM or less, there may be several genes that reside in the area. There are several mechanisms that can be used to identify the causative gene, even in the absence of any information about the gene product or its function. This approach has been possible since the 1980s and has accelerated rapidly with the mapping tools provided by the Human Genome Project. Candidate genes The availability of sequence databases means that once a disease is mapped to a particular chromosomal region, genes located in or near the region can be scrutinized closely as potential candidate genes. Confirmation or exclusion of specific mutations in the gene can then rapidly establish its status as the causative gene. This ‘positional candidate gene’ approach is rapidly replacing the longer gene mapping strategies. The time interval between mapping a locus to a chromosomal site and identification of the disease-causing gene and pathogenic mutations is considerably shortened as a result. Similarly, searching databases for genes already cloned and that may have functions likely to be involved in the pathogenesis of the disorder under consideration, may be another way of identifying a likely candidate gene. Observing a similar disease phenotype in another species, such as the mouse, may suggest other candidate genes and allows human–mouse homologies to be investigated. Computer databases There are a number of robust computer resources for accessing information about human genes. These include the following.

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1. Online Mendelian Inheritance in Man (OMIM) (http://www.ncbi.nlm.nih.gov/Omim): Lists and catalogues all known inherited human disorders and currently contains entries for approximately 7720 distinct genetic loci. 2. The London Dysmorphology Database (LDDB) and London Neurogenetics Database (LNDB) (http://www.lmdatabases.com): Contains information on over 3000 nonchromosomal multiple congenital anomaly syndromes with references and a photo-library on CD Rom. The companion LNDB covers clinical neurological features, including neuroradiology and neuropathology with comprehensive referencing. 3. Genome Database (GDB) (http://gdbwww.gdb.org): Contains information about genetic markers and location of known genes. 4. The Dysmorphology Human–Mouse Homology Database (DHMHD) (http://www. hgmp.mrc.ac.uk/DHMHD/dysmorph.html): Lists phenotypic features of human dysmorphic syndromes, mouse syndromes, and human cytogenetic abnormalities as well as having search facilities for human–mouse homologies. Functional cloning This approach correlates the basic biochemical defect with the causative gene. If the protein product of a gene has been characterized, the responsible gene can be identified using the cloning methods described (Fig. 1.11); however, this process has limited applicability, as there are only a minority of single-gene disorders where a biochemical defect has already been identified. Cytogenetic abnormality

Families Genetic markers

Finer genetic mapping

Physical mapping and cloning

Mutation search Transcript identification

Normal mutation

YACs and BACs Positional candidate approach

Fig. 1.11 Functional versus positional cloning. (Reprinted with the kind permission of the publisher from Mueller, R. F. and Young, I. D. (1998). Emery’s elements of medical genetics, Churchill Livingstone, London.)

Mutational analysis Once a disease gene has been identified, it must fulfil a number of criteria before it is confirmed to be responsible for that particular disorder. The first is to identify mutations in the candidate gene. A variety of mutation scanning methods have been developed (Grompe

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1994) and the mutation then requires confirmation by sequencing; however, any detectable mutation requires careful interpretation. For example, large deletions that remove part of the coding sequence of the gene, or sequence changes that are likely to have a functional effect by causing premature termination of translation by generating a stop codon, are likely to be particularly convincing. However, a missense mutation (which alters a single amino acid residue) may be a genuine mutation or a normal variant, the latter having no effects on protein function. The pathogenic credentials of an identified mutation are strengthened further if it is shown to co-segregate with the disease in the family, be absent in 100 ethnically matched controls and predicted to have a significant effect on the protein structure. Further support is provided if the candidate gene can be shown to be expressed in the appropriate tissues and at the relevant stages of development. In vitro restoration of the normal phenotype, by transfecting the normal gene in a cell line or the creation of a transgenic model by introducing the mutation in a homologous gene, adds further evidence for the relevance of a mutation. Genetic counselling Introduction Genetic counselling is a communication process that deals with the human problems associated with the occurrence, or risk of occurrence, of a genetic disorder in a family. It may also be defined as what happens when an individual, a couple or a family ask questions of a genetic counsellor about a medical condition or disease that is, or may be, genetic in origin (Clarke 1994). It involves an attempt to help the individual or family: ● ●

● ● ● ●

Comprehend the medical facts about a disorder. Appreciate the manner in which heredity contributes to the disorder and to the risk of recurrence. Understand the choices and options available for dealing with the risk of recurrence. Choose a course of action that seems most appropriate to them. Make the best possible adjustment to the disorder in an affected member. It has a strong communicative and supportive element.

Genetic counselling is practised within clinical genetics, a unique branch of medicine that draws upon the resources of high technology and applies them to the problems of those who are at risk of genetic disease, either in themselves or their offspring (Berry 1994). The goal of genetic counselling is to improve the quality of life of the families that seek such help (Twiss 1979). The ethos is for the professional to respond to the concerns of the clients and not to impose his/her own understanding on solutions to the problem. The manner in which the information provided might be received by a consultand and utilized in arriving at a decision, will depend on a number of factors. These include previous experiences, prior knowledge and understanding, beliefs, values and social obligations and a sense of personal identity (Clarke, A. 1997). Organization of clinical genetics services In some countries, regional genetic centres tend to be based in main teaching centres, with most holding central clinics on site, as well as peripheral or satellite clinics in other hospitals in their regions. Specialist laboratories providing molecular, cytogenetic, and biochemical services are an integral part of the provision of a clinical genetics service (Table 1.6).

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Table 1.6 The main sources of referrals to clinical genetic services Paediatricians Obstetricians General practitioners Community child health services Other hospital specialities Adoption agencies Self-referrals

Clinic staff The clinical team normally includes a number of health professionals, often with both NHS and academic affiliations. Apart from clinical geneticists and specialist registrars, genetic counsellors or associates are an increasingly important part of the clinical team. Many have a specialist nursing background, with others having a postgraduate qualification in genetic counselling in addition, or as an alternative, to a nursing qualification. Clinic environment The personal appointment, which is likely to last 40 min or longer, needs a quiet surrounding, conducive to the nature of the consultation, ensuring privacy and allowing ample time for discussions and questions. It is generally inappropriate for more than one observer to be present and then only with the permission of the family. The consultation An accurate diagnosis is the essential basis of genetic counselling. As in any field of medicine, reaching a diagnosis involves taking a history, carrying out an examination and, if appropriate, undertaking necessary investigations. Referral letters may contain varying amounts of information, which will require clarification. The standard way of recording genetic information is by constructing a family tree (pedigree). The main symbols/notations used and a sample pedigree are shown in Fig. 1.12. Specific enquiries pertaining to miscarriages, stillbirths, infant deaths, and consanguinity need to be made with sensitivity, as this information may not always be volunteered. A clinical examination may yield diagnostic information about the disorder in the individual seeking advice (e.g. neurocutaneous signs of tuberous sclerosis) and may require further investigation. An estimation of the genetic risk may be possible and is dependent on the pattern of inheritance of the disorder in the family. The availability of an accurate test and interpretation of the findings is obviously desirable when advising the family. It may be possible to quantify both the risk of developing and transmitting the disorder on the basis of the family tree, whereas specific tests may be required to identify gene carriers of other conditions (e.g. a renal ultrasound scan in an individual at risk of polycystic kidney disease). As most genetic diseases are individually rare, detailed literature searches and the use of computerized databases are often required. With the rapid pace of gene identification, conditions for which molecular tests were not possible until a few months ago may now be available. In many instances, it may be necessary to examine the affected person in the family or obtain the medical records.

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Shehla Mohammed Male

Female

Sex unknown

Pregnancy

Individual Key: example Affected individual

Colour blindness

Two or more conditions

cystic fibrosis

Multiple individuals

5

2

6

d.35 yr

d.4 mo.

d.1936

SB 30 wk

SB 34 wk

Number inside symbol

Deceased individual

Stillbirth SB 28 wk Consultand (individual seeking genetic counselling) Proband (First affected member coming to medical attention) P Obligate carrier (Will not develop condition) ∆F508

P

Record any information for example, known mutation under symbol

45,XX,t(14 : 21)

Spontaneous abortion 11wk Affected spontaneous abortion

8wk

Termination of pregnancy Affected termination of pregnancy Partnership Previous Twins

Consanguineous ?

Monozygotic Dizygotic Unknown (id i l) ( id i l) Fig. 1.12 Pedigree symbols and sample pedigree. (Reprinted with the kind permission of the publisher. From Bonthron D., FitzPatrick D., Porteus M., and Trainer A. (1998). Clinical genetics: a case-based approach, W. B. Saunders, London.)

It is prudent to establish whether there may be a current pregnancy in the individual seeking advice, or in a family member, who may be at genetic risk and to discuss the need and organization of family studies if appropriate. Who attends? The need for genetic counselling is well recognized for a number of common genetic disorders; however, the need for genetic advice in many other situations may be equally important.

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Table 1.7 Indications for referral to a genetics clinic Those who have had a child affected with a genetic condition Those who have a family history of a genetic condition Those who are, themselves, at risk of developing a genetic condition Investigation and diagnosis of possible genetic disease Diagnosis or family history of learning difficulties/physical handicap History of neonatal abnormalities or stillbirths Related couples, e.g. first cousins Investigation of recurrent pregnancy loss Advice regarding management of pregnancies at an increased genetic risk Interpretation of an abnormal prenatal result Those whose ethnic background indicates an increased chance of carrying a specific gene Pregnant couples/individuals who fall into any of the above categories

For example, a birth defect or learning difficulties can occur as part of a single gene disorder, chromosomal abnormality or from an interaction of environmental and genetic factors. Some of the indications for referral to a genetics clinic are outlined in Table 1.7. Information giving The nature and needs of the person requesting information need to be explored, so that the technical aspects of the condition can be communicated in an appropriate and sensitive manner, without resorting to jargon. Many genetic conditions, particularly those inherited in an autosomal dominant manner, show a variable phenotype. Portrayal of the variable nature of the condition is an important part of the counselling process. For example, an individual who may exhibit only mild signs of neurofibromatosis needs to be aware of the full range of potential manifestations, so that they can decide whether or not to take the 50 per cent risk of transmitting the disorder to future offspring, in whom it will be impossible to predict the severity of the disease. The diagnosis of a genetic disorder is often accompanied by feelings of grief, denial and/or anger in the individual or family and such feelings need to be acknowledged (see below). Follow-up arrangements Most genetic consultations are followed up with a summary letter, sent to the individual/ couple, as well as correspondence with the referring clinician. Any outstanding issues are covered in a follow-up appointment and, where appropriate, by a home visit. Concerns/issues for families The diagnosis of a genetic disorder can often be accompanied by considerable emotional stress and trauma for the family. The impact and long-term burden of a disorder need to be discussed, particularly the natural history and prognosis of the condition and its medical management, as well as the psychosocial implications. Inevitably, the lack of specific treatment and cure for most genetic conditions can be particularly difficult. For many families, an important question concerns the recurrence risk. If a clear diagnosis has been established, then talking to families about their risk is usually straightforward, at least mathematically (Table 1.8). For other families, however, the recurrence risk is less clear, either owing to the lack of a clear diagnosis, an unknown inheritance pattern or the unavailability of samples from other family members. In such situations, the best that may be available is an empirical risk. Many families are faced with making decisions about future reproduction, often not only in the light of worrying recurrence risks, but in the knowledge that a prenatal test may lead

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Shehla Mohammed Table 1.8 Offspring risk for carriers Inheritance

Risks to offspring

Autosomal recessive Autosomal dominant X-linked recessive

Very low (unless partner also a carrier) 50% (risk of overt disease may vary) 50% (if male offspring)

Table 1.9 Problems arising from genetic counselling Guilt and loss of self-esteem Anxiety regarding personal health Marital stress Family disruption and ill-feeling Risks to future children and grandchildren Ethical decisions

to difficult decisions about the future of the pregnancy. Prenatal diagnosis is available for an increasing number of genetic conditions; however, for many individuals, testing is not an option due to religious or personal reservations about termination of pregnancy. It is important for the clinician to remember this when dealing with families who may be considering extending their family in order to prevent assumptions being made and to ensure that all avenues are explored with the couple. For some, the answer lies in the use of other techniques such as donor insemination, ovum donation, or preimplantation genetic diagnosis (PGD). The geneticist is exposed to complex feelings of guilt and loss, which may take a variety of forms in numerous circumstances. As well as some of the predictable emotions experienced by those who pass on ‘disease causing’ genes to their offspring or experience the loss of an affected child or pregnancy, the guilt of the ‘survivor’ is often felt by family members who find that they do not carry the gene for the disorder in the family, whilst others experience a significant loss of ‘normality’ after learning they carry a ‘faulty’ gene (Table 1.9). Many families may find a specific genetic diagnosis helpful, whilst for others, a ‘label’ is associated with a negative experience of other peoples’ assumptions about what the condition will entail. This is particularly difficult for affected individuals where the clinical phenotype is widely variable or where there is a significant degree of public ignorance about the genetic condition in question. Professional concerns Although clinical genetics is often viewed as being at the forefront of a rapidly developing discipline, testing for genetic disorders brings with it many ethical dilemmas. For example, it is now possible to test a healthy individual for a late-onset incurable condition like Huntington disease. Whilst such testing can dispel uncertainty and enable individuals to make appropriate plans for the future, it can result in significant emotional turmoil and has implications not only for the individual seeking testing, but also for the wider family. These issues and those surrounding pre-symptomatic testing of children are covered in more detail in the Section on ‘Ethical issues and presymptomatic testing’. As already mentioned, the aim of genetic counselling is to provide information and to help support individuals and families to make choices. Most geneticists would view their roles as

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providers of information, rather than dispensers of definitive advice about emotive and highly personal issues (Berry 1994). This ‘non-directive’ approach can, however, prove difficult for some families who may request specific direction and advice from the counsellor. Providing an opportunity for careful reflection on different options available may be helpful in such situations. This is particularly important when there are reproductive issues to consider, with potentially irreversible decisions, such as sterilization. The inherent nature of genetic disorders means that there are often ramifications for the wider family. Varying family relationships and long-term estrangements in some families can sometimes lead to conflicting rights of individuals when access to genetic information is needed. Whilst many such situations can be resolved, it is worth remembering that while individuals have wider moral concerns for others with whom they have close ties, these are for the individual to consider, rather than for professionals to enforce (BMA 1995). In the meantime, geneticists must continue to remain sensitive to the interests of those who consult them as well as to their extended family (Berry 1994). Prenatal diagnosis Introduction Over the past three decades there has been a considerable increase in the use of prenatal diagnosis, the ability to detect abnormalities in an unborn child. Many couples facing a high genetic risk will consider embarking on a pregnancy only if a prenatal test is available. Until very recently, such couples would have had to choose between accepting the risk, having no further children or considering gamete donation or adoption. The availability of a prenatal test means that couples facing the risk of having a child with a genetic disorder have the option of preparing for the birth of an affected child and, if appropriate, opt for in utero treatment. It also enables them, if they wish, to choose termination of pregnancy. Equally, prenatal testing may be appropriate for low-risk pregnancies in the presence of parental anxiety. Apart from pregnancies that are at known genetic risk, some may be identified following the screening, which is often offered as a routine part of antenatal care. In many situations, as this is accepted with a view to ‘reassurance’ and with the expectation of a normal result, the detection of fetal abnormality can be particularly difficult to cope with. Many of the common developmental renal abnormalities may be detected in this manner. Specific fetal renal abnormalities are mentioned elsewhere, so that the general principles of prenatal diagnosis are outlined below. Indications for prenatal diagnosis There are approximately 100 genetic disorders with renal involvement (Winter and Baraitser 2001), which are not detailed here, but many are mentioned in relevant chapters. Broadly speaking, these comprise chromosomal disorders (e.g. trisomy 13, 18, and 21, Turner syndrome), Mendelian conditions (Alport disease, polycystic kidney disease, and tuberous sclerosis), and non-Mendelian or developmental disorders (Lowe syndrome, renal agenesis, Prune-Belly syndrome, and VATER association). Indications for prenatal diagnosis are summarized in Table 1.10 Methods for prenatal diagnosis A variety of techniques exist by which prenatal diagnosis can be achieved (Table 1.11).

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Shehla Mohammed Table 1.10 Indications for prenatal diagnosis Advanced maternal age Previous history of chromosomal abnormality Single gene disorder Neural tube defect Congenital structural abnormalities Other high risk factors: consanguinity, maternal diabetes

Table 1.11 Non-invasive techniques Technique type

Timing

Miscarriage risk (%)

Detection of abnormality

Non-invasive techniques Ultrasonography Nuchal scan

2nd trimester 11 weeks

No risk

Structural abnormalities Marker for chromosomal/ structural abnormality

Invasive techniques Amniocentesis

14–19 weeks

1

Chorion biopsy

11–12 weeks

2

Cordocentesis

2nd trimester

3

Chromosomal, biochemical disorders DNA, enzyme analysis, chromosomal disorders DNA, chromosomal, haematological disorders

Placental biopsy Fetoscopy

2nd trimester 2nd trimester

2–3 4–5 2–3

Fetal tissue sampling Viewing

Ultrasonography Fetal ultrasonography is an increasingly sensitive method for detecting developmental abnormalities. The sensitivity of the method is operator-dependent and increased in known highrisk cases than on routine screening (Chitty et al. 1991). Accordingly, the interpretation of an abnormality may not be straightforward and may require further detailed assessments in a specialist unit. Some of the urinary tract abnormalities detectable on scanning include: ● ● ● ● ● ● ●

Renal agenesis Hydronephrosis, secondary to pelviureteric junction obstruction or urethral valves Polycystic kidneys Dysplastic kidneys Bladder exstrophy Duplication abnormalities Horseshoe kidney

In addition to structural abnormalities, oligohydramnios or polyhydramnios may also indicate an underlying renal abnormality.

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Amniocentesis Amniocentesis is a widely available and long-established procedure, which is normally performed between 14 and 19 weeks gestation. Although a relatively safe procedure, it does carry a miscarriage risk, generally thought to be around 0.5–1 per cent. Around 20 ml of amniotic fluid is aspirated under ultrasonic control, following placental localization (Fig. 1.13). The fluid is normally clear and straw-coloured, with suspended amniotic (fetal) cells, which require culturing. A bloodstained sample indicates damage to the placenta, while discoloration may indicate fetal death. The sample is spun and duplicate cell cultures established which normally require between 2 and 3 weeks of growth to obtain sufficient cells. The main indication for amniocentesis is for chromosomal or biochemical analysis. Although generally thought to have a lower associated miscarriage risk, the timing of the test and results necessitates a second trimester termination in the event of a detectable abnormality. Probe Placenta

Uterus

Amniotic fluid Symphysis pubis

Sacrum

Cervix Vagina

Fig. 1.13 Amniocentesis.

Chorion biopsy/chorionic villus sampling Chorionic villus sampling (CVS) allows first trimester prenatal diagnosis, using fetal tissue obtained from chorionic villi. The sample is normally obtained around 11–12 weeks of pregnancy by a transcervical or transabdominal approach under ultrasonic guidance (Fig. 1.14). The maternal decidual tissue is dissected away microscopically, leaving villi, consisting of tissue that is representative of the fetus for analysis. Uncultured chorionic villi are the most suitable source of fetal DNA and can be used for molecular testing for any disorder for which such molecular testing is feasible. Chorion biopsy is also used for the detection of chromosomal disorders, either following cultured preparations, but increasingly, for rapid analysis using FISH. Prior to pregnancy Pre-implantation diagnosis Pre-implantation genetic diagnosis (PGD) is now becoming feasible. This technique relies on in vitro fertilization and embryo culture, followed by biopsy at the 6–8 cell stage of

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Shehla Mohammed Probe Speculum

Chorion

Cannula

Fig. 1.14 Chorion villus sampling.

embryonic development. Analysis of a single cell, by molecular testing or chromosomal in situ hybridization, is followed by the reimplantation of normal embryos. This method may be more acceptable to some, as it obviates the dilemmas of termination of pregnancy. However, the procedure is associated with a low rate of successful pregnancies because of the technical complexities and the need for IVF. Refinement of the techniques involved should enable PGD to be offered for many genetic disorders in the future. Prenatal treatment Experience of prenatal treatment is limited, but some cases of urinary tract obstruction are amenable to intrauterine treatment by the insertion of a vesicoamniotic shunt (Freedman et al. 1999). Termination of pregnancy The increasing availability of genetic and ultrasound technology can confer many benefits to those who face pregnancies with potential genetic problems with the relief that comes from a reassuringly normal result; however, the availability of such testing also presents couples and clinicians with often complex decisions. The detection of a serious fetal abnormality may be unexpected and the decision to terminate or continue the pregnancy is usually exceedingly difficult. Couples may experience disbelief, shock, and anger and it is, therefore, not surprising that decisions made in this frame of mind can be followed by doubts about the wisdom of such an emotive decision. After termination of pregnancy, grief and guilt may be compounded by low self-esteem and awareness of a personal contribution to the loss, particularly when there are painful reminders of other, continuing pregnancies in friends and relatives. Individuals or couples will need continuing help and support in coping with a loss that is ‘chosen’, but unavoidable. Regaining equilibrium takes time, with continuing sadness about the loss of a wanted pregnancy and anxiety about recurrence in the future (White van Mourik 1994). Professionals have a responsibility to understand the grief such pregnancy loss brings and to recognize the need for professional counselling if appropriate. Discussions about prenatal diagnosis should include preparation for an abnormal result. Although painful, it is appropriate

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to allude to the nature of termination that may be available if requested. This might entail a surgical first-trimester termination under an anaesthetic, but with a later termination, a woman would experience labour and delivery. It is worth remembering that whilst the majority of couples may opt for termination in the face of a serious abnormality, for some, continuation is a better option than termination, with nature being allowed to take its course. There is some evidence to suggest that those who choose this option have less guilt and distress than those who choose termination (Chitty et al. 1996). Ethical issues and presymptomatic testing Recent advances in medical genetics bring with them potentially huge benefits for those at risk of genetic disease, giving them greater control over their lives. All disciplines of medicine encounter ethical issues, but none more so than clinical genetics, which arise because a genetic diagnosis impinges not only on an individual, but also on the wider family. Inevitably, the benefits to one individual may generate conflict for other relatives who may be the unwilling recipients of information they had not sought. For example, an identical twin at 50 per cent risk of autosomal dominant polycystic kidney disease, by clarifying his/her own genetic status and receiving a positive result, will inevitably have established that his/her twin is also at risk, when this individual may not choose to have this information. Whilst such advances may offer more informed choices, it might bring burdens for others. The diagnosis of a lethal X-linked recessive disorder in a boy, impacts on his female relatives, who may not only have concerns for the health of their own sons, but may be faced with difficult reproductive choices. Some of the common situations where ethical issues often arise are those around informed consent, confidentiality, paternity, and pre-symptomatic testing. These are discussed briefly here, but are also the subject of many excellent reviews (Harper and Clarke 1997, Clark 1998, Marteau and Richards 1996). Informed consent There is general agreement that any form of testing or treatment in medicine is based upon the individual concerned giving free and informed consent. This necessitates the provision of accurate information, so that consent can be valid. In a research setting, the nature of consent is usually stipulated by ethical committees approving the research project. More recently, the issue of written consent has received a higher profile and it is now recommended that a consent form should be adopted as a means of formalizing and recording consent to share family information (Genetic Interest Group 1998). Initially, these will focus on molecular and other laboratory data, but eventually they may cover other aspects of the counselling session. Clinicians need to be aware that individuals may be consenting to a genetic test under pressure from partners, relatives or even other health professionals. Where there are grounds to suspect that undue pressure is being applied, there is a need to address and resolve these issues before proceeding with such testing. If it emerges that valid consent has not been given, testing should be postponed, at least until the conditions under which consent has been ‘given’ can be properly explored.

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Confidentiality Confidentiality has been a long-standing cornerstone of good medical practice. In clinical genetics, as testing one individual can have direct relevance to the extended family, difficulties and conflicts with regard to confidentiality often arise. As the ability to give accurate information to those seeking advice relies on an accurate diagnosis, access to medical records is often essential. Although, ideally, the release of medical information should require consent from the affected individual, in practice, this can be difficult. The affected person may have died or be incapable of giving consent. This may be compounded by the client’s wish not to disclose to other family members that they are seeking genetic advice, particularly if there is an ongoing pregnancy. At present, there is a degree of medical acceptance that information can be passed from one medical practitioner to another, but this may change in the future with the demise of medical paternalism (Berry 1994). In some situations, information may be deliberately withheld. Whilst most couples or individuals will recognize their responsibilities to other family members, others may have an immediate and understandable desire to keep the information a ‘secret’. It is unusual, however, for individuals to refuse to transmit information to relevant family members once they have had a chance to think through carefully the implications of not disclosing this information. Withholding information, or refusing to supply key biological samples, may reflect longstanding family disputes or a reluctance to exacerbate already strained relationships. In such situations, clinicians may have an obligation to override confidentiality and pass on such information. Reports from the Royal College of Physicians (1991), the Nuffield Council of Bioethics (1993), and the Genetic Interest Group (1998) suggest that there may be situations where the breaching of confidentiality is justified. Others argue against the principle of involuntary disclosure on the premise that it diminishes medical respect for genetic privacy and that the imposition of duty on families and professionals to pass on genetic information, rather than recommending it as good practice, would lend support to the concept of genetic harm (Clarke, A.J. 1997). Issues surrounding inadvertent disclosure can sometimes arise during the course of genetic testing. This is particularly likely if the laboratory needs to undertake linkage studies to answer a diagnostic or pre-symptomatic question, which will often require samples from both affected and unaffected family members. To avoid such studies revealing the genetic status of relatives who may have given samples only to assist with molecular studies, it is important to ensure that the laboratory only generates information that has been requested. With the advent of direct mutational analysis, such situations arise less often, but need to be borne in mind. Paternity issues Non-paternity is sometimes discovered during the course of a routine genetic test or is potentially recognized if there is a puzzling pedigree for a condition with a recognized inheritance pattern. This is particularly likely if linkage analysis is used which then shows lack of transmission of a genetic marker by the father. Results can sometimes still be given independent of paternity, but the reason for difficulty in interpreting results may well have to be discussed in confidence with the appropriate individual. Predictive testing Individuals at risk of a late-onset disorder, for which genetic testing is possible, now have the choice to clarify their own risk so that they can make appropriate life plans for the future. The

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extensive experience of pre-symptomatic testing for Huntington disease is now being adapted for use in other dominantly inherited late-onset disorders and testing for conditions predisposing to cancer susceptibility. As most disorders for which such testing is now possible are progressive in nature (and often untreatable), careful consideration is required of a number of issues prior to testing. Accordingly, protocols for such testing have been designed to include a minimum of two counselling sessions some months apart. Apart from covering the inheritance and medical facts about the disorder, these sessions also enable discussions about the reasons for requesting testing, ways of coping with the result, reviewing professional and social support, as well as practical implications for life insurance and careers. One important dilemma that is raised follows the request for testing by individuals who are at 25 per cent risk of being gene carriers, that is, those who have an affected grandparent, but the intervening parent remains healthy. By inference, the identification of a mutation in the individual at 25 per cent risk means that an inadvertent predictive result will have been made on the intervening parent, when they may not have wished to have this information. These situations can be resolved by appropriate discussions including all parties. If not, then a decision has to be made as to whether the right of the individual at 25 per cent risk to know their genetic status outweighs the right of the intervening parent ‘not to know’. Similar issues surround the pre-symptomatic testing of children. The genetic testing of children can be uncontroversial, for example, the identification of a particular faulty gene can lead to life-saving surgery in a child with a potentially lethal inherited condition. Equally, if a genetic test proves that a young person does not carry the disease-causing mutation for an inherited type of bowel cancer, the child is spared many distressing medical investigations. The central problems with the use of genetic testing in childhood lie in two main areas. First, in situations where the medical benefits of testing are unproven. Second, and perhaps most controversially, where the medical benefits of testing are clearly non-existent. In the latter situation, testing can only be justified if it will result in psychological or social benefit and, at the very least, do the child no harm. However, there is little evidence as to whether carrier testing is harmful or beneficial to the child or the family and research studies in this area are scarce. One central anxiety arises from potential damage caused by possible changes in the attitude of a parent towards a child who is identified as genetically ‘different’. Childhood testing raises many issues to do with parental rights and responsibilities. It also brings into question such issues as the autonomy of a child as a future adult and medical power in allowing or withholding tests requested by parents on behalf of their children (Clarke, A. 1997). References Berry, A. C. (1994). Genetic Counselling: a medical perspective. In Genetic counselling: practice and principles (ed. A. Clarke), pp. 1–28. Routledge, London and New York, NY. British Medical Association (1995). Human Genetics; choice and responsibility. Oxford University Press, Oxford. Chitty, L. S., Barnes, C. A., and Berry, A. C. (1996). Continuing with pregnancy after a diagnosis of lethal abnormality: experience of five couples and recommendations for management. British Medical Journal, 313, 478–80. Chitty, L. S., Hunt, G. H., Moore, I., and Lobb, M. O. (1991). Effectiveness of routine ultrasonography in detecting foetal structural abnormalities in a low-risk population. British Medical Journal, 303, 1165. Clarke, A. (1997). Introduction. In Culture, kinship and genes. Towards cross-cultural genetics (ed. A. Clark and E. Parsons). Macmillan Press, London and St Martins Press, New York, NY.

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Clarke, A. (1994). Introduction. In Genetic counselling: practice and principles (ed. A. Clarke), pp. 1–28. Routledge, London and New York, NY. Clarke, A. J. (1997). Challenges to genetic privacy. In Genetics, society and clinical practice (ed. P.S. Harper and A.E. Clarke). BIOS Scientific Publishers Ltd, Oxford. Clarke, A. J. (1998). The genetic testing of children. BIOS Scientific Publishers, Oxford. Collins, F. S. (1992). Positional cloning: let’s not call it reverse any more. Nature Genetics, 1, 3–6. Cooper, D. N., Ball, E. V., and Krawczack, M. (1998). The human gene mutation database. Nucleic Acids Research, 26, 285–7. Freedman, A. L., Johnson, M. P., Smith, C. A., Gonzales, R., and Evans, M. I. (1999). Long-term outcome in children after antenatal intervention for obstructive uropathies. Lancet, 354(9176), 374–7. Genetic Interest Group (1998). Confidentiality guidelines—Confidentiality and medical genetics. Grompe, M. (1994). The rapid detection of unknown mutations in nucleic acids. Nature Genetics, 5, 111–7. Harper, P. S. and Clarke, A. J. (1997). Genetics, Society and Clinical Practice. BIOS Scientific Publishers, Oxford. Herrera, L., Kakati, S., Gibas, L., Pietzrak, E., and Sandberg, A. A. (1986). Gardner syndrome in a man with an interstitial deletion of 5q. American Journal of Human Genetics, 25(3), 473–6. Jacobs, P. A., Browne, C., Gregson, N., Joyce, C., and White, H. (1992). Estimates of the frequency of chromosome abnormalities detectable using moderate levels of banding. Journal of Medical Genetics, 29, 103–8. Kessler, S. (1990). Current psychological issues in genetic counselling. Journal of Psychosomatic Obstetrics and Gynaecology, 11, 5–13. Ledbetter, D. H., Rich, D. C., O’Connell, P., Leppert, M., and Carey, J. C. (1989). Precise localization of NF1 to 17q11.2 by balanced translocation. American Journal of Human Genetics, 44(1), 20–4. Marteau, T. M. and Richards, M. (1996). The troubled helix-social and psychological impact of the new human genetics. Cambridge University Press, Cambridge. Paris Conference (1971). Birth defects original article series (1972) 8, 7: Paris Conference 1971: Standardization in Human Cytogenetics. National Foundation – March of Dimes, New York, NY. Sampson, J. R., Maheshwar, M. M., Aspinall, R., Cheadle, J. P., Ravine, D., Roy, S., et al. (1997). Renal cystic disease in tuberous sclerosis: the role of the polycystic kidney disease 1 gene. American Journal of Human Genetics, 61, 843–51. The European Polycystic Kidney Disease Consortium (1994). The PKD1 gene encodes a 14 kb transcript and lies within a duplicated region of chromosome 16. Cell, 77, 881–94. Twiss, S. B. (1979). Problems of social justice in applied human genetics. In Genetic counselling: facts, values and norms (ed. A. M. Capron, R. F. Laper, T. M. Murray, T. M. Powledge, and D. Bergsma). Alan R. Liss, Inc., New York, NY. White van Mourik, M. (1994). Termination of a second trimester pregnancy for fetal abnormality— psycho-social aspects. In Genetic counselling: practice and principles (ed. A. Clarke), pp. 1–28. Routledge, London and New York. Winter, R. and Baraitser, M. (2001). Oxford Medical Database. Version 3.0. www.oup.com/uk/omd

Suggested reading Abramsky, L. and Chaple, J. (1994). Prenatal diagnosis—the human side. Chapman and Hall, London. Strachan, T. and Read, A. P. (1999). Human molecular genetics. BIOS Scientific Publishers Limited, Oxford. Gardner, R. J. M. and Sutherland, G. R. (1966). Chromosome abnormalities and Genetic Counselling. Oxford University Press, Oxford. Kingston, H. M. (1996). ABC of Clinical Genetics, Chapter 1. BMJ Publishing Group, London.

2 Renal function and management of renal disease A. David Makanjuola and John E. Scoble

Introduction The kidney inspires awe in all those to attempt to understand its functions. The concepts of its actions are simple. Blood is passed through a filter which has some specific size and charge characteristics. This produces an ultrafiltrate which is then reabsorbed. Figure 2.1, shows a physician and his glomerular filtrate to his left. It can immediately be seen that with normal function we each filter more than twice our body weight each day. In fact, the physician holds the urine volume which is less than 2 per cent of the amount he has filtered in a 24-h period. Conceptually, it has been easy to imagine a pump such as the heart involved in work of expelling a certain volume every minute. However, because the kidney is silent in its functions it is more difficult to imagine the ‘work’ involved in the reabsorption of 178 kg of fluid

Fig. 2.1 A physician and his glomerular filtrate. This shows a renal physician on the right with peritoneal dialysis fluid of 180 litres which is equal to his glomerular filtrate, and he holds in his hands 2 litres which represents his urinary output. This shows both the volume filtered and the work performed by the kidney each day and the small proportion which results in urine production.

A. David Makanjuola and John E. Scoble

Kidney function (%)

28

100 90 80 70 60 50 40 30 20 10 0 0

20

40 60 Time (min)

80

100

Fig. 2.2 Renal function and symptoms. Patients rarely exhibit symptoms until they enter the grey zone below a GFR of 20 ml/min but as soon as the GFR passes below 10 ml/min it is potentially lethal.

under normal conditions. However, the vast volumes that are filtered and reabsorbed suggest the important impact that small changes in the reabsorptive processes can have in producing pathophysiological changes. At the same time, it can be appreciated that significant changes in excretory function can occur without a change in urine volume or significant symptoms. Symptoms rarely occur until the overall renal function has decreased to less than 20 per cent whereas this fall in function for the heart would result in severe and disabling symptoms, Fig. 2.2. This results in many patients with progressive renal disease of whatever cause presenting as a medical emergency with less than 10 per cent of normal renal function. In practice, the planning of renal replacement therapy in many patients with genetic renal disease occurs when their glomerular filtration rate does not give them any symptoms. Renal physiology The kidney provides a highly efficient mechanism for waste product excretion which cannot be reproduced by even the most modern dialysis mechanisms. This is because it filters an enormous volume of plasma per day and has exquisitely effective mechanisms for the reabsorption of all but the smallest amount. Figures 2.3–2.8 show that the functions of the kidney can be divided into simple parameters. The proximal tubule is an isotonic reabsorptive segment with the consequent sodium transport process and water permeability. However, the thick ascending limb is a sodium reabsorbing segment without water permeability. As will become clear with the subsequent chapters, these specific mechanisms are the consequence of specific transport proteins which have biological variation. The glomerular filtrate is the consequence of the hydrostatic forces within the glomerulus which is a hemi-arteriole and the physical characteristics of the glomerular basement membrane. In certain pathophysiological situations these forces can be altered by extrinsic factors. For instance, in the renovascular disease associated with neurofibromatosis, the decreased afferent arteriolar pressure can be offset by increased angiotensin II production and increased efferent arteriolar tone. However, once the filtrate is formed it is under the control of the tubular reabsorptive mechanisms.

Renal function and management of renal disease

H2O permeable H2O permeable + Na transporting H2O permeable + Na transporting +/– H2O permeable + Na transporting

29

Fig. 2.3 The renal tubule and the function of the individual segments with the hypertonicity of the inner medulla generated by the thick ascending limb.

H+ Na+ Gl Na+

ATP K+ Na+

Phos Na+ AA Na+

Cl– HCO3–

H2O

Fig. 2.4 The proximal tubular cell and its transport mechanisms.

Loop diuretics

ATP K+ Na+

Na+ K+ 2Cl– Cl– K+

Fig. 2.5 The thick ascending limb and its transport mechanisms.

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Thiazide diuretics

ATP K+ Na+

Na+ Cl– Cl–

Fig. 2.6 The early distal tubule and its transport mechanisms.

ATP K+

Na+

Aldost.

Na+ K+

Principal cell

Cl– Band 3 protein

ATP

ATP K+ Na+

H+ Intercalated cell

Fig. 2.7 The distal tubule and its transport mechanisms.

Although the array of reabsorptive mechanisms is enormous when individually analysed the number of processes in each individual segment of the renal tubule is small as shown in Fig. 2.2. Figures 2.3–2.8 show the mechanisms which are important in the individual segments of the renal tubule.

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ADH cAMP

H2O

Fig. 2.8 The collecting tubule and its transport mechanisms.

The proximal tubule is responsible for bulk reclamation of the ultrafiltrate. This is achieved by sodium transport with anion transport of bicarbonate (Fig. 2.4). Also obvious from Fig. 2.4 is the fact that all the substances which require absolute reabsorption under normal circumstances have sodium co-transporters. Thus glucose and amino acid reabsorption is linked to sodium reabsorption. Although previously thought to depend on paracellular water transport, it is now clear that water movement is through the aquaporin 1 (CHIP) water channels through the proximal tubular cell (Yamamoto and Sasaki 1998). It is important to note that at the end of the proximal tubule the tubular osmolality is unaltered even though twothirds of the filtered fluid volume has been reabsorbed. In spite of bicarbonate reabsorption there is little alteration in tubular pH. Thus the proximal tubule reabsorbs the precious commodities and two-thirds of the salt and water without altering tubular fluid osmolality or pH. At the end of the proximal tubule, the tubular fluid osmolality is unchanged at approximately 300 mosm/kg. The tubular fluid pH is also unchanged. Thus bulk fluid reclamation has occurred with specific reabsorption of essential moieties. Tubular fluid then descends to the thin ascending limb to the centre of the osmotic gradient and the tip of the renal medulla. In humans, the interstitial osmolality reaches 1200 mosm/kg we believe. As can be seen from Fig. 2.3 the interstitial osmolality increases with the nearing of the tip of the medulla. In the thin ascending limb, there are no active transport mechanisms but at the same time this segment is entirely permeable to both salt and water. However, as can be seen from Fig. 2.5 in the thick ascending limb there are active reabsorptive mechanisms. At this point there is the NaK/2Cl co-transporter. This segment is also known as the diluting segment. Because, as is shown in Fig. 2.4, in this segment the tubule is impermeable to water, any electrolyte reabsorption results in a decrease in the tubular fluid osmolality. The maximum that a human can achieve is about 70 osmo/kg. As can be seen there is a leak back of K into the tubular fluid via the renal outer medullary K (RomK) channel (Kurtz 1998). The early distal tubule, Fig. 2.6, has a sodium reabsorptive transporter in the Na/Cl co-transporter. It is unclear at the present time how this transporter is related to calcium and magnesium reabsorption (Kurtz 1998). However it is clinically apparent that ‘Loop diuretics’ which inhibit the NaK/2Cl co-transporter also result in a loss of urinary calcium and magnesium. It is also clear that thiazide diuretics which inhibit the Na/Cl co-transporter are also associated with both an increase in urinary magnesium excretion but also an increase in calcium reabsorption. This has been used as a diagnostic test for hyperparathyroidism.

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If a patient’s plasma calcium increases on a thiazide diuretic, then this is seen as a diagnostic criterion for primary hyperparathyroidism. The later distal tubule (Fig. 2.7), is the segment responsible for pH alteration and also for potassium regulation. This is a segment which was recently thought to possibly be responsible for essential hypertension (Stokes 1999). The transport in this segment revolves around the Na channel. Abnormalities of this may result in Liddle syndrome (Stokes 1999) but are also physiologically controlled by aldosterone and thus abnormal in Addison disease and Conn syndrome. It is also the segment responsible for acidification of the urine which is achieved by the H-ATPase. Abnormalities of this or the basolateral HCO3/Cl can result in a deficiency of urinary acidification, distal renal tubular acidosis. It is also clear that extra sodium reabsorption, as in Conn syndrome, will result in hypertension and because of the resultant intraluminal positive electrical gradient, an alkalosis and hypokalaemia. The intriguing feature is whether minor defects short of those in Liddle syndrome might be responsible for essential hypertension. The collecting tubule’s essential function is water homeostasis. The inner medullary hypertonicity is connected to the contents of the collecting tubule if there is water channel insertion in this segment. In the absence of water channel insertion, there is the syndrome of diabetes insipidus. Classically, this is due to the lack of pituitary derived vasopressin. However, this syndrome can also be produced by defects in the effector mechanisms in the collecting duct in nephrogenic diabetes insipidus. Various genetic abnormalities of the insertion of the water channels have been described as well as damage due to drugs such as lithium (Bichet 1998). Figure 2.3 shows the properties of the various segments of the renal tubule with regard to a small number of parameters. The proximal tubule is sodium transporting and water permeable. The thick ascending limb is sodium transporting but water impermeable. The collecting duct is variably water permeable. The action of the renal tubule is dependent on the insertion and action of a small number of transport proteins. It is not surprising that genetic defects in these result in a large number of clinical syndromes.

Non-excretory renal function Although the kidney is an organ of excretory function as well as controlling the fluid status of the individual there are a number of other functions. The kidney is the site of erythropoeitin production (Macdougall and Eckardt 1998). The association of anaemia and renal failure has long been recognized. There have been many suggestions as to the basis metabolic problem ranging from a uraemic toxic effect on the bone marrow to gut iron loss from the prolonged bleeding time in renal failure. It was also recognized that in certain forms of renal disease anaemia was less common. In autosomal dominant polycystic kidney disease (APKD) even patients on dialysis could have well-maintained haemoglobins. The introduction of the recombinant erythropoeitin and its dramatic effect in correcting the anaemia seen in patients with renal failure showed that this was the most important defect. It appears that in adults erythropoeitin production occurs in cells within the interstitium. The importance of this site for production is unclear but the relatively large blood supply to the kidney may be a reason. However, it is clear that as renal function deteriorates in most forms of renal disease then anaemia is an absolute consequence. It is now routine practice to start patients on erythropoeitin prior to dialysis if they are iron-replete and anaemic.

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33

The kidney is the site of the hydroxylation of 25-hydroxycholecalciferol to the active metabolite 1,25-dihydroxycholecalciferol. This is vitally important to calcium phosphate homeostasis and its lack leads to the complex process of renal osteodystrophy. Activation of the hepatically produced 25-hydroxycholecalciferol occurs in the proximal tubular cells. With the decline in renal function the lack of 1,25-dihydroxycholecalciferol produces significant changes in calcium homeostasis. Bone matrix calcification fails with the increase in uncalcified osteoid resulting in osteomalacia. Gut absorption of calcium is reduced leading to low plasma calcium levels. At the same time 1,25-dihydroxycholecalciferol has a direct effect in inhibiting the release of parathyroid hormone from the parathyroid cells. The low plasma calcium levels and the lack of negative feedback result in the increase in parathyroid hormone excretion. In health, this hormone has many effects including the specific stimulation of the 1-hydroxylase in the proximal tubular cell to increase production of 1,25-dihydroxycholecalciferol. As with erythropoeitin production synthetic 1,25-dihydroxycholecalciferol can be administered to patients with renal failure and abrogate the hyperparathyroidism seen in endstage renal failure. The kidney is also the organ of metabolism of all the small peptide hormones. These are filtered and degraded in the brush border of the proximal tubular cell. The most clinically important effect of this is in diabetes mellitus where a consequence of progressive renal impairment is a decreased insulin requirement. This may mean that the first sign of renal impairment in a stable diabetic is the new occurrence of hypoglycaemic attacks. Most small polypeptides are found in high concentrations in patients on haemodialysis. The kidney is intimately involved in acid–base homeostasis. The predominant organ which controls acid–base homeostasis however is the liver (Atkinson and Bourke 1987). This alters the degradation of amino acids to produce either urea which does not lead to bicarbonate generation or glutamine which can be metabolized in the proximal tubule to generate bicarbonate. This results in bicarbonate which can be reclaimed to the circulation and excretion of ammonium into the urine. A consequence of this metabolic function is the production of Acetyl CoA. This is then synthesized into glucose. The kidney is the only organ other than the liver to have the property of gluconeogenesis. The common fallacy that metabolism results in the net generation of an acid load although much discussed in older text books can no longer be justified (Atkinson and Bourke 1987).

Investigations of renal disease The investigations of renal disease can be divided into two avenues. The first is the gross anatomy and function of the kidney. The second is the renal parenchymal architecture and includes examination of the renal histology. Imaging procedures can provide data on the size and configuration of each kidney. These are shown in Table 2.1. The advantages and disadvantages of the various techniques are shown. Each technique provides complementary information about the renal vasculature as well as renal parenchyma. At present, there are no techniques which provide all the information required. An example of the change in practice is APKD. Previously, radiological investigations involving the use of radiocontrast agents and radiation were required to diagnose this condition. However, with the advent of ultrasound it is now simple and non-invasive to diagnose this condition after the age of 20 years. Figure 2.9 shows the detail a CT scan can

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A. David Makanjuola and John E. Scoble

Table 2.1 Imaging procedures for determining kidney size and configuration Technique

Advantage

Disadvantage

Plain abdominal film

Simple Low X-ray dose Good delineation of collecting system and ureter Non-invasive Good delineation of intrarenal lesions and dilatation Excellent delineation of renal artery

Only relevant in radio-opaque renal stone disease Contrast load Poor delineation of intrarenal lesions Poor views of ureter Operative dependent

Intravenous urogram Renal ultrasound Intra-arterial angiography Spiral CT scan MRI angiography

No intra-arterial instrumentation Three dimensional images of renal vasculature No contrast load

DTPA scanning DMSA* scanning

Dynamic view of renal function Static view of functional renal tissue

*DMSA  Dimercapto

Contrast load dependent on angle of view Intra-arterial instrumentation Contrast load Over-interpretation of lesions Patient compliance Dependent on initial bolus Not related to renal blood flow

succinic acid.

Fig. 2.9 A CT scan of a patient with both polycystic kidney disease and a horseshoe kidney.

Renal function and management of renal disease

35

A

B

Fig. 2.10 Intra-arterial angiography, A: in the same patient compared with MRI angiography, B: in between the first and second scan the patient had occluded his aorta below the level of the renal arteries.

provide in APKD with the congenital variant of a horseshoe kidney. Figure 2.10 shows the use of magnetic resonance imaging (MRI) in atherosclerotic renal artery stenosis compared with conventional angiography in the same patient. It is apparent that at present a number of investigations will provide anatomical clarity of the renal tract. With time those investigations which provide the greatest clarity with the least radiation exposure will become the most widely used.

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A. David Makanjuola and John E. Scoble

The advantages and disadvantages of the various tests to determine renal function are shown in Table 2.2 (Cameron and Greger 1998). The nuclear medicine investigations do not provide excellent anatomical clarity but do reflect renal function. The diethylene triamine penta acetate (DTPA) nuclear medicine scan can show obstruction to urinary flow. It can also confirm renal artery stenosis when combined with administration of an angiotensin converting enzyme inhibitor (ACEI) such as captopril as shown in Fig. 2.11. Other parameters which are important include 24-h urinary protein excretion. Specific investigations relating to the Table 2.2 Techniques for determining renal function Technique

Advantage

Disadvantage

Plasma creatinine

Simple Single test Estimation of glomerular filtration rate Cheap No radiation Single blood test Precise Not dependent on complete urine collection Precise Not dependent on complete urine collection No chemical assay

Dependent on muscle mass

Creatinine clearance

Inulin clearance Cr–EDTA clearance

Incomplete collection common

Needs multiple blood tests Needs specific laboratory analysis Radioactive Needs multiple blood tests

Fig. 2.11 The DTPA scan shows the effect of a single administration of 25 mg of the ACEI captopril on excretion in a patient with bilateral renal artery stenosis. This shows that the renal artery stenosis was critical to function. The patient had ‘flash pulmonary oedema’ and was cured by bilateral renal angioplasty.

Renal function and management of renal disease

37

disease causing the renal disease will be important for patient management but do not relate to renal function. In many conditions after the initial diagnosis is made the most important parameter is the plasma creatinine as this is an imperfect but reproducible measure of renal function. However, this does presume that creatinine is filtered and neither excreted nor reabsorbed by the renal tubule. This may not be true in situations of low renal function. In normal clinical practice, the plasma creatinine is used as a simple measure of renal function. However, as the relationship between plasma creatinine and creatinine clearance is a reciprocal one it can be difficult to grasp the changes in renal function. For instance, every time the plasma creatinine doubles the creatinine clearance halves. For an individual with a creatinine clearance of 100 ml/min, a plasma creatinine of 100 mol/l then a rise of plasma creatinine to 200 mol/l represents a halving of renal function. That is a loss of 50 ml/min. If, however, the creatinine then doubles to 400 mol/l this only represents the loss of a further 25 ml/min of glomerular filtration rate. The use of the creatinine clearance will provide a measure of glomerular filtration but incomplete 24-h urinary collection will lead to underestimation of renal function. The most specific measures of renal function use either inulin clearance or Cr–EDTA clearance (Maisey and Britton 1998) as shown in Table 2.2. These techniques require repeated blood samples and in the case of Cr–EDTA use of a radionuclide. This is because most renal disease is bilateral and affects both kidneys equally. The new technique of single kidney glomerular filtration rate has proved useful in asymmetrical renal disease (Scoble and Cook 1998). The measurement of overall renal function can be achieved by a number of techniques. These tests are applicable as most causes of renal dysfunction affect both kidneys equally. Table 2.2 shows the advantages and disadvantages of each. However, the difficulty with this parameter is that it does not correlate with symptoms until very late in the disease course, Fig. 2.2. Newer investigations such as MRI may provide non-invasive imaging in conditions affecting the renal vasculature as well as functional assessment in a single test. The single test which will provide evidence of specific changes in the glomerular or tubular compartment is the renal biopsy (Ponticelli and Mihatsch 1998). This technique is universally used and provides very precise recognition of patterns of renal disease. There is however a risk of bleeding produced by the biopsy and rarely nephrectomy can be required. The risks of bleeding increase with the worsening of renal function as there is prolongation of bleeding time with significant renal dysfunction. A single histological pattern may result from a number of different causes. For instance focal glomerulosclerosis can be related to a familial condition, renal artery stenosis, gross obesity, or an idiopathic cause! The importance of the various patterns of renal histology will be discussed below but this investigation remains the cornerstone of the investigation of most forms of renal disease.

Management of renal disease The major issue with renal physicians is that symptoms with renal disease are rare below a glomerular filtration rate (GFR) of 30 ml/min, Fig. 2.2. It is unclear why cardiac disease will produce symptoms at marginal changes in function whereas in renal disease the vast majority of renal function has to be lost before the patient feels any abnormality. This explains why

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A. David Makanjuola and John E. Scoble

in a number of conditions abnormalities of urine testing, such as haematuria in Alport syndrome, are common presentations of renal disease. Although changes in glomerular filtration present late, changes in tubular electrolyte handling will present much earlier as is seen in Bartter syndrome. The management of renal disease can be divided into the general aspects of the management of a patient with a reduced glomerular filtration rate and the specific management of the pathological processes in a specific condition. There are a number of conditions such as focal and segmental glomerulonephritis where there are a number of aetiological factors including a genetic element where the general management is the same irrespective of the cause. There are also a number of conditions where a genetic element in the aetiology has only recently been recognized such as reflux nephropathy. Presumably, in the future there will be a number of idiopathic conditions described here which will be found to have a genetic element. The general management of patients with renal disease has evolved from simple dietary restriction or alteration to focus on factors which may lead to progression (Locatelli and Del Vecchio 2000). The most important of these is the management of hypertension (Dworkin and Weir 2000). Renal disease can obviously cause hypertension and hypertension can cause renal disease. In its most severe form hypertension can cause acute dialysis dependence which may not be reversible. However, it is now clear that in all forms of renal disease, control of hypertension is the single most effective treatment to halt the progression. The work that has established this has come from the investigation of patients with juvenile onset diabetes mellitus. The work of Parving et al. (1983) has shown that tight control of hypertension can cause a decrease in the rate of decline of renal function. This group of patients has been the most easy to study given its homogeneity. The results appear applicable to all forms of renal damage including post-transplantation. In the clinical setting, for conditions such as APKD where there are no specific ameliorative gene therapies at present, we can offer the patient the possibility of altering the rate of decline of renal function by tight blood pressure control. This may however mean the use of multiple agents to achieve the blood pressure guidelines equal to diabetic patients of 130/85 which are now advised. The work of Lewis et al. (1993) clearly showed that for patients with juvenile onset diabetes mellitus (JODM) there was an additive effect of the use of ACEI to decrease the reaching of a combined end-point of death or renal failure. The important feature to note is that in this trial this was achieved against a placebo group with good blood pressure control. A subsequent study in a large group of patients with maturity onset diabetes mellitus (MODM) has suggested that control of blood pressure with or without an ACEI is the most important therapeutic manoeuvre (UK Prospective Diabetes Study Group 1998). However, other studies have suggested that in renal disease the use of an ACEI in the management of all forms of progressive renal disease does confer a beneficial outcome (Maschio et al. 1996). In proteinuric states ACEI will also beneficially reduce the proteinuria in an effect separate from control of hypertension which has been linked to the reduction in intraglomerular pressure. In all forms of renal disease, the management of hypertension is a primary aim of therapy and it is probable that in most forms the use of an ACEI will also be beneficial. The general management of renal disease will also include dietary manipulation. There has been considerable experimental evidence from animal models of a beneficial effect of protein restriction on the progression of renal disease. However, in clinical practice it is difficult to severely protein restrict many of our patients. The results of a large trial have been disappointing in not showing an overall effect of protein restriction in the progression of renal

Renal function and management of renal disease

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disease (Klahr et al. 1994). However in individual patients control of protein, potassium, and phosphate intake will prolong the time to dialysis. Irrespective of the progression of renal disease, these individual parameters may require urgent dialysis in individual patients. General management of patients with progressive renal disease now include measures to improve symptomatology. The use of erythropoeitin prior to dialysis is now widespread in patients approaching end-stage renal failure. At the same time, the use of 1,25-dihydroxycholecalciferol or an analogue will also be routine practice in pre-dialysis patients to prevent renal osteodystrophy. However, at present there are no routine measures to try to prevent the extremely high initial cardiovascular mortality of patients entering the end-stage programme compared with age matched controls (Parfrey and Foley 1999). This is a frequent cause of both morbidity and mortality in this group of patients. It is important to state that the 5-year survival of patients over the age of 64 years entering a dialysis programme is worse than similar patients with colonic cancer (Parfrey and Foley 1999)! The management of renal disease in specific conditions will depend on the condition. The causes of renal disease can be simply analysed as pre-renal, parenchymal renal, and post-renal disease. Pre-renal disease Pathological processes affecting the renal artery or more rarely the renal venous supply will effect renal function. The causes of renal artery disease are shown in Table 2.3. The various conditions are very disparate. The original work of Goldblatt et al. (1934) suggested that the relief of renal artery narrowing will cure the hypertension the renal ischaemia induces. A major focus on the management of these conditions is to re-establish the normal blood flow to the kidney. Initially the early reports were of nephrectomy or renal artery surgery. The introduction of renal angioplasty and more recently renal artery stenting has meant that renal revascularization can be achieved without a significant surgical operation. However, the use of this procedure has shown how complex the processes involved in renal artery narrowing are. It is clear from the artery reports that acute renal artery occlusion can be tolerated with the recovery of renal function even after anuria. The situation in chronic renal artery narrowing is less clear. In conditions where there is a systemic component such as Takayasu syndrome or atherosclerotic management of the non-renal manifestations may be most important in patient outcome. The group also represents a heterogeneous pathology of renal disease. The cure of hypertension was an early goal and the use of angioplasty has produced good results in fibromuscular dysplasia even in the early reviews of data. However, later studies have shown a less good result for atherosclerotic disease. The results from intervention in atherosclerotic disease which is the most common in the UK and the USA suggest a variable response with only about a third of patients benefiting from intervention. The medical management of these patients is made more difficult by the inability to use ACEI as this causes a decline in renal function. The major causes of renal vascular disease are discussed. Fibromuscular dysplasia This condition appears to affect the renal arteries and less commonly other territories including the carotid and iliac arteries (Stanley 1995). It has a predominantly female bias in

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A. David Makanjuola and John E. Scoble Table 2.3 Causes of renal artery diseases Renal artery occlusion Acute Embolus from central source; thrombus, bullet Trauma Acute on chronic with pre-existing renal artery stenosis, e.g. with ACEI inhibitors and diuretics Renal artery aneurysm Aortic dissection Aortic occlusion and retrograde thrombosis leading to renal artery occlusion Secondary to intervention e.g. angioplasty Clotting disorder Antiphospholipid antibody syndrome Acute vascular transplant rejection Spontaneous Chronic Atherosclerotic Fibromuscular dysplasia Takayasu disease Middle Aortic syndrome Renal transplant renal artery stenosis Renal vein occlusion Acute Nephrotic syndrome Clotting disorders Antiphospholipid antibody syndrome Secondary to intervention e.g. renal venography or surgery Trauma Inferior vena cava occlusion Acute vascular transplant rejection Spontaneous Chronic Nephrotic syndrome especially membranous glomerulonephritis Tumour Retroperitoneal fibrosis Veno occlusive diseases

incidence and is also very rare in certain ethnic groups such as the Afro-Caribbean population. Its presentation is of hypertension at an early age and must be suspected in malignant hypertension at an early age. It appears that the lesions progress rarely in later life although the long-term data are unclear on this aspect. A decline in renal function appears also to be rare although patients may present with another manifestation of renal artery disease, acute unprovoked pulmonary oedema. Management of this condition will include control of hypertension as well as angioplasty. In a number of patients the renal artery lesions may be very distal, and require bench surgery and auto-transplantation. There are no systemic manifestations of this disease as is the case of the rarer condition of the Middle Aortic syndrome.

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Takayasu syndrome This condition was first completely described during the Second World War in Japan. It is an arteritis affecting the aorta and major arteries resulting in its description as the pulseless disease. The renal artery lesion is progressive and can lead to dialysis requirement. There are manifestations of systemic disease with a raised erythrocyte sedimentation rate (ESR). The management of this condition involves the suppression of the arteritis with steroids and then the revascularization of the arterial tree affected. Renal angioplasty has been effective and more recently it has been suggested that mycophenolate mofetil is useful in the long-term management (Daina et al. 1999). Atherosclerotic renal artery stenosis This is the most common cause of renal artery stenosis in the UK and USA (Scoble 2000). It is predominantly a disease of the older population and is intimately related to peripheral vascular disease. It can present with hypertension, renal impairment, or flash pulmonary oedema. It is also a marker of enormously high cardiovascular mortality. Although it is possible to revascularize the kidneys the results for hypertension are disappointing and the results for renal function suggest only a minority of patients, who cannot be identified prior to intervention can be identified. However, in anecdotal reports and in the minority of patients in series of intervention, dialysis-dependent patients may be rendered dialysis-independent. Glomerular disease The spectrum of glomerular disease extends from patients with mild urinary abnormalities to those with the full-blown nephritic or nephrotic syndrome, chronic glomerulonephritis, and end-stage renal failure. The major clinical syndromes of glomerular disease are acute glomerulonephritis, rapidly progressive glomerulonephritis, chronic glomerulonephritis, nephrotic syndrome, and asymptomatic haematuria with or without proteinuria. Acute glomerulonephritis These patients have an abrupt onset of haematuria, proteinuria, oedema, oliguria, hypertension, with a reduction in GFR, and uraemia. Haematuria refers to the excretion of abnormal amounts of erythrocytes in the urine. This is often first brought to light on dipstick examination of the urine (microscopic haematuria), or it might be obvious to the naked eye either as dark brown or bright red urine (macroscopic haematuria). The haematuria may either be persistent or intermittent, and it might be symptomatic or asymptomatic (painless). Patients with acute glomerulonephritis often have red cell casts in the urinary sediment, and the proteinuria is usually less than 3 g per day. Some of the causes of acute glomerulonephritis are shown in Table 2.4. There is often an immunological basis to the glomerular injury, which then triggers secondary events such as complement activation, fibrin deposition, platelet aggregation, and

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A. David Makanjuola and John E. Scoble Table 2.4 Some causes of acute glomerulonephritis Familial Alport syndrome Nail-patella syndrome Fabry disease Primary Fibrillary glomerulonephritis Post-streptococcal glomerulonephritis IgA nephropathy Idiopathic rapidly proliferative glomerulonephritis Membranous nephropathy Focal and segmental glomerulosclerosis Secondary Systemic lupus erythematosus Goodpasture syndrome Henoch–Schonlein purpura Mixed essential cryoglobulinaemia

activation of kinin systems. These result in increased capillary permeability and glomerular damage. The kidneys, in acute glomerulonephritis, are usually of normal size, but may be enlarged and oedematous, and the surface of the kidney may show punctate haemorrhages. In the clinical setting, it is often important to distinguish between the patient who has an acute exacerbation of an underlying chronic renal disorder from the patient with previously normal renal function. It would be reasonable to expect the latter patient to regain normal excretory renal function perhaps with residual asymptomatic haematuria or proteinuria; although it is possible that they may develop chronic renal impairment, or pursue a rapidly progressive course, leading to end-stage renal failure. Rapidly progressive glomerulonephritis (RPGN) These patients are characterized by a rapid, often inexorable progression towards end-stage renal failure without (and often in spite of) treatment. Chronic glomerulonephritis This is the persistence and/or progression of an initial glomerular insult, which will ultimately lead to end-stage renal failure. It may take years before patients end up requiring renal replacement therapy, and the need for this is often precipitated by other factors, such as poorly controlled hypertension. In chronic glomerulonephritis, the kidneys are often small, with fine granular scarring. Nephrotic syndrome Protein is normally present in only small amounts in the urine (Cameron 1998). This is largely due to the fact that in spite of the huge amounts delivered to the nephrons the glomerulus significantly restricts the passage of proteins across its membrane, and the tubules reabsorb most of the protein that manages to traverse the glomerular basement membrane. About 0.5–1 g of

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albumin is filtered by the glomeruli each day, but after tubular reabsorption, only 20–40 mg is passed in the urine. In the healthy individual, 24-h protein excretion is usually less than 150 g, but glomerular and tubular diseases often lead to a significant increase in the amount of protein excreted. In the absence of urinary tract infections, it is fair to say that proteinuria is almost always of glomerular origin, whereas haematuria may be due to lesions anywhere along the renal tract. The nephrotic syndrome is the consequence of persistent heavy proteinuria. Its cardinal features are proteinuria, which by definition is in excess of 3.5 g/1.73 m2 body surface area or 50 mg/kg body weight, hypoalbuminaemia, oedema, hyperlipidaemia, and hypercoagulability (Cameron 1998). These patients also have some degree of immunodeficiency. Proteinuria secondary to glomerular disease is believed to arise as a result of compromise of the filtration barrier, which alters its charge selectivity (minimal change nephropathy), or alters the pore sizes (proliferative glomerulonephritis). This allows plasma proteins, especially albumin to leak into the filtrate in quantities that exceed the tubules’ reabsorptive capacity, and thus end up in the urine. Hypoalbuminaemia is due to a variety of factors, which include excessive loss of albumin in the urine; increased renal tubular catabolism of albumin, and an inappropriately low rate of albumin synthesis by the liver. Oedema is partly due to the reduced plasma oncotic pressure as a consequence of the hypoalbuminaemia, but this is by no means the sole explanation. These patients exhibit a significant avidity for sodium, with consequent sodium retention by the kidneys, the cause/s of which have not been fully elucidated. Hyperlipidaemia is due to increased liver synthesis of lipoproteins. The hypoalbuminaemia and increased albumin synthesis by the liver seem to be accompanied by increased delivery of the cholesterol precursor mevalonic acid to the liver, and stimulation of lipoprotein synthesis, especially very low density lipoprotein (VLDL). Reduced lipoprotein catabolism also contributes to the hyperlipidaemia. Lipiduria is another common feature. It is possible that these patients are at increased risk of ischaemic heart disease, and in patients with other risk factors for ischaemic heart disease, lipid-lowering therapy ought to be instituted. Hypercoagulability usually manifests as thrombo-embolism. This is predominantly venous, but can also affect the arterial tree. The most common manifestation is renal vein thrombosis, but close to one-fifth of all patients with the nephrotic syndrome develop deep venous thromboses and pulmonary emboli. Low levels of antithrombin III seem to correlate closely with the increased risk of thrombo-embolism. Some nephrologists advocate prophylactic anticoagulation once the serum albumin levels fall below 20 mg/dl. Immunodeficiency is believed to be due to losses of immunoglobulins in the urine, as well as increased catabolism and depressed synthesis. Serum immunoglobulin (Ig) levels are reduced, and there are defects in cell-mediated immunity. These people are prone to serious infections, and this is often compounded by the fact that treatment regimes often involve the use of steroids and other immunosuppressive agents. Virtually any glomerular lesion, if it generates enough proteinuria, will produce the features of this syndrome but only a small number of disorders constitute the bulk of the cases encountered in clinical practice. Secondary causes of the nephrotic syndrome include diabetic nephropathy, amyloidosis, systemic lupus erythematosus, and neoplasia. Primary (idiopathic) causes include minimal change nephropathy, focal and segmental glomerulosclerosis, and membranous nephropathy.

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Patients with the nephrotic syndrome may also have features of acute glomerulonephritis, and may progress to chronic glomerulonephritis. Tubular proteinuria is usually of lesser magnitude, and in a patient known to have tubulointerstitial disease, development of nephrotic range proteinuria strongly suggests co-existent glomerular pathology. Histopathological types of glomerular diseases The histological response to immunological insults varies, and is nonspecific. Identical histological appearances may be the result of very different insults, but on the other hand, a condition such as systemic lupus erythematosus is associated with many different histological changes. Clinical manifestations do not correlate closely with the histological changes, but nonetheless, it is often possible to predict with some degree of accuracy what the clinical outcome is likely to be after reviewing the renal biopsy, by light microscopy, electron microscopy, and immunofluorescence. Minimal change nephropathy (minimal change disease) As the name suggests, the glomerular appearances are almost normal (Broyer et al. 1998). On light microscopy, there is usually no abnormality of note, and electron microscopy shows fusion of the epithelial cell foot processes. Immunofluorescence is negative. Its aetiology is unknown, but an immunological basis is suggested by the fact that it is usually steroid responsive, there is an increased incidence of atopy in patients as well as family members, and depressed levels of IgG subclasses and elevated IgM levels have been noted. The fact that it occurs in some patients with T-lymphocyte dysfunction such as Hodgkin disease and that it remits on treatment of the malignancy. Minimal change disease is the most frequent chronic renal disease in childhood, and its relative incidence decreases with advancing age in patients which manifest clinically with the nephrotic syndrome. The proteinuria is believed to be due to increased glomerular permeability, and some authors have suggested that one or more lymphocyte-derived factors could cause this by inducing electrostatic changes on the glomerular capillaries. There is a possibility that a more generalized increase in capillary permeability is present in children with minimal change disease, as they often have more oedema than other patients with comparable degrees of hypoproteinaemia due to other glomerular lesions. The clinical course is characterized by relapses of varying frequency. Eighty per cent of children have a rapid response to an initial course of steroid therapy, but relapses occurred in the majority of them. The frequency of relapses is unpredictable for the individual patient, but in general, relapses are more common if the onset of disease was in early childhood. Progressive disease leading to end-stage renal failure is uncommon in children. Mild to moderate degrees of renal impairment is seen in some patients and is often due to intravascular volume depletion, or sepsis. Idiopathic focal and segmental glomerulosclerosis (FSGS) This condition, described initially is seen in 10–15 per cent of people with idiopathic nephrotic syndrome, and is the most common cause of steroid-resistant nephrotic syndrome

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in children. It has been suggested that there is a circulating factor able to elute from the serum of these patients which might be responsible for the increased glomerular permeability, and this might explain the tendency for this condition to recur in kidneys transplanted into affected individuals sometimes within hours of transplantation (Korbet 1998). Light microscopy in FSGS shows focal and segmental glomerular sclerosis, varying degrees of mesangial proliferation and often, subendothelial areas of hyalinosis and lipid droplets. Other portions of the glomeruli appear normal. The process seems to begin in the juxta-medullary glomeruli and then progresses to involve the more peripheral glomeruli. The tubules may be atrophic and there might be an interstitial mononuclear cell infiltrate. Immunofluorescence shows mesangial deposits of IgM and C3, usually within sclerotic segments. These histological findings are indistinguishable from those seen in patients with secondary FSGS, suggesting that the histologic picture is a common final pathway for several kinds of glomerular damage (Korbet 1998). These patients, as mentioned earlier, tend to be steroid unresponsive. There is also a greater incidence of hypertension, azotaemia, and progression to chronic renal impairment than is seen in patients with minimal change disease. Diffuse proliferative glomerulonephritis This histological lesion is often seen in patients with acute post-streptococcal glomerulonephritis, and can also be seen in systemic lupus erythromatosus (SLE). It usually manifests clinically as an acute nephritis, with haematuria, proteinuria, hypertension, oedema, and azotaemia. On light microscopy, the glomerulus appears swollen, with proliferation of endothelial and mesangial cells and several polymorphonuclear leucocytes. Electron microscopy shows subepithelial humps on the glomerular basement membrane (GBM), and immunofluorescence shows granular deposits of immunoglobulins and C3. Focal proliferative glomerulonephritis This condition is seen SLE, IgA nephropathy, Henoch–Schonlein purpura, anti-neutrophil cytoplasmic antibody (ANCA)-positive vasculitis, and infective endocarditis. Only some of the glomeruli are involved, and the affected glomeruli show segmental proliferative changes. Clinical manifestations are varied, and range from minor urinary abnormalities to acute nephritis, or the nephrotic syndrome. Crescentic (rapidly progressive) glomerulonephritis Extensive glomerular crescent formation (usually more than 50 per cent) is the main histological finding (Cameron and Rees 1998). It is a particularly severe form of many glomerular diseases, and is quite often seen in anti-GBM disease, microscopic polyarteritis and Wegener granulomatosis. The crescents may be partially or totally circumferential, and may be cellular, fibrocellular, or fibrotic. Prominent endocapillary proliferation suggests a post-infectious or multisystem disease. The presence of a vasculitis and necrotizing glomerular lesions suggests systemic disease. Immunofluorescence will show linear staining of the GBM with IgG, in anti-GBM disease. Idiopathic RPGN is associated with granular deposits of IgG and C3 in mesangial areas and along the subendothelial aspect of the capillary wall.

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As the name suggests, renal function declines rapidly, with end-stage renal failure ensuing within anything from 1 week to 3 months from diagnosis or the onset of symptoms in the absence of treatment. The outlook has improved significantly since the advent of treatment with high dose steroids, plasma exchange, and cytotoxic agents such as cyclophosphamide. Membranous glomerulonephritis This condition occurs mainly in adults and predominantly in males (Zucchelli and Pasquali 1998). These patients typically present with proteinuria, and in the great majority, the nephrotic syndrome. Light microscopy shows diffuse and uniform thickening of the GBM. Mesangial proliferation and cellular infiltration may occur in patients with underlying disease, but are often absent in the idiopathic form. Immunofluorescence in well-developed cases shows diffuse granular staining with IgG and usually C3 in capillary walls. Silver staining outlines the GBM but not immune deposits, and displays the characteristic ‘spikes’. Electron microscopy reveals subepithelial deposits. One half of the patients will achieve partial or complete remission, and the other half will have chronic or end-stage renal failure. Young age, female sex, normal renal function, and the absence of the full-blown nephrotic syndrome at presentation are associated with a good prognosis. The presence of tubular atrophy and interstitial fibrosis on renal biopsy are adverse prognostic features. Mesangio-capillary (membrano-proliferative) glomerulonephritis (MCGN) This glomerular lesion is associated with a chronic, progressive glomerulonephritis, which tends to affect adolescents and young adults (Williams 1998). Secondary forms occur, usually in association with systemic, infectious, and neoplastic disorders. Idiopathic MCGN is divided into three types based on histo- and immuno-pathologic features. In Type 1, there are subendothelial and mesangial deposits of immunoglobulin and C3. This is the most common form. It is also the histological pattern most commonly seen in association with secondary forms. The most common clinical manifestation is the nephrotic syndrome, though some patients may present with an acute nephritis. C3 levels are low, and both the classical and alternative pathways of complement activation are involved. Type 2 is characterized by electron-dense deposits and linear C3 deposits situated in intramembranous and mesangial areas. It is also known as dense deposit disease. These patients tend to have a younger age of onset, and the typical presentation is with acute nephritis. Partial lipodystrophy may be seen. C3 levels are low, and a unique IgG auto-antibody— the C3 nephritic factor—causes complement activation via the alternative pathway. In Type 3 there is a rare group of patients who exhibit features of both Type 1 and Type 2. This category is however, not universally accepted. Secondary causes of MCGN include hepatitis C, SLE, and mixed essential cryoglobulinaemia. Type 1 MCGN is slowly progressive, but Type 2 is much more aggressive, with patients progressing to end stage renal failure from 5 to 10 years following diagnosis. Immunoglobulin A (IgA) nephropathy (Berger disease) Originally described in 1968, this is an idiopathic glomerular disease characterized by diffuse mesangial deposition of IgA (Topham et al. 1994). It appears to be the most frequently

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encountered glomerular disease worldwide. It may have a genetic component as the incidence is much lower in blacks in the USA, but seems to be much more common in the American Indians of New Mexico and familial clustering has been reported. The HLA B35 is reported to be more frequent in a large population of French patients, in whom it seems to correlate with progression to end-stage renal failure. The diagnostic hallmark of the disease is mesangial deposition of IgA. Immunofluorescence on renal biopsies shows IgA to be the predominant or sole immunoglobulin, although in most cases, C3 and IgG are also present, though with a lesser degree of immunofluorescence. Light microscopy shows mesangial enlargement, usually in a focal and segmental distribution, but in some cases, the appearance may be that of a diffuse proliferative process. The pathogenesis is not clear, but polymeric IgA1 of mucosal origin is believed to be the immunoglobulin acting as antibody. Clinically, patients manifest with asymptomatic microscopic haematuria with or without proteinuria. These patients are usually detected by chance at routine medical examinations. Macroscopic haematuria, when it occurs, is usually temporally related to a respiratory, gastro-intestinal, or less commonly a urinary tract infection. Proteinuria is often mild, but may occasionally be severe enough to cause the nephrotic syndrome. As many of these patients are asymptomatic, the true onset of the disease is not often known. Renal survival rates are estimated at 78–91 per cent at 10 years and 75–80 per cent at 20 years from apparent onset in some large series. The rate of progression from diagnosis to end-stage renal failure varies from a few months to over 30 years. Poor prognostic factors include male sex, elevated serum creatinine at time of diagnosis, hypertension, protracted severe proteinuria, and absence of a history of recurrent macroscopic haematuria.

Tubulo-interstitial disease Tubulo-interstitial disease is any disorder resulting in structural and/or functional derangements of the tubulo-interstitial constituents of the kidney (Michel and Kelly 1998). The causes of this histological pattern are many. Tables 2.5–2.7 show the drug, infectious, and metabolic causes of this pattern of renal disease. Tubulo-interstitial disease can be divided into acute and chronic types. About 15 per cent of acute renal failure and 25 per cent of end-stage renal failure is caused by primary tubulointerstitial disease. The pathogenesis of tubulo-interstitial disease can involve a variety of mechanisms, which include direct invasion by microbial organisms, direct toxicity as with heavy metal poisoning, immunological insults, metabolic diseases, and hereditary disorders such as cystic diseases and Alport syndrome. Acute tubulo-interstitial nephritis Councilman used this term in 1898 as a histopathological diagnosis for the acute renal inflammatory changes seen in autopsy specimens of patients with diphtheria and scarlet fever (Michel and Kelly 1998). Infections are less commonly responsible for acute interstitial nephritis nowadays and most cases are drug induced. In a few cases, no definite cause is found and in this ‘idiopathic’ group, no clear sex or age predisposition has been found.

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A. David Makanjuola and John E. Scoble Table 2.5 Drugs associated with acute interstitial nephritis Antibiotics Beta-lactam Ampicillin Methicillin Flucloxacillin Carbenicillin Amoxycillin Cefotaxime Cephalexin Cephloridine Cefoxitin

Anti-convulsants Phenytoin Sodium valproate Carbamazepine Phenobarbitone

NSAIDs* Ibuprofen Indomethacin Fenoprofen Naproxen Diclofenac Piroxicam

Others Rifampicin Sulphonamides Septrin Ciprofloxacin Erythromycin Vancomycin Ethambutol

Diuretics Thiazides Frusemide Triamterene

Others Cimetidine Allopurinol Haloperidol Captopril Diazepam Propranolol Amphetamines Aspirin Clofibrate

*NSAIDs  Non-steroidal anti-inflammatory drugs.

Table 2.6 Infectious causes of acute interstitial nephritis Bacteria Escherichia coli Proteus species Enterococci Klebsiella aerobacter Staphylococci Leptospira species Brucella species Legionella species Viruses Epstein Barr virus Coxsackie B virus Fungi Candida species Coccidioidomycosis Protozoa Falciparum malaria Toxoplasmosis

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Table 2.7 Causes of chronic interstitial nephritis Metabolic abnormalities Hyperuricaemia Oxalosis Hypercalcaemia Hypokalaemia Cystinosis Hereditary disorders Sickle cell disease Alport syndrome Polycystic kidney disease Others Balkan nephropathy Radiation Allograft rejection Tuberculosis Leprosy Sjogren syndrome Obstructive uropathy Reflux nephropathy Chronic pyelonephritis

Neoplasia Multiple myeloma Lymphoma Leukaemia

Systemic disorders Systemic lupus erythematosus Polyarteritis nodosa Wegener granulomatosis Cryoglobulinaemia Exogenous toxins Lead Cadmium Gold Cyclosporin cis-Platinum Lithium

Clinically, patients with acute tubulo-interstitial nephritis manifest with an abrupt deterioration in renal function characterized histologically by inflammation and oedema of the renal interstitium. Drug-induced acute interstitial nephritis There are a significant number of drugs, which have been reported as causative agents. The beta lactam antibiotics and non-steroidal anti-inflammatory drugs are well recognized causes. The clinical manifestations depend on the drug producing the adverse reaction. With beta lactam antibiotics, the hypersensitivity triad of rash, fever, and eosinophilia dominates the picture. Patients may also have arthralgia, eosinophilluria, lymphadenopathy, and hepatitis. Sterile pyuria and haematuria may occur. These manifestations usually occur in patients who have received high doses of the beta lactam for a prolonged period of time and can occur either in the presence of active infection or when the drug is being used prophylactically. Non-steroidal anti-inflammatory drugs are frequently associated with very heavy proteinuria and minimal change nephrotic syndrome. The onset of the nephrotic syndrome typically coincides with the onset of the acute interstitial nephritis and acute renal failure, all of which tend to settle on cessation of the drug. Infectious causes The causative agents, Table 2.6 may be bacterial, viral, fungal, or parasitic in nature. Acute renal failure is prominent, but the hypersensitivity triad is not common. The manifestation of

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the basic infective process often predominates, and the renal failure usually remits as the infection settles. The diagnosis of acute tubulo-interstitial nephritis is conclusively made on renal biopsy. However, a classical clinical picture in association with non-oliguric renal failure in a patient on a medication known to cause this is highly suggestive, especially if the renal failure improves following cessation of the drug. Chronic tubulo-interstitial nephritis This is characterized by progressive renal insufficiency, associated with non-nephrotic range proteinuria and functional tubular abnormalities. The primary site of injury is the tubule and surrounding interstitium. In far advanced disease, peri-glomerular fibrosis, and glomerulosclerosis may be seen. The tubules are often atrophic and dilated. Clinical manifestations vary, and depend to some degree on the causative factor. Conditions such as heavy metal poisoning and multiple myeloma affect the proximal tubule primarily, and may present with renal tubular acidosis, glycosuria aminoaciduria, and uricosuria. Chronic obstructive uropathy or amyloidosis may cause isolated distal tubular damage; while sickle cell disease, analgesic nephropathy, or polycystic disease of the kidney may affect the medulla and lead to a defect in concentrating ability. Hypertension is usually a late finding and oedema is often absent. Avoidance of the nephritogenic agent or correction of the predisposing factor may prevent progression or even reverse the renal insufficiency. Hypertension where present should be treated aggressively. Post-renal disease This is an important area where the causes of renal dysfunction are geographically very different. Table 2.8 shows that the causes of this are many and can be divided into malignant and non-malignant. Just as in renal arterial obstruction relief of the obstruction is important, then even more so in renal outflow obstruction, relief is vitally important and may enable patients to come off dialysis. Pelvi-ureteric obstruction is usually not complete and often is precipitated by fluid challenge. Its relief is by surgical plication of the pelvi-ureteric junction. Renal stones may cause obstruction to the urinary tract from the calyx to the urethra but most commonly lodge at the pelvi-calyceal junction or at the uretero-vesical junction. The causes of renal stones will be discussed elsewhere. However, once formed their management is by their removal. This can be achieved via lithotripsy or surgical removal directly or endoureterically. The management of malignant obstructive disease depends on the cause. In prostatic malignancy, obstruction can occur at both the urethral level as well as ureteric level. Hormone manipulation can provide a dramatic response. For other malignancies such as cervical cancer the outcome is more gloomy and hormonal manipulation is not possible. In the infective causes of obstruction the principle of therapy is treatment of the disease and then surgical treatment of the urinary tract to provide adequate drainage. The management of congenital obstructive disease is to reverse the obstruction at the earliest possible opportunity although the outcome of this is variable. It is clear that complex

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Table 2.8 Causes of obstructive uropathy Non-malignant Pelvi-ureteric obstruction Urethral valves Benign prostatic hypertrophy Stone disease Retroperitoneal fibrosis Ureteric fibrosis—tuberculosis Bladder fibrosis—Bilharzia Malignant Retroperitoneal fibrosis—lymphoma Prostatic malignancy Cervical malignancy Urothelial malignancy

pathological processes are unleashed by urinary obstruction and it is not a simple matter of mechanical obstruction (Klahr 1983). Treatment of end-stage renal failure This book seeks to enlighten us about the genetic causes of renal failure. It is also about the prevention of end-stage renal failure by treatment of the genetic diseases if possible. Although it is unnecessary to document the technicalities of the treatment of end-stage renal failure, there are some specific points which need to be made. The first patient to receive renal dialysis treatment was Sophia Schafstadt in the Second World War in Nazi occupied Holland. Kolff performed this first dialysis and Fig. 2.12 shows a later version of the original machine. Although there has been considerable development of dialysis machines the basic principles remain as described by Kolff. Initial use of dialysis was for conditions which would recover. Regular haemodialysis only became possible with the development of the Scribner shunt and this was then superseded by the Cimino fistula which remains to this day the optimum vascular access. Peritoneal dialysis which originated as a form of acute dialysis with a ‘hard catheter’ developed into chronic ambulatory peritoneal dialysis (CAPD) with the use of the soft Tenkhoff catheter. It is important to underline that both haemodialysis and CAPD provide only the equivalent to 10 per cent of normal glomerular filtration rate. This fact relates to Fig. 2.1. This shows a healthy nephrologist and his glomerular filtrate. If he had end-stage renal failure he may be assigned to CAPD and would do four exchanges a day totalling 8 l. This would prevent him from dying due to hypervolaemia or hyperkalaemia but would not give him by any estimation normal plasma biochemistry. The only form of treatment of end-stage renal failure which provides for treatment which will reproduce normal renal function is renal transplantation. It is probably best to describe dialysis as preventing death rather than replacing normal renal function which can today only be achieved by transplantation. Renal transplantation has evolved from the procedure only applicable to identical twins to a process which may provide excellent life expectancy to many potential recipients. In the majority of instances transplantation is from cadaveric donors who have died due

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Fig. 2.12 A Kolff dialysis machine similar to the original machines used for haemodialysis.

100 95 90

LRDT-identical Parent LURT CAD-identical CAD-all

85 80 75 70 65 60 Fig. 2.13 The outcome of 3-year renal graft survival where LRDT = live related donor transplant; LURT = live unrelated donor transplant; and CAD = cadaveric donor. UCLA data of 28,945 patients in the USA.

to a number of reasons. Figure 2.13 shows the outcome for renal transplantation in the USA published in 1995 (Terasaki et al. 1995). The use of initially Azathioprine and Prednisolone achieved useful results. The introduction of more powerful agents such as cyclosporin A and more recently Tacrolimus and Mycophenalate moefitil have enabled the results shown in Fig. 2.13 to be achieved. However, what is clear from these data is that the donor origin is an important determinant of graft outcome. This is especially relevant in inherited renal disease as related donors must be excluded from having renal disease. There is a lack of cadaveric

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donors and this means that the waiting list for transplantation in the UK increases year by year. The only country in Europe which has not had an increase in its transplant waiting list is Norway. This has been achieved by 40 per cent of transplants occurring from living donors. This compares with the rate for 1998 in the UK of 15 per cent. However with regard to endstage renal failure due to genetic disease this is a very difficult area as the most common donors are first degree relatives. The only group unaffected by potential genetic disease is spousal donors. The figures in Fig. 2.11 show that organ donation from living donors has a better outcome even in genetically unrelated spousal donors with the best figures being in HLA identical siblings. This is obviously a very difficult area for patients with end-sage renal disease due genetic causes. Renal transplantation in spite of the risks of malignancy and opportunistic infection still provides the best replacement of renal function. At the same time, if successful it also provides the longest life expectancy for the individual. Conclusion There are many forms of genetic disease which affect the renal tact and may lead to total failure of excretory function. A few have specific treatments which may help but for the majority of patients the investigations and treatments will be the same as for any renal disease. It is clear that with time the proportion of diseases which have a genetic basis or altered susceptibility on the basis of genetic factors will increase. However the management of most conditions revolves around tight blood pressure control prior to end-stage renal failure and then renal replacement therapy by transplantation.

References Atkinson, D. E. and Bourke, E. (1987). Metabolic apsects of the regulation of systemic pH. American Journal of Physiology, 252, F947–56. Bichet, D. G. (1998). Nephrogenic diabetes insipidus. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 2, pp. 1095–109. Oxford University Press, Oxford. Broyer, M., Meyrier, A., Niaudat, P., and Habib, R. (1998). Minimal changes and focal segmental gloemruloslerosis. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 493–536. Oxford University Press, Oxford. Cameron, J. S. (1998). The nephrotic syndrome: management, complications and pathophysiology. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 461–93. Oxford University Press, Oxford. Cameron, J. S. and Greger, R. (1998). Renal function and testing of function. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 39–69. Oxford University Press, Oxford. Cameron, J. S. and Rees, A. J. (1998). Crescentic glomerulonephritis. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 625–46. Oxford University Press, Oxford. Daina, E., Shieppati, A., and Remuzzi, G. (1999). Mycophenolate mofetil for the treament of Takayasu arteritis: report of three cases. Annals of Internal Medicine, 130, 422–6.

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Dworkin, L. D. and Weir, M. R. (2000). Hypertension in renal parenchymal disease: role in progression. In Mechanisms and clinical management of chronic renal failure (ed. A. M. El Nahas, K. P. G. Harris, and S. Anderson), pp. 173–210. Oxford University Press, Oxford. Goldblatt, H., Lynch, J., Hanzal, R. F., and Summerville, W. W. (1934). The production of persistent elevation of systolic blood pressure by means of renal ischemia. Journal of Experimental Medicine, 59, 347–78. Klahr, S. (1983). Pathophysiology of obstructive nephropathy. Kidney International, 27, 690–702. Klahr, S., Levey, A. S., Beck, G. J., et al. (1994). The effects of dietary protein restriction and blood pressure control on the progression of chronic renal disease. New England Journal of Medicine, 330, 877–84. Korbet, S. M. (1998). Primary focal segmental glomerulosclerosis. Journal of American Society of Nephrology, 9, 1333–40. Kurtz, I. (1998). Molecular pathogenesis of Barrter’s syndrome and Gitelman’s syndromes. Kidney International, 54, 1396–1410. Lewis, E. J., Hunsicker, L. G., Bain, R. P., and Rohde, R. D. (1993). The effect of angiotensinconverting-enzyme inhibition on diabetic nephropathy. New England Journal of Medicine, 329, 1456–62. Locatelli, F. and Del Vecchio, L. (2000). Natural history and factors affecting the progression of human renal diseases. In Mechanisms and management of chronic renal failure (ed. A. M. El Nahas, K. P. G. Harris, and S. Anderson), pp. 20–79. Oxford University Press, Oxford. Macdougall, I. C. and Eckardt, K. U. (1998). Haematological disorders. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 3, pp. 1935–53. Oxford University Press, Oxford. Maisey, M. and Britton, K. E. (1998). Nuclear imaging in nephrology. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 137–49. Oxford University Press, Oxford. Maschio, G., Alberti, D., Janin, G., Locatelli, F., Mann, J. F., Motolese, M. et al. (1996). Effect of the angiotensin-converting-enzyme inhibitor benazapril on the progression of chronic renal insuffficiency. New England Journal of Medicine, 334, 939–45. Michel, D. M. and Kelly, C. J. (1998). Acute interstitial nephritis. American Journal of Kidney Diseases, 9, 506–15. Parfrey, P. S. and Foley, R. N. (1999). The clinical epidemiology of cardiac disease in chronic renal failure. Journal of American Society of Nephrology, 10, 1606–15. Parving, H. H., Andersen, A. R., Smidt, U. M., and Svendsen, P. A. (1983). Early aggressive antihypertensive treatment reduces the rate of decline in kidney function in diabetic nephropathy. Lancet, 1, 1175–9. Ponticelli, C. and Mihatsch, M. J. (1998). Imbasciata. Renal biopsy: performance and interpretation. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 157–71. Oxford University Press, Oxford. Scoble, J. E. (2000). The natural history and management of renovascular disease. In Mechanisms of chronic renal failure (ed. A. M. El Nahas, K. P. G. Harris, and S. Anderson), pp. 265–301. Oxford University Press, Oxford. Scoble, J. E. and Cook, G. J. R. (1998). Individual kidney function in atherosclertotic nephropathy. Nephrology, Dialysis and Transplantation, 13, 842–4. Stanley, J. C. (1995). Renal artery fibrodysplasia. In Renal vascular disease (ed. A. Novick, J. Scoble, and G. Hamilton), pp. 21–33. W. B. Saunders, London. Stokes, J. B. (1999). Disorders of the epithelial sodium channel: insights into the regulation of extracellular volume and blood pressure. Kidney International, 56, 2318–33. Terasaki, P. I., Cecka, J. M., Gjertson, D. W., and Takemoto, S. (1995). High survival rates of kidney transplants from spousal and living unrelated donors. New England Journal of Medicine, 333, 333–6.

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Topham, P. S., Harper, S. J., Furness, P. N., Harris, K. P. G., Walls, J., and Feehally, J. (1994). Glomerular disease as a cause of isolated microscopic haematuria. Quarterly Journal of Medicine, 87, 329–35. UK Prospective Diabetes Study Group. (1998). Tight blood pressure control and risk of macrovascular and microvascular complications in type 2 diabetes: UKPDS 38. British Medical Journal, 317, 703–13. Williams, D. G. (1998). Mesangiocapillary glomerulonephritis. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 591–612. Oxford University Press, Oxford. Yamamoto, T. and Sasaki, S. (1998). Aquaporins in the kidney: emerging new aspects. Kidney International, 54, 10141–51. Zucchelli, P. and Pasquali, S. (1998). Membranous nephropathy. In Oxford textbook of clinical nephrology (ed. A. M. Davison, J. S. Cameron, J. P. Grunfeld, D. N. S. Kerr, E. Ritz, and C. G. Winerals), Vol. 1, pp. 571–90. Oxford University Press, Oxford.

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3 Renal development Paul J. D. Winyard

Introduction Mammalian kidneys perform a number of functions that are essential for normal postnatal life including excretion of nitrogenous waste products, homeostasis of water, electrolytes and acid–base balance, and the production of hormones. Mature human kidneys contain around a million nephrons, each consisting of specialized segments including glomerulus, proximal tubule, loop of Henle, and distal tubule, connected to the tree-like collecting duct system and intimately associated with the vascular supply. The precursor of the adult organ, the metanephros, arises in the fifth week of human development and consists of only two cell types: epithelia of the ureteric bud and mesenchyme of the metanephric blastema. Mutual interactions between these two tissues are essential for normal kidney development, or nephrogenesis, which involves precisely coordinated cell proliferation and death, morphogenesis and differentiation. A number of key nephrogenic molecules have recently been identified using descriptive studies of normal and abnormal development and, increasingly, from mice with targeted null mutations which disrupt nephrogenesis. These include the transcription factors PAX2 and WT1, growth factor glial cell line-derived neurotrophic factor (GDNF), and survival factor BCL2. The majority of this chapter is taken up with a review of the cell biology of kidney development, with particular emphasis on these important nephrogenic molecules, after first considering the structure of the mature organ and the anatomy of normal human development.

Structure A brief review of normal mature renal structures is given below. Readers requiring a more comprehensive account should refer to standard anatomical or pathological texts (Ventatachalam and Kriz 1998). Mature human kidneys from adult males are around 11  6  2.5 cm3 in size and weigh up to 170 g. Females have slightly smaller organs. The paired kidneys are located in the retroperitoneum and extend between, but lateral to the twelfth thoracic and third lumbar vertebrae. The renal pelvis lies medially and tapers into the ureter, which connects inferiorly to the bladder. The kidney parenchyma consists of nephrons, collecting ducts, blood vessels, lymphatics, nerves, and interstitium. Each nephron consists of a glomerulus, proximal tubule, loop of Henle, and a distal tubule, which is joined to a collecting duct via the connecting tubule (Fig. 3.1A). All of the glomeruli are located in the cortex, a 1-cm thick strip which forms the

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A

B 1

6 7

2

Cortex

Epithelial cells (podocytes)

7 3

5

2

6 1 3

4

Medulla

4

5

Endothelial cell Fenestrated endothelium

Podocyte foot processes

Basement membrane Fig. 3.1 Schematic of nephron and glomerular structure. A: schematic view of nephron and glomerular structures. A: Short superficial glomerulus (left) and a longer juxtamedullary glomerulus (right), with cortex in the upper part of the panel and medulla in the lower part, separated by the dotted line. Segments are: 1—glomerulus, 2—proximal convoluted tubule, 3—proximal straight tubule, 4—loop of Henle, 5—distal straight tubule, 6—distal convoluted tubule, and 7—connecting tubule. Macula densa in distal straight tubule is shown in black and collecting ducts are shaded grey. B: Glomerulus showing intimate relationship between fenestrated endothelium of endothelial cells, basement membrane, and foot processes of visceral epithelial/podocyte cells. Not drawn to scale.

outermost part of the kidney, whereas other nephron components extend into the medulla towards the centre of the organ. In humans, the cortex is continuous, whereas the medulla consists of around 14 discrete pyramids. This is termed ‘multipapillary’ and contrasts with the ‘unipapillary’ kidneys found in rats and rabbits. Nephrons Glomerulus Human glomeruli are around 200 m in diameter. They consist of mesangial cells and matrix supporting a tuft of capillaries directly surrounded by glomerular basement membrane and visceral epithelial cells (podocytes) separated from the parietal epithelium lining the inner surface of Bowman’s capsule by Bowman’s space (Ventatachalam and Kriz 1998) (Fig. 3.1B). The intimate relationship between the fenestrated capillary endothelium, basement membrane, and podocyte foot processes facilitates glomerular filtration but restricts protein losses in healthy glomeruli. Disruption of any of these components has significant effects on urine composition, as described in Chapters 4–7. Proximal tubule The proximal tubule consists of a convoluted portion, the pars convoluta, followed by a straight portion, the pars recta. Bowman’s space and the lumen of the proximal tubule are continuous, but there is an abrupt transition in cellular phenotype from flattened parietal

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epithelium to tall proximal tubule cells. The latter have a characteristic appearance with a well-developed brush border, to increase the surface area for reabsorption, and numerous mitochondria and lysosomes which reflect their high metabolic rate. Basolateral surface area is also increased by interdigitaiting cytoplasmic protrusions and ridges and this is the major location for NA, K–ATPase activity. The main role of the proximal convoluted tubule is to   , PO3), water, and organic solutes such reabsorb minerals, ions (Na, HCO 3 , Cl , K , Ca 4 as glucose and amino acids. Approximately two-thirds of the ultrafiltrate is reabsorbed in the proximal tubule. Loop of Henle The descending thin limb of the loop of Henle arises from the proximal tubule at the junction between the inner and outer stripes, of the outer medulla; loop epithelial cells can be easily distinguished by their flattened appearance and lack of brush border. Some ‘long’ loops descend deep into the medulla and turn back as ascending thin limbs before becoming the thick ascending limb, whereas this transition occurs in the vicinity of the bend in ‘short’ loops. At least four different types of thin limb segments have been described in the rat but there are only a few detailed studies in humans (Kriz 1981). The main role of the loops is to generate an osmolar gradient which facilitates reabsorption of water by the collecting ducts. Distal straight tubule (thick ascending limb of Loop of Henle) The distal straight tubule arises, in humans, as a gradual transition from the thin limb at the junction of the inner and outer medulla; straight tubule epithelial cells are taller and basolateral cell processes interdigitate with neighbouring cells. Rabbits also have a gradual transition, whereas there is a sharp change in the rat kidney. The straight tubule extends into the cortex just beyond the macula densa (described in ‘juxtaglomerular apparatus’ below). The main role of this segment is active, Na, K–ATPase-driven ion transport, particularly sodium chloride via the Na, K, 2Cl co-transporter. Defects in this frusemide sensitive transporter have been implicated in some cases of Bartter syndrome (see Chapter 14) (Guay-Woodford 1998). Distal convoluted tubule Distal convoluted tubules arise in the cortex and the junction of distal straight and convoluted tubules marked by an abrupt increase in epithelial height; cells in the initial segments are otherwise similar to the distal straight tubule, but increasing numbers of intercalated cells are observed in the terminal portion. Connecting tubule The distal tubule is attached to the collecting duct by the connecting tubule. This segment is well defined in the rabbit, with cells which are intermediate in phenotype between distal convoluted tubule and collecting duct cells, but is not so easily distinguishable in other species. Collecting duct Human collecting ducts arise in the cortex as a gradual transition from the connecting tubule and consist of cortical, outer medullary, and inner medullary segments. The initial collecting ducts drain an average of 11 nephrons in humans and successive fusion of these structures generates larger calibre ducts as they pass into the medulla. The largest collecting ducts, the terminal papillary collecting ducts of Bellini, open out into the renal calyces at the papillary tips. Collecting duct epithelia consist of principal and intercalated cells. Principal cells have

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characteristic basal infoldings, rare apical microvilli, and few mitochondria. The apical cytoplasm contains numerous vesicular structures containing aquaporin 2 water channels which increase water permeability by fusing with the apical plasma membrane following vasopressin binding to V2 receptors. Most cases of congenital nephrogenic diabetes insipidus are caused by mutations of these receptors or aquaporin 2 (Bichet 1998; see Chapter 14). Intercalated cells have few, if any, basal infoldings, variable numbers of apical microfolds, and contain dark mitochondria-rich cytoplasm. These cells contain H–ATPase in either the apical or basolateral plasma membranes, and are termed alpha- and beta intercalated cells, respectively. Alpha intercalated cells are responsible for proton secretion whereas beta intercalated cells mediate bicarbonate secretion. Juxtaglomerular apparatus This composite structure comprises the terminal segment of the afferent arteriole and the proximal segment of the efferent arteriole, with the extraglomerular mesangium between them plus the macula densa of the same nephron. The distinctive tall, columnar cells of the macula densa detect sodium chloride delivery to the distal tubule and modulate renin secretion via specialized granular myoepithelial cells in the afferent arteriole. Regulation of glomerular haemodynamics by the juxtaglomerular apparatus is influenced by this local mechanism, along with systemic blood pressure, hormones, and autonomic nerves. Development The process of renal development is conventionally called ‘nephrogenesis’, although this term more correctly refers to differentiation of the nephron tubules alone. The anatomy of normal human nephrogenesis has been described in detail by a number of authors (Kampmeier 1926; Potter 1972), but much less is known about its molecular basis. Paradoxically, recent studies of abnormal development in congenital malformations such as renal dysplasia (Woolf and Winyard 2000) and genetic syndromes (reviewed by Woolf in Chapter 4) have increased our understanding of the normal processes and molecules expressed during human nephrogenesis. There is still, however, significantly more information and a greater understanding of nephrogenesis in mice and other animals because large quantities of embryonic/fetal material are available and it is possible to use a variety of experimental strategies to define the roles of specific genes. This section will therefore consider methods used to study nephrogenesis, basic mechanisms of development, the anatomy of normal nephrogenesis, and molecules expressed during nephrogenesis in both human and non-human species. Methods for studying nephrogenesis There are two complementary ways in which nephrogenesis has been investigated: descriptive studies have traditionally been used to define gross anatomy, but can now also be used to determine the temporal and spatial expression of mRNA and protein, whilst functional studies establish the role of specific genes using in vivo and in vitro methodologies. Descriptive studies Most of the information on human renal development has been derived from descriptive studies since it is difficult to obtain sufficient material for repeated functional studies and

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there are ethical limits on human experiments. In their simplest form, descriptive studies document the gross anatomy and developmental stage of the normal kidney and renal malformations. They also describe patterns of gene expression by defining the distribution of mRNA by in situ hybridization and protein by immunohistochemistry. This may be at a specific timepoint, or at a series of different times/stages. These experiments may identify candidate genes which potentially play an essential role in nephrogenesis, by virtue of their temporal and spatial expression patterns, although animal functional studies are often required to confirm this role. Functional studies in vitro Isolated metanephroi can be grown for several days in organ culture. These organs develop in a morphologically normal manner with serial branching of the ureteric bud and new nephron formation, but the rate of development is slower than normal and glomeruli are avascular (Bernstein et al. 1981). Grobstein used this technique nearly 50 years ago to demonstrate that the mesenchyme and ureteric bud fail to develop when cultured separately, although some nephrons do form when mesenchyme is co-cultured with spinal cord (Grobstein 1955)— hence proving that interactions between the bud and mesenchyme are essential for normal development and that other tissues have the potential to induce normal development. It is possible to manipulate cultured metanephroi by adding exogenous substances to the culture medium. Examples are growth factors, blocking antibodies to growth factors or cell adhesion molecules, and antisense oligonucleotides which block expression of specific genes (Rogers et al. 1992, 1993; Rothenpieler and Dressler 1993). Individual cells have also been labelled using retroviral mediated gene transfer to follow their subsequent fate using in vitro lineage analysis (Herzlinger et al. 1993; Qiao et al. 1995). Cell lines can also be generated from the developing (and mature) kidney. These can be studied in primary culture or transduced with potentially immortalizing constructs such as the SV 40 T antigen to facilitate long-term studies. Cells can then be characterized on the basis of their morphology, membrane constituents, intracellular proteins, synthesis, and release of specific products such as growth factors, and extracellular matrix proteins cells (Burrow and Wilson 1993; Woolf et al. 1995). In this way it is possible to isolate homogeneous populations of cells which have a defined lineage, and these are the ideal populations to assess the response of specific cell types to exogenous factors (as above). Biochemical pathways can also be investigated such as ligand binding, receptor kinetics, and intercellular signalling pathways. Functional studies in vivo Nephrogenesis can be manipulated in vivo in animals using a number of techniques including exposure to teratogens (Gage and Sulik 1991) and obstruction of the developing urinary tract (Beck 1971; Attar et al. 1998). Increasingly, however, transgenic technology is being used to either ablate or overexpress specific molecules. These techniques may well become the standard tools for studying nephrogenesis over the next few years and a detailed description of this technology can be obtained from recent text books and review articles (Rossant and Nagy 1995; Gilbert 1997). A list of null mutant (‘knock out’) mice with abnormal renal development is shown in Table 4.2. Interpretation of functional studies Functional studies must be interpreted with caution since conflicting results can be generated using different techniques. A classic example occurs when organ culture studies implicate a molecule in nephrogenesis in vitro, yet null mutant mice have normal kidney development

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in vivo. There are several possible explanations for this discrepancy. Organ culture, for example may be considered a ‘stressful’ situation which is reliant on molecules that are of little significance in vivo. Alternatively, there may be circulating or maternal factors which compensate for the deficient factor and rescue the phenotype in vivo (Letterio et al. 1994). There may also be functional redundancy in vivo (Sariola 1996) and more than one gene may need to be knocked out to produce a phenotype (Mendelsohn et al. 1994; Davis et al. 1995). It should also be noted that many mice experiments are performed on inbred strains. This may be relevant when extrapolating murine results to out bred human populations since genetic background can influence the final kidney phenotype, suggesting that multiple genes may modulate the same developmental pathway (Threadgill et al. 1995). Anatomy of nephrogenesis There are three pairs of ‘kidneys’ in the mammalian embryo: the pronephros, mesonephros, and metanephros. These arise sequentially from intermediate mesoderm on the dorsal body wall (Larsen 1993). The pronephros and mesonephros degenerate during mammalian fetal life whilst the metanephros develops into the adult kidney. In contrast, the pronephros is the functioning kidney in the adult hagfish and some amphibians whereas the mesonephros is the excretory organ in adult lampreys, some fish, and amphibians. The pronephros It is possible to detect the first evidence of the pronephros at the 10 somite stage on day 22 after fertilization in humans, which is morphologically equivalent to embryonic day 9 in mice. At this stage, it comprises a small group of nephrotomes with segmental condensations, grooves, and vesicles between the second and sixth somites. The nephrotomes are nonfunctional and most likely represent vestiges of the pronephric kidney of lower vertebrates. The pronephric duct develops from the intermediate mesoderm lateral to the notochord (Gilbert 1997) from around the level of the ninth somite. The duct elongates caudally and reaches the cloacal wall on day 26. It is renamed as the mesonephric, or Wolffian duct, as mesonephric tubules develop. The nephrotomes and pronephric part of the duct involute and cannot be identified by day 24 or 25 after fertilization (Gilbert 1997). The mesonephros In humans, the long sausage-shaped mesonephros develops from around 24 days of gestation and consists of the mesonephric duct and adjacent mesonephric tubules (Fig. 3.2A and B). The mesonephric duct begins as a solid rod of cells which canalizes in a caudocranial direction after fusion with the cloaca. Mesonephric tubules develop from intermediate mesoderm medial to the duct by ‘mesenchymal to epithelial’ transformation, a process which is subsequently reiterated during nephron formation in metanephric development. In humans, a total of around 40 mesonephric tubules are produced (several per somite), but the cranial tubules regress at the same time as caudal ones are forming so that there are never more than 30 pairs at any time. Each human mesonephric tubule consists of a medial cup-shaped sac encasing a knot of capillaries, respectively analagous to the Bowman’s capsule and glomerulus of the mature kidney, and a lateral portion in continuity with the mesonephric duct. Other segments of the tubule resemble mature proximal and distal tubules histologically but there is no loop of Henle. The human mesonephros is reported to produce small quantities of urine between

Renal development A

go

63

B

go gl

gl

C

go meso

meta n E

D

l c b b

c F

G

b

c b

s

b b c gl

com

Fig. 3.2 Histology of normal human renal development. Haematoxylin and eosin stained sections of developing human kidneys at A: 38; B–E: 42; F and G: 56 days of gestation. A: Transverse section showing neural tube (n), mesonephroi (enclosed by dotted lines) and gonadal ridges (go). B: High power of mesonephros showing large glomeruli (gl). C: Close anatomical relationship between gonad, mesonephros (meso) and metanephros (meta). D and E: Early metanephros showing central ureteric bud (arrow) with peripheral branches (b) surrounded by condensing mesenchyme (c). F: and G: More mature metanephros showing nephron precursors including comma (com) and S-shaped bodies (s), and an immature glomerulus (gl). Bar (m) corresponds to 500 in A, 250 in C, 150 in D and F, and 60 in the remainder.

weeks 6 and 10 which drains via the mesonephric duct, whereas the murine organ is much more rudimentary and does not contain well-differentiated glomeruli. Mesonephric structures involute during the third month of gestation in humans, although caudal mesonephric tubules contribute to the efferent ducts of the epididymis and the mesonephric duct forms the duct of the epididymis, the seminal vesicle and ejaculatory duct is retained as the vas deferens (Moore and Persaud 1998).

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The metanephros The adult human kidney develops from the metanephros which consists of two cell types at its inception: the epithelial cells of the ureteric bud, and the mesenchyme cells of the metanephric mesenchyme. A series of reciprocal interactions between these tissues cause the ureteric bud to branch sequentially to form the ureter, renal pelvis, calyces, and collecting tubules whilst the mesenchyme undergoes an epithelial conversion to form the nephrons from glomerulus to distal tubule. This process is depicted graphically in Fig. 3.3. In addition, a third cell type, the interstitial cells are also thought to be derived from the mesenchyme.

A

B m u

u

mc

d

C

D c

u

s

gl

Fig. 3.3 Early branching of the ureteric bud and nephron formation. Schematic view with mesonephric duct (d) and ureteric bud (u) shown in black, uninduced mesenchyme (m) in white, and mesenchymal condensates (mc) and nephron precursors, including comma shapes (c), S-shaped bodies (s) and immature glomeruli (gl), in grey. A: In the fifth week of gestation the ureteric bud grows out from the mesonephric duct into metanephric mesenchyme. B: In week 6 the bud branches once and mesenchyme condenses around the ampullae. C: Comma and S-shaped bodies are formed by the eighth week. D: The first glomeruli are formed by the ninth week; further branching of the ureteric bud and mesenchymal condensation continues in the nephrogenic cortex.

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Timing of nephrogenic events In humans, metanephric kidney development begins at day 28 after fertilization when the ureteric bud sprouts from the distal part of the mesonephric duct (Larsen 1993). By day 32, the tip (ampulla) of the bud penetrates a portion of sacral intermediate mesenchyme called the metanephric blastema, and this condenses around the growing ampulla. The first glomeruli form by 8–9 weeks and nephrogenesis continues in the cortex of the fetal kidney until 34 weeks (Potter 1972). Nephrons elongate and continue to differentiate postnatally but no new nephrons are formed. In mice, the ureteric bud enters the metanephric mesenchyme by embryonic day 11.5, the first glomeruli form by embryonic day 14 and nephrogenesis continues for 14 days after birth. The timing of nephrogenesis in humans/mice is shown in Table 3.1. Differentiation of the ureteric bud and its derivatives Branching of the ureteric bud. As the ureteric bud grows into the metanephric blastema it becomes invested with condensed mesenchyme and the ampullary tip begins to divide. This process of growth and branching occurs repeatedly during nephrogenesis, particularly in the outer or nephrogenic cortex where new nephric units are formed until around 34 weeks of human gestation (Potter 1972). It leads to an arborialized (tree-like) collecting duct system connected to nephrons which develop concurrently from mesenchymal condensates adjacent to the ampullary tips (see below). There is great inter-species variation in the number of nephrons which reflects the number of branches of the ureteric bud required to both induce these nephrons and form the collecting ducts to drain urine from them. It has been estimated that it requires 9–10 branching generations in mice to obtain 10–20,000 nephrons and a further 10 generations to give rise to one million nephrons in each human kidney (Ekblom et al. 1994). Formation of the renal pelvis and calyces. The mature collecting ducts drain into minor calyces which in turn connect to the major calyces of the renal pelvis before the ureter. These intervening structures are formed by the coalescence of many of the early branches of the ureteric bud. This concept of remodelling has been recognized since Kampmeier (1926) Table 3.1 Timing of nephrogenic events Structure Pronephros Appears Regresses Mesonephros Appears Regresses Metanephros Renal pelvis Collecting tubules/nephrons Glomeruli Nephrogenesis ceases Length of gestation *Time

Human

Mouse

22 days 25 days

9 days 10 days

24 days 16 weeks 32 days 33 days 44 days 9 weeks 34–36 weeks 40 weeks

10 days 14 days 11.5 days 12.5 days 13 days 14 days 14 days after birth 20 days

of first appearance of renal structures during human and murine nephrogenesis, unless otherwise stated.

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described ‘vestigial’ and ‘provisional’ zones in the human medulla in which nephrons initially developed but subsequently degenerated. The exact number of generations of branches which are remodeled is unknown although Potter (1972) estimated that the first 3–5 generations form the pelvis and the next 3–5 give rise to the minor calyces and papillae. Nephrons which were initially attached to these early branches were described by Potter to either transfer to a later branch or degenerate during development. Differentiation of the mesenchyme and its derivatives. Each nephron develops from mesenchyme adjacent to an ampullary tip of the ureteric bud. The mesenchyme is initially loosely arranged but the cells destined to become nephrons condense around the bud tips and undergo phenotypic transformation into epithelial renal vesicles. Each vesicle elongates to form a comma shape which folds back on itself to become an S-shaped body. The distal portion of the S-shape elongates and differentiates into proximal convoluted tubule, the descending and ascending limbs of the loop of Henle and the distal convoluted tubule, which fuses with the adjacent branch of the ureteric bud to form a continuous functional unit. The proximal S-shape is destined to become the glomerular epithelium and capillaries develop in the glomerular crevice, probably by a process known as vasculogenesis rather than angiogenesis (see below; Woolf and Loughna 1998). The primitive multilayered visceral glomerular epithelium subsequently forms a monolayer of podocytes, separated from glomerular capillary loops by glomerular basement membrane, which is synthesized by both the endothelium and epithelium (Abrahamson and Leardkamolkarn 1991). The first glomeruli are formed by 9 weeks of human gestation, nephron formation ceases around 34 weeks of gestation. Basic mechanisms of development Nephrogenesis involves a balance between key events at a cellular level including proliferation, death, differentiation, and morphogenesis (Table 3.2). Disruption of this balance has been implicated in the pathogenesis of kidney malformations, cystic diseases, and tumours (Winyard et al. 1996a,b; Woolf and Winyard 1998).

Table 3.2 Proliferation, apoptosis, PAX2, WT1, and BCL2 expression in normal developing human kidneys

Undifferentiated mesenchyme Mesenchymal condensates and vesicles S-shaped bodies Glomerular podocytes Tips of ureteric bud (ampullae) Immature collecting ducts Mature collecting ducts

Proliferation

Apoptosis

PAX2

WT1

BCL2

rare  

rare  

 

 

 

 rare    rare 

    

    rare 

    

    

This describes the extent of proliferation and apoptosis as assessed by PCNA immunohistochemistry and propidium iodide staining respectively, and the distribution of PAX2, WT1, and BCL2 proteins using immunohistochemistry. ‘’ Indicates not detected/no staining, ‘rare ’ indicates that rare cells were apoptotic/less than 10 per cent of cells showed positive immunostaining and ‘’ to ‘’ indicates increasing levels of apoptosis/increasing percentage of positive cells or intensity of staining.

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Cell proliferation The adult mammalian kidney develops from less than a thousand cells at its inception to many millions in the mature organ and it is self evident that this process requires extensive cell proliferation. The most actively proliferating areas of the metanephros are the tips of the ureteric bud branches and the adjacent mesenchymal cells in the nephrogenic cortex (Fig. 3.4A) (Winyard et al. 1996b). Stem cells are also thought to reside in the undifferentiated renal mesenchyme and it is postulated that these divide to generate a copy of themselves and another cell which differentiates into either nephron epithelia or interstitial cells (Burrow and Wilson 1993). These cells are potentially important in the context of renal malformations since, if they are still present, it may be possible to stimulate elements of normal nephron formation if the appropriate inductive signals can be defined. True stem cells are not considered to be present in the mature adult kidney, although some cells can dedifferentiate after insults such as acute tubular necrosis to regenerate some parts of the nephron (Kawaida et al. 1994).

Fig. 3.4 Proliferation, apoptosis, and protein distribution in normal human renal development. Sections of the nephrogenic cortex from early to mid gestation, counterstained with methyl green or haematoxylin. A and C–E show immunohistochemistry. A: proliferation marker PCNA; B: in situ end labelling; C and D: transcription factors PAX2 and WTI; E: survival factor BCL2. A: Active proliferation in cells at the tips of the ureteric bud (u), renal vesicles (v) and S-shaped bodies (s). B: Apoptosis (arrows) in early nephron precursors such as comma shaped bodies (c). C: Strong PAX2 protein expression in the tips of the ureteric bud (b) and in mesenchymal condensates and vesicles (v); PAX2 expression is not detected in the uninduced mesenchyme. D: Weak WT1 expression in condensates and renal vesicles. WT1 expression is increased as glomerular podocytes are formed (not shown). E: Marked BCL2 expression in condensed mesenchyme, rapidly downregulated by the S-shaped body stage of nephron formation. Bar (m) corresponds to 60 in A–C and 100 in D and E. (See Plate 4 of the Colour Plate Section at the centre of this book.)

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Cell death/apoptosis Kampmeier (1926) reported ‘vestigial’ and ‘provisional’ zones in the human metanephros in which the first three generations of nephrons are destined to die before birth. Potter (1972) also suggested that early nephrons may die unless they moved their attachments to later branches of the ureteric bud. It is only recently, however, that the extent and mechanism of cell death has been investigated: In the rat, Coles et al. (1993) estimated that as many as 50 per cent of the cells which are born in the developing kidney will die by a process known as apoptosis, or programmed cell death. Prominent apoptosis also occurs in human renal development (Winyard et al. 1996a). The major sites of apoptosis are early nephron precursors (comma and S-shaped bodies; Fig. 3.4B) and the medulla, locations in which cell death may be important for morphogenesis and collecting duct remodelling, respectively (see above). Interestingly, therapy with some growth factors, including epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2), reduces renal cell death in both the whole animal and organ culture (Koseki et al. 1992; Coles et al. 1993; Perantoni et al. 1995). This finding is consistent with the possibility that the cells normally die during development because they lack sufficient survival factors. Excess apoptosis has also been reported in congenital renal malformations (Winyard et al. 1996a) which raises the tantalizing possibility that normal development could be rescued, at least in part, by blocking apoptosis. Differentiation The process by which cells acquire a specialized phenotype is known as differentiation. A classic example occurs in the renal mesenchyme where some cells undergo epithelial differentiation to form nephrons while others differentiate into stromal cells, or interstitial fibroblasts. Terminal differentiation occurs at a later stage and includes the specialization of cells within nephron precursors into glomerular parietal and visceral epithelia, proximal tubule, and loop of Henle cells. The term ‘lineage’ describes the series of phenotypes as a precursor differentiates into a mature cell. Morphogenesis The processes outlined above occur in individual cells whereas ‘morphogenesis’ describes the developmental process by which groups of cells acquire complex three-dimensional shapes. Examples include the formation of nephron tubules from renal mesenchymal cells, serial branching of the ureteric bud to form collecting ducts, and capillary formation. Molecular biology of nephrogenesis The molecular biology of nephrogenesis can be reviewed in several ways. One approach is to define critical stages of development and then consider which molecules control these stages. Using this scheme, crucial stages should include, for example, outgrowth of the ureteric bud, initiation of bud branching, elongation of bud derivatives, formation of the renal pelvis, mesenchymal condensation, nephron formation, and terminal differentiation. Several excellent recent reviews have used this format (Davies and Davey 1999; Woolf 1999). An alternative approach is to consider the role of individual molecules over the whole of nephrogenesis. This method is preferred here because it integrates the roles of critical molecules expressed at different stages and it is more accessible to readers with a genetic research background.

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Several classes of molecules are expressed during nephrogenesis, including transcription factors, growth factors, survival factors, and adhesion molecules. These categories are not mutually exclusive since certain molecules fall into two categories, an example being epidermal growth factor which appears to act as both a survival factor for renal mesenchyme (see above) and a growth and differentiation factor. Transcription factors Transcription factors regulate the expression of other genes, acting as ‘master regulators’ to set up basic embryonic pattern formation. They contain DNA binding domains which recognize specific sequences and hence modulate the transcription of mRNA from the target gene. The specific genetic targets of many of the transcription factors are, however, currently unknown. PAX2, WT1, EYA1, HOX, MYC, and BF2 transcription factors are reviewed below, whilst other important factors include EMX2 (Miyamoto et al. 1997), LIM1 (Shawlot and Behringer 1995), SOX9 (Bell et al. 1997), POD1 (Quaggin et al. 1998), and LMX1B (Chen et al. 1998). PAX2 PAX genes are a family of transcription factors with roles in control of embryonic patterning and cell specification in a number of organisms including Drosophila, zebrafish, frog, turtle, chick, mouse, and human (Halder et al. 1995). Only PAX2 and PAX8 are expressed in the developing kidney and there is increasing evidence that PAX2 has a critical role in normal and abnormal renal development particularly during mesenchymal condensation and epithelial transformation (Rothenpieler and Dressler 1993). Little is known about the role of PAX8, in contrast, although it is unlikely to have a major role in nephrogenesis since lack of this molecule perturbs thyroid, rather than kidney development (Mansouri et al. 1998). In the mouse, PAX2 mRNA is expressed in the mesonephric duct, the tips of the ureteric bud and the condensing mesenchyme (Dressler et al. 1990). This pattern is repeated in the outer cortex throughout nephrogenesis. Expression of the protein persists in nephron precursors such as the comma and S-shaped bodies but then decreases as these epithelia mature (Dressler and Douglas 1992). In contrast, there is little expression in ureteric bud derivatives aside from low levels in the collecting ducts. In early development of the human excretory system, PAX2 protein is observed in the mesonephric duct and distal-type mesonephric tubules, and in both the central ureteric bud stalk and peripheral bud tips (Winyard et al. 1996a). Later in human development, the distribution pattern is similar to the mouse, with PAX2-positive cells confined to ureteric bud tips, and condensing mesenchyme, comma, and S-shaped bodies (Fig. 3.4C). A series of experiments in mice and mutation analysis in humans have highlighted the critical importance of PAX2 in renal development and, taken collectively, suggest that this transcription factor has roles in mesenchyme to epithelial transformation and cell proliferation. Mice with decreased levels of PAX2 have aberrant kidney development: heterozygous mutations causes hypoplastic kidneys with reduced branching of the ureteric bud, reduced numbers of nephrons and cortical thinning whilst homozygous null mutants lack mesonephric tubules and the metanephroi fail to form because the ureteric buds are absent (Torres et al. 1995). In organ culture, addition of antisense PAX2 DNA-oligonucleotides leads to repression of PAX2 protein (Dressler et al. 1993) which causes inhibition of mesenchymal condensation and blocks the transition of mesenchyme to epithelium. Interestingly, the mesonephric tubules

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are also formed by mesenchyme to epithelium conversion which implicates PAX2 as an essential molecule in this process (Torres et al. 1995). PAX2 mutations involving a single nucleotide deletion within the conserved octapeptide sequence in exon 5 have been described in the human ‘renal-coloboma’ syndrome, which consists of optic nerve colobomas, renal anomalies, and vesicoureteral reflux (Sanyanusin et al. 1995). A similar mouse mutation (PAX2 (1Neu)) has also been described (Favor et al. 1996). Overexpression of PAX2 causes kidney abnormalities including cystic tubular changes, proteinuria and renal failure in mice (Dressler et al. 1993) and PAX2 is also overexpressed in some highly proliferative human cystic epithelia (Winyard et al. 1996a). Several other lines of evidence linking PAX gene expression and proliferation, including: high levels of PAX2 in renal clear cell carcinoma lines and the decline in rates of proliferation when these cells are treated with PAX2 antisense (Gnarra and Dressler 1995), cell transformation by overexpression of PAX (Maulbecker and Gruss 1993), and persistent expression of PAX genes in human Wilms tumours (Dressler and Douglas 1992). One of the downstream genetic targets of PAX2 during nephrogenesis may be the WT1 transcription factor (see below) since human PAX2 binds to two sites in the WT1 promoter sequence and causes up to a 35-fold increase in expression, as assessed with a reporter gene fused to the PAX2 binding sites on WT1 (McConnell et al. 1997). Interestingly, there is also evidence that WT1 downregulates PAX2, which raises the possibility that PAX2-mediated upregulation of WT1 during nephrogenesis leads to negative feedback and decreased PAX2 expression (Ryan et al. 1995). WT1 WT1 gene, which is mutated in a small percentage of Wilms tumours, encodes a transcription factor protein containing four zinc-finger DNA binding motifs (Pritchard-Jones and Hawkins 1997). Several studies have documented expression of both WT1 mRNA and protein during general mammalian development (Armstrong et al. 1993) and specifically in the urogenital system and Wilms tumours (Pelletier et al. 1991; Winyard et al. 1996a). During early human nephrogenesis, WT1 mRNA is expressed in the mesonephric glomeruli and at low levels in condensing metanephric mesenchyme. As the comma and S-shaped bodies develop, levels of WT1 increase and become restricted to the visceral glomerular epithelia (Winyard et al. 1996a) (Fig. 3.4D). Expression is restricted to podocytes in the adult kidney. Increased expression of WT1 has also been reported in Wilms tumours (Pritchard-Jones et al. 1990). The metanephros does not form in WT1 null mutant mice, and animals die around embryonic day 14 from defects in mesothelial-derived components in the heart and lungs. Small numbers of normal appearing mesonephric tubules form earlier in development but the ureteric bud fails to branch from the Wolffian duct and the intermediate mesoderm, which would normally give rise to the nephrons, dies by apoptosis (Kreidberg et al. 1993). Initial reports suggested that explants of mutant blastema do not form nephrons following heterologous culture with spinal cord and that PAX2 is not expressed in this mesenchyme (Kreidberg et al. 1993). Recently, however, Donovan et al. (1999) have demonstrated that mRNA for PAX2, and several other early markers of induction, are present in mutant WT1 / mesenchyme. This suggests that WT1 is essential for ureteric bud outgrowth and mesenchyme survival during early development, but is not required for initial mesenchymal differentiation. Several human syndromes are associated with WT1 mutations. Denys–Drash syndrome consists of genitourinary abnormalities, including ambiguous genitalia in 46 XY males,

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nephrotic syndrome with mesangial sclerosis leading to renal failure, and a predisposition to Wilms tumour (Little and Wells 1997). This is caused by point mutations of WT1, predominantly affecting the zinc finger DNA-binding domains and there is now a mouse model of this syndrome (Patek et al. 1999). The WAGR syndrome consists of Wilms tumour, aniridia, genitourinary abnormalities including gonadoblastoma, and mental retardation. Frasier syndrome is characterized by focal glomerular sclerosis with progressive renal failure and gonadal dysgenesis. This is caused by intronic point mutations of WT1, which affect the balance between different WT1 splice isoforms (Klamt et al. 1998). WT1 acts as a transcriptional repressor during nephrogenesis and several regulatory targets have been proposed including the insulin-like growth factor axis, early growth response gene 1 (EGR1) (Rackley et al. 1995). Ryan et al. (1995) moreover, demonstrated high-affinity WT1 binding sites in the 5 untranslated sequence of PAX2 and that WT1 represses expression of a reporter gene fused to a fragment of the PAX2 gene including these potential regulatory sequences. This experiment, taken together with the overlapping metanephric expression patterns of PAX2 and WT1, suggests that WT1 may downregulate PAX2 during nephrogenesis. In addition, WT1 may act as a powerful autorepressor since there are multiple WT1 binding sites in its own promoter (Rupprecht et al. 1994). EYA1 EYA1 is the mammalian homologue of the transcriptional co-activator ‘eyes absent’ gene, which is required for normal eye specification in Drosophila. In mice, EYA1 is widely expressed in the ear, branchial arches, and metanephric mesenchyme during development. Homozygote EYA1 null mutant mice have a correspondingly wide variety of abnormalities including craniofacial and skeletal defects, and absent ears (Xu et al. 1999). Affected mice, which die at birth, also lack kidneys because of defective ureteric bud outgrowth leading to failure of metanephric induction and increased mesenchymal apoptosis. Rare, straindependant, renal defects occur in EYA1 / heterozygotes, including hypoplasia and unilateral agenesis. Mutations of the human EYA1 gene occur in 20–25 per cent of patients with branchio-oto-renal syndrome (Abdelhak et al. 1997). This is characterized by a combination of hearing loss, preauricular pits, branchial fistulae, and variable renal anomalies including agenesis, hypoplasia, and dysplasia. HOX Vertebrate HOX genes encode homeodomain transcription factors which specify positional information along the anterior posterior axis. HOXA11 mRNA is normally expressed in metanephric mesenchyme (Hsieh-Li et al. 1995) but individual null mutations of these genes do not have kidney abnormalities. In contrast, double knockouts generated by interbreeding of HOXA11 and HOXD11 mutants have renal agenesis or hypoplasia (Davis et al. 1995). MYC C-myc RNA is expressed in uninduced mesenchyme and in very early epithelial structures in the mouse. Its expression is downregulated as the kidney matures and its expression pattern appears to correlate with areas of cellular proliferation during nephrogenesis (Schmid et al. 1989). N-myc is upregulated during the mesenchyme to epithelium

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conversion, where it appears to be a marker of induction, and expression is confined to the condensates, comma, and S-shaped bodies (Mugrauer and Ekblom 1991). Homozygous nullmutant mice for N-myc have mesonephroi which are hypoplastic, with increased apoptotic cells and the embryos die at around embryonic day 11 (Stanton and Parada 1992). The L-myc is restricted to the ureter of the mature kidney and it is not expressed by proliferating or embryonic cells (Mugrauer and Ekblom 1991). BF2 BF2 is one of the few genes which have been implicated in formation of interstitial cells from the mesenchyme lineage. It is a member of the winged helix family of transcription factors which are related to the Drosophila fork head gene (Lai et al. 1993). In mice, BF2 is expressed in the cells immediately surrounding condensed mesenchyme cells which express PAX2. Mice with null mutations have rudimentary, fused kidneys and die soon after birth (Hatini et al. 1996). Interestingly, the mesenchyme condenses in the null mutant mice but does not develop any further and neither comma nor S-shaped bodies are formed. The ureteric bud also fails to branch normally and RET (see below) is widely distributed in the bud epithelium, rather than confined to the bud tips. It therefore seems likely that BF2 modulates expression of a factor, or factors, from the ‘uninduced’ cells which is essential for both ureteric bud growth and maturation of pretubular aggregates. This factor has not yet been identified. Growth factors/growth factor receptors Growth factors and their cell surface receptors play an important role in nephrogenesis. There are three modes of action: paracrine factors are secreted by one cell and act on neighbouring cells, autocrine factors act on the producing cell, and juxtacrine factors become inserted into the plasma membrane of the producing cell in order to interact with receptors on adjoining cells. The growth factors bind to specific cell surface receptors, mainly receptor tyrosine kinases, which dimerize and become autophosphorylated and transduce signals into the cell. These signals may stimulate many different processes including cell division, cell survival, apoptosis, differentiation, and morphogenesis. The glial cell line-derived neurotrophic factor (GDNF), hepatocyte growth factor (HGF), epidermal growth factor (EGF), transforming growth factor (TGF) beta, vascular endothelial growth factor (VEGF), and WNT signalling systems are reviewed below. Readers are referred to alternative sources for accounts of fibroblast growth factors (FGF) (Perantoni et al. 1995; Qiao et al. 1996), insulin-like growth factors (IGF) (Rogers et al. 1991), platelet-derived growth factor (PDGF) (Leveen et al. 1994), TGF alpha (Rogers et al. 1992), and vascular endothelial growth factor (Tufro et al. 1999). GDNF/RET GDNF is the ligand for the receptor tyrosine kinase RET (Vega et al. 1996), although binding also requires the adapter molecule, GDNF receptor  (GDNFR). RET is expressed in the mesonephric duct and the ureteric bud arising from it in early development, and then at the branching tips of the ureteric bud throughout metanephrogenesis (Schuchardt et al. 1994). GDNF is expressed by condensing renal mesenchyme adjacent to these bud tips, while GDNFR is expressed in the same cells as RET and, at lower levels, in renal mesenchyme (Towers et al. 1998).

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GDNF and RET are absolutely essential for normal renal development since transgenic mice with homozygous null mutations of either factor fail to develop kidneys or have severe dysplasia (Schuchardt et al. 1994; Pichel et al. 1996). The major postulated role of this signalling system is in the initial outgrowth of the ureteric bud from the mesonephric duct and its subsequent branching; levels of GDNF appear to be critical for this process. Excess GDNF, for example, promotes formation of primary ureteric buds from the mesonephric duct and increases ureteric bud branching (Sainio et al. 1997) and exogenous GDNF stimulates branching morphogenesis of a ureteric bud-derived cell line (Qiao et al. 1999). Neutralizing antibodies against GDNF, in contrast, inhibit branching morphogenesis and decreased ureteric bud branches are observed in GDNF/ heterozygous mice (Vega et al. 1996). A further possible role for GDNF may be modulation of cell survival since GDNF prevents apoptosis of ureteric bud cultured as a monolayer (Towers et al. 1998). Other members of the GDNF family have recently been implicated in collecting duct morphogenesis including persephin (Milbrandt et al. 1998) and neurturin (Davies et al. 1999). HGF/MET Hepatocyte growth factor, or scatter factor as it is also known, is the ligand for the MET receptor tyrosine kinase (Bottaro et al. 1991). Expression patterns of HGF and MET are similar in human and murine nephrogenesis: HGF is expressed in the renal mesenchyme, particularly in the cortex as the kidney matures, whilst MET mainly localizes to developing epithelia (Kolatsi-Joannou et al. 1997; Sonnenberg et al. 1993). Mice with homozygous HGF or MET null mutations die around embryonic day 13–14 with placental, liver, and muscle abnormalities (Schmidt et al. 1995). Early nephrogenesis appears grossly normal in these animals, which is surprising since blockade of this signalling system perturbs metanephric development in organ culture (Santos et al. 1994; Woolf et al. 1995). Exogenous HGF, moreover, causes branching morphogenesis of the Madin Darby Canine Kidney (MDCK) collecting duct-derived cell line in culture (Montesano et al. 1991) and overexpression leads to prominent tubular cystic disease and progressive glomerulosclerosis (Takayama et al. 1997). This system is therefore likely to be important, but not critical, for stimulating cell proliferation and organ growth during nephrogenesis. EGF Epidermal growth factor (EGF) and its embryonic homologue transforming growth factor alpha (TGF) both bind to the epidermal growth factor receptor (EGFR). Rogers et al. (1992), showed that E13 rat metanephroi produce tgf and nephrogenesis is perturbed by blocking antibodies against tgf. EGF is a potent inhibitor of cell death within the developing kidney in vivo (Coles et al. 1993) and rescues isolated renal mesenchyme from apoptosis in vitro (Koseki et al. 1992). EGF has also been reported to increase PAX2 levels in rabbit proximal tubule cells (Liu et al. 1997). Interestingly, mice with null mutations of egf receptors have different renal phenotypes depending on their genetic background (Threadgill et al. 1995). TGF Transforming growth factor beta 1 (TGF1) is the prototypic molecule of a large family of growth factors, and signalling is transduced via cell surface Type I and Type II receptors

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(TGFR1 and TGFR2) (Wrana 1998). In early mouse kidney development, TGF1 mRNA is expressed in the metanephric mesenchyme (Lehnert and Akhurst 1988; Schmid et al. 1991) and the protein is distributed in both the mesenchyme and ureteric bud (Rogers et al. 1993). At similar stages, receptor transcripts are expressed in both renal mesenchyme and immature epithelia (Mariano et al. 1998). Both the ligand and receptors are also expressed in the developing vasculature. Homozygous null mutants of TGF1 have normal kidneys, which is presumed to result from transplacental transfer of maternal circulating growth factor (Letterio et al. 1994), but perturbation of TGF1 in organ culture markedly disrupts nephrogenesis: exogenous TGF1 inhibits tubulogenesis whereas blocking antibodies enhance nephron formation (Rogers et al. 1993). Tubulogenesis of MDCK cells in tissue culture is also inhibited by TGF1 (Sakurai et al. 1997). This suggests a role for TGF1 in controlling nephron number, which is intriguing for several reasons. First, urinary tract obstruction disrupts the programme of nephrogenesis (Attar et al. 1998) and this could potentially be modulated by the upregulation of TGF1 reported in this condition (Medjebeur et al. 1997). Second, this cytokine may modulate PAX2 levels, which is critical for nephrogenesis as described above, since TGF1 decreases PAX2 levels in cultured rabbit proximal tubule cells by decreasing PAX2 mRNA stability (Liu et al. 1997). And finally, our group have recently implicated TGF1 in the pathogenesis of human dysplastic kidneys, which characteristically lack normal nephrons (Yang et al. 2000). Knockout of the closely related TGF2 ligand also causes urogenital abnormalities, although only a limited number of animals were described in the initial report (Sanford et al. 1997). Other members of the TGF superfamily, such as the bone morphogenetic proteins (BMPs) are also important during nephrogenesis. The BMP7 is expressed by the branches of the ureteric bud and is upregulated in primitive nephrons, whilst nephrogenesis is markedly impaired in BMP7 null-mutant mice, which form only a few nephrons and ureteric bud branches (Dudley et al. 1995; Luo et al. 1995). In early development, BMP4 expression is essential for nephric duct formation from intermediate mesoderm (Obara-Ishihara et al. 1999). Later in nephrogenesis, BMP4 is expressed in the non-condensed mesenchyme around the stalk of the ureteric bud, heterozygote BMP4/ knockouts develop hypoplastic or dysplastic kidneys and up- or downregulation of the BMP4 axis modulates ureteric bud elongation/growth in organ culture (Miyazaki et al. 1999).

WNT The WNT gene family, which consists of around 20 members, encodes secreted signalling molecules and several of these have been implicated in nephrogenesis. WNT4 is upregulated in renal mesenchymal cells as they differentiate and nephrogenesis is ‘frozen’ at the condensate stage in WNT4 null mutant mice (Stark et al. 1994). WNT4 is, moreover, sufficient to trigger tubulogenesis in isolated metanephric mesenchyme, in a process which is dependent on cell contact and sulphated glycosaminoglycans (Kispert et al. 1998). Isolated dorsal spinal cord also induces tubulogenesis and it may act by mimicking the normal mesenchymal action of WNT4 since spinal cord mediated induction still occurs with WNT4 mutant mesenchyme (Kispert et al. 1998). The WNT11 is expressed at the tips of the ureteric bud (Lako et al. 1998), but is not sufficient to induce tubulogenesis (Kispert et al. 1998). The WNT1-transfected

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fibroblasts induce nephron formation when co-cultured with isolated renal mesenchyme, although WNT1 is not an endogenous metanephric molecule (Herzlinger et al. 1994). Survival/proliferation factors Survival and proliferation are key cellular events and a number of genes have been identified in renal development which are linked to these processes. BCL2 The BCL2 gene, originally discovered in human follicular B-cell lymphomas, is the prototypic member of an evolutionarily conserved gene family involved in the control of apoptotic cell death (Knudson and Korsmeyer 1997). BCL2 is gene expressed in a wide variety of tissues during both murine and human development including the nervous system, immunological tissues, and epithelia (LeBrun et al. 1993). In the kidney, BCL2 protein is upregulated in the mesenchyme as it condenses around the ureteric bud tips (Fig. 3.4E) but is then rapidly down regulated in the comma and S-shaped bodies and is barely detectable in the adult organ (LeBrun et al. 1993; Winyard et al. 1996a). Homozygous BCL2 null mutants have a number of defects in haematopoiesis, hair, and kidney development. BCL2/ kidneys were initially reported to resemble polycystic kidneys with epithelial hyperproliferation and dilation of proximal and distal tubular segments (Veis et al. 1994). Further analysis, however, suggests that the abnormalities are more complex: prior to birth ‘fulminant’ apoptosis leads to small hypoplastic kidneys, with fewer nephrons and smaller nephrogenic zones (Sorenson et al. 1995), whilst cysts develop postnatally accompanied by increased proliferation in both cortex and medulla and apoptosis of interstitial cells (Sorenson et al. 1996). Hence, primary deregulation of cell survival in the BCL2 null mutant mice appears to cause a secondary upregulation of proliferation in the kidney. p53 Mutations of p53 lead to tumours in humans and it is therefore thought to act as a tumour suppresser gene. The p53 mRNA is normally expressed in comma and S-shaped bodies in the mouse but homozygous deletion of p53 has no affect on murine renal development (Donehower et al. 1995). In contrast, overexpression of p53 leads to smaller kidneys with fewer nephrons which is thought to be caused by incomplete mesenchymal differentiation (Godley et al. 1996). p57-KIP2 (CDKN1c) Cyclin-dependent kinases are essential for regulation of the cell cycle and proliferation. Cyclin kinase inhibitors such as p57-KIP2 block proliferation by binding to the kinases in G1/S phase. The p57-kip2 gene is expressed in podocytes in glomeruli and stromal cells between the renal tubules during renal development and null mutants have fewer renal tubules and small inner medullary pyramids (Zhang et al. 1997). Poorly formed medullary pyramids are also seen in human Beckwith–Wiedemann syndrome which is caused by loss of the imprinted, expressed maternal allele on chromosome 11p15.5. This site is close to the p57KIP2 gene and heterozygous mutations have been reported in some Beckwith–Wiedemann patients. The mechanism for aberrant development in the null mutant mice has not been

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reported but it seems logical that cell proliferation will be more prominent in null mutants and this may perturb apoptotic remodelling of the medulla.

Cell adhesion molecules Adhesion molecules mediate two forms of attachment, namely cell–cell and cell–matrix adhesion. Examples of cell–cell adhesion proteins are the calcium independent neural cell adhesion molecule (NCAM) (Klein et al. 1988a; Bellairs et al. 1995) and calcium-dependent E-cadherin (also known as uvomorulin) (Vestweber and Kemler 1985). Molecules involved in cell–matrix adhesion include the collagens, fibronectin (Bellairs et al. 1995), galectin 3 (Bao and Hughes 1995; Winyard et al. 1997), KAL (Duke et al. 1995; Soussi-Yanicostas et al. 1996), laminins (Klein et al. 1988b), nidogen (Ekblom et al. 1994), tenascin (Aufderheide et al. 1987), and integrin cell surface receptors (Kreidberg et al. 1996; Muller et al. 1997). Adhesion molecules may also have additional roles since some molecules, particularly proteoglycans, are able to sequester growth factors, hence modulating binding of growth factors to their receptors (Vainio et al. 1989a). It is difficult to separate the specific roles of most individual adhesion molecules so a general overview is given here, followed by a more detailed description of integrins, galectin 3 and KAL. Wallner et al. (1998) have also recently reviewed this topic. Overview Uninduced metanephrogenic mesenchyme expresses collagen I, III, and fibronectin in the mouse (Ekblom et al. 1981). The cell surface neural cell adhesion molecule (NCAM) and the proteoglycan syndecan, which acts as a receptor for interstitial matrix molecules are also expressed by the uninduced mesenchyme (Klein et al. 1988a; Vainio et al. 1989b). Following condensation, the mesenchymal cells begin their transition to polarized epithelia. Levels of syndecan increase transiently but then decrease, NCAM decreases and cells stop expressing interstitial collagens and fibronectin and begin to express uvomorulin and collagen type IV, and laminin A chain (Ekblom 1981; Ekblom et al. 1981; Vestweber and Kemler 1985; Klein et al. 1988a; Vainio et al. 1989b). Laminin is a large multidomain cruciform glycoprotein found in basement membranes, consisting of three different polypeptide chains: A, B1, and B2. Uninduced metanephric mesenchyme expresses B1 and B2 whilst the laminin A chain is expressed at the onset of epithelial polarization. Mesenchymal cells that do not undergo epithelial transformation continue to express interstitial collagens and fibronectin and upregulate tenascin as the cells around them are condensing (Aufderheide et al. 1987). Tubule formation is perturbed in mouse metanephric organ culture by antibodies to fragments E3 and E8 of the laminin A chain, because the mesenchymal cells are unable to convert to polarized epithelial cells (Klein et al. 1988b). The cell-surface receptor for the E8 fragment of laminin is the 61integrin, described below. The laminin E3 fragment binds the dystroglycan complex and antibodies that interfere with this binding also inhibit the conversion of mesenchyme to epithelia (Durbeej et al. 1995). Another basement membrane glycoprotein known as either nidogen or entactin is produced by mesenchymal cells and binds to domain III on the laminin B2 chain. This laminin B2/nidogen binding is critical for the production of epithelial basement membranes of epithelial structures formed during early nephrogenesis (Ekblom et al. 1994).

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Proteoglycans, such as heparan and chondroitin sulphate, bind/sequester diverse factors and are crucial for normal nephrogenesis (Davies et al. 1995). Proteoglycans, for example, are postulated to bind the N-glycosylated KAL protein mutated in X-linked Kallmann syndrome (Soussi-Yanicostas et al. 1996). KAL is expressed in the cortex during nephrogenesis (Duke et al. 1995) and up to 40 per cent of Kallmann syndrome patients have renal agenesis (Kirk et al. 1994). Heparan sulfate binds fibroblast growth factors, hence preventing their degradation and facilitating receptor binding (Kiefer et al. 1990), syndecan binds basic fibrobast growth factor (FGF2) (Elenius et al. 1992), and betaglycan binds TGF (Ruoslahti and Yamaguchi 1991). Integrins Integrins are transmembrane heterodimeric glycoprotein complexes consisting of  and  chains with diverse roles in cell–cell and cell–matrix adhesion, polarity, migration, and angiogenesis (Wallner et al. 1998). Specific ligand binding is determined by the particular combination of  and  chains. Integrin subunits expressed during nephrogenesis include: 1 in uninduced mesenchyme (Korhonen et al. 1990a), 2 in glomerular endothelium and distal tubules (Korhonen et al. 1990a), 3 in maturing podocytes (Ekblom 1996), 6 and 8 in the mesenchyme undergoing condensation and epithelial transformation adjacent to the ureteric bud (Falk et al. 1996; Muller et al. 1997), 1 in undifferentiated mesenchyme and glomerular endothelial cells, 3 in Bowman’s capsule and 4 in fetal collecting ducts (Korhonen et al. 1990b). Co-expression of different subunits includes 11 and 41 in uninduced mesenchyme, 21 in endothelia, and 61 in epithelia. Homozygous integrin mutant mice have a variety of defects but 3 and 8 knockouts have marked disruption of kidney development. Mice with mutations in the 3 subunit have very abnormal glomeruli containing wide capillaries, disorganized basement membrane and aberrant podocytes foot processes, plus microcystic proximal tubules and decreased branching of the medullary collecting ducts, hence suggesting roles for 3 in basement membrane organization and branching morphogenesis (Kreidberg et al. 1996). Other integrins have also been functionally implicated in tubulogenesis: blocking antibodies to the 61integrin, which is the receptor for the E8 fragment of laminin, perturb tubule formation in vitro (Sorokin et al. 1990; Falk et al. 1996) and antisense to the 2 subunit of the 21 integrin perturb cystogenesis and HGF-induced tubulogenesis of MDCK cells cultured in three dimensional collagen type I gels (Saelman et al. 1995). Mice lacking the 8 subunit exhibit profound kidney deficits with aberrant branching of the ureteric bud and formation of nephrons, which suggests, in conjunction with the expression data (Muller et al. 1997), that this molecule is essential for mesenchymal–epithelial transformation (Kreidberg et al. 1996). Aberrant expression of 1, 2, and 6 integrin subunits has also been described in human dysplastic kidneys (Daikha-Dahmane et al. 1997) Galectin 3 Galectin 3, or Mac 2 as it was formerly known, is a calcium-independent water soluble -galactoside binding lectin with a number of postulated roles in pre-mRNA splicing, oncogenic transformation, and prevention of apoptosis (Hughes 1994). Its major role, however, is likely to be in cell–matrix interactions: it binds embryonic glycoforms of laminin and fibronectin which have polylactosamine side chains and modulates laminin/integrin interactions (Sato and Hughes 1992).

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Galectin 3 is expressed in the ureteric bud lineage during human nephrogenesis (Winyard et al. 1997), but subcellular localization changes from an apical position in the actively branching tips of the ureteric bud to the basolateral domain in maturing collecting ducts. Two pieces of evidence suggest that galectin 3 may have an important role in the latter location, possibly via its interaction with laminin. First, in human cystic kidney diseases, galectin 3 is mislocalized to the apical domain (Winyard et al. 1997). The protein is unable to interact with the extracellular matrix in this location which, along with other potential roles in proliferation and in preventing apoptosis, may contribute to cyst expansion. And second, in three dimensional cell culture, MDCK cyst growth is influenced by galectin 3 expression: blocking antibodies increase cyst growth and exogenous galectin-3 slows cyst growth (Bao and Hughes 1995). These results are intriguing, but it should be noted that the kidneys appear normal in the galectin 3 knockout (Colnot et al. 1998), which suggests that other molecules must be able to compensate for lack of galectin 3 in vivo. Other molecules There are a number of functionally important metanephric molecules which do not fit comfortably into any of the categories outlined above. These include the renin–angiotensin system, described below; cyclo-oxygenase2 (cox2) an enzyme involved in synthesis of prostaglandins (Morham et al. 1995); formins, molecules of uncertain function without which the embryonic kidneys fail to form (Maas et al. 1994); mpv17, a peroxisomal protein implicated in the integrity of the glomerular filtration barrier; retinoic-acid receptors (RAR), which are important in morphogenesis (Mendelsohn et al. 1994). Renin–angiotensin system The renin–angiotensin system has an important role in renal development. Renin, which converts angiotensinogen to angiotensin I, is widely expressed in perivascular cells in the arterial system during early nephrogenesis, but is restricted to the juxtaglomerular apparatus in the mature kidney (Gomez et al. 1988). Angiotensin II, generated from angiotensin I by angiotensin converting enzyme, binds to two types of G protein-coupled receptors, AT1 and AT2. AT1 mediates the majority of the traditionally recognized functions of angiotensin II such as vasoconstriction and stimulation of cell growth, while AT2 antagonizes some of these actions and has postulated roles in the control of apoptosis. Both receptors are expressed in the developing kidney: AT1 is expressed in S-shaped bodies, developing tubules and mature glomeruli whereas AT2 is restricted to mesenchymal cells, initially around the stalk of the ureteric bud but then extending to just outside the nephrogenic cortex, and cells between collecting ducts (Kakuchi et al. 1995). This distribution of AT2 receptors corresponds to areas with high levels of apoptosis. The wide spectrum of renal/urological abnormalities that result from targeted mutation of different components of the renin–angiotensin system are discussed in Chapter 4 by Woolf (Kidney and Lower urinary tract malformations) and were recently reviewed by Pope et al. (1999). New issues Cell lineage In conventional descriptions of nephrogenesis, there is a very clear distinction between nephrons, which are said to be derived from the metanephric mesenchyme, and collecting

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ducts, which reportedly arise from the ureteric bud. Several reports of in vitro lineage tracing experiments by Herzlinger, Qiao and colleagues suggest, however, that renal cell lineages may be more complex (Herzlinger et al. 1993; Qiao et al. 1995). They isolated pure populations of mesenchymal cells or ureteric bud epithelia from embryonic day 13 rat embryos, tagged them with lacZ and co-cultured these with non-tagged ureteric buds and mesenchyme respectively. Most cells differentiated along conventional lines but some mesenchymal cells were recruited into collecting system epithelia (Qiao et al. 1995) and a small proportion of ureteric bud cells underwent an epithelial–mesenchymal transition to form part of the metanephric blastema (Herzlinger et al. 1993). There are several other examples of epithelial to mesenchymal transformations during general development, including neural crest and endocardial cushion formation from neuroepithelium and cardiac endothelia respectively (Gilbert 1997), but these were the first reports of this process in renal development. This may become particularly relevant in the future because there is a growing body of evidence that epithelial to mesenchymal transdifferentiation is a critical event in both human kidney malformations (Yang et al. 2000) and the pathogenesis of end stage renal disease (Okada et al. 1996). Vascular development Embryonic blood vessels arise by either vasculogenesis, in which mesenchyme differentiates in situ to form capillary endothelia, or angiogenesis, which involves ingrowth of existing capillaries (Woolf and Loughna 1998). Renal capillaries were initially hypothesized to arise by angiogenesis, based on experiments showing that the glomeruli that develop in organ culture are avascular (Bernstein et al. 1981) and capillary loops formed when mouse metanephroi were grafted onto avian chorioallantoic membranes are of host origin (Sariola et al. 1983). This hypothesis has been challenged, however, by grafting experiments into the anterior eye chamber and under the capsule of neonatal mouse kidneys where glomerular endothelia are derived from the donor (Hyink et al. 1996; Loughna et al. 1997). Further support for vasculogenesis comes from recent reports that molecules which characterize endothelial precursors, such as the receptors for VEGF, are expressed by non vascularized uninduced metanephric mesenchyme and cell lines derived from this tissue (Loughna et al. 1997) and the formation of cellular masses with molecular characteristics of vessels, albeit with poorly defined lumina, in hypoxic organ culture (Loughna et al. 1998). These results suggest that vasculogenesis definitely occurs during capillary formation in the metanephros, although they do not completely exclude formation of some vascular components by angiogenesis.

References Abdelhak, S., Kalatzis, V., Heilig, R., Compain, S., Samson, D., Vincent, C., et al. (1997). A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family. Nature Genetics, 15(2), 157–64. Abrahamson, D. R. and Leardkamolkarn, V. (1991). Development of kidney tubular basement membranes. Kidney International, 39, 382–93. Armstrong, J. F., Pritchard-Jones, K., Bickmore, W. A., Hastie, N. D., and Bard, J. B. (1993). The expression of the Wilms’ tumour gene, WT1, in the developing mammalian embryo. Mechanisms of Development, 40(1–2), 85–97.

80

Paul J. D. Winyard

Attar, R., Quinn, F., Winyard, P. J., Mouriquand, P. D., Foxall, P., Hanson, M. A., et al. (1998). Shortterm urinary flow impairment deregulates PAX2 and PCNA expression and cell survival in foetal sheep kidneys. American Journal of Pathology, 152, 1225–35. Aufderheide, E., Chiquet-Ehrismann, R., and Ekblom, P. (1987). Epithelial–mesenchymal interactions in the developing kidney lead to expression of tenascin in the mesenchyme. Journal of Cell Biology, 105(1), 599–608. Bao, Q. and Hughes, C. (1995). Galectin-3 expression and effects on cyst enlargement and tubulogenesis in kidney epithelial MDCK cells cultured in three-dimensional matrices in vitro. Journal of Cell Science, 108, 2791–800. Beck, A. D. (1971). The effect of intra-uterine urinary obstruction upon the development of the fetal kidney. Journal of Urology, 105(6), 784–9. Bell, D. M., Leung, K. K., Wheatley, S. C., Ng, L. J., Zhou, S., Ling, K. W., et al. (1997). SOX9 directly regulates the type-II collagen gene. Nature Genetics, 16(2), 174–8. Bellairs, R., Lear, P., Yamada, K. M., Rutushauser, U., and Lash, J. W. (1995). Posterior extension of the chick nephric (Wolffian) duct: the role of fibronectin and NCAM polysialic acid. Developmental Dynamics, 202(4), 333–42. Bernstein, J., Cheng, F., and Roszka, J. (1981). Glomerular differentiation in metanephric culture. Laboratory Investigations, 45, 183–90. Bichet, D. G. (1998). Nephrogenic diabetes insipidus. American Journal of Medicine, 105(5), 431–42. Bottaro, D. P., Rubin, J. S., Faletto, D. L., Chan, A. M. L., Kmiecik, T. E., Vande Woude, G. F., et al. (1991). Identification of the hepotacyte growth factor as the c-met proto-oncogene product. Science, 251, 802–4. Burrow, C. and Wilson, P. D. (1993). A putative Wilms tumor-secreted growth factor activity required for primary culture of human nephroblasts. Proceedings of the National Academy of Sciences, USA, 90, 6066–70. Chen, H., Lun, Y., Ovchinnikov, D., Kokubo, H., Oberg, K. C., Pepicelli, C. V., et al. (1998). Limb and kidney defects in Lmx1b mutant mice suggest an involvement of LMX1B in human nail patella syndrome. Nature Genetics, 19(1), 51–5. Coles, H. S. R., Burne, J. F., and Raff, M. C. (1993). Large-scale normal death in the developing rat kidney and its reduction by epidermal growth factor. Development, 118(3), 777–84. Colnot, C., Fowlis, D., Ripoche, M. A., Bouchaert, I., and Poirier, F. (1998). Embryonic implantation in galectin 1/galectin 3 double mutant mice. Developmental Dynamics, 211(4), 306–13. Daikha-Dahmane, F., Narcy, F., Dommergues, M., Lacoste, M., Beziau, A., and Gubler, M. C. (1997). Distribution of alpha-integrin subunits in fetal polycystic kidney diseases. Pediatric Nephrology, 11(3), 267–73. Davies, J., Lyon, M., Gallagher, J., and Garrod, D. (1995). Sulphated proteoglycan is required for collecting duct growth and branching but not nephron formation during kidney development. Development, 121(5), 1507–17. Davies, J. A. and Davey, M. G. (1999). Collecting duct morphogenesis. Pediatric Nephrology, 13(6), 535–41. Davies, J. A., Millar, C. B., Johnson, E. M., Jr., and Milbrandt, J. (1999). Neurturin: an autocrine regulator of renal collecting duct development. Developmental Genetics, 24(3–4), 284–92. Davis, A. P., Witte, D. P., Hsieh-Li, H. M., Potter, S. S., and Capecchi, M. R. (1995). Absence of radius and ulna in mice lacking hoxa-11 and hoxd-11. Nature, 375(6534), 791–5. Donehower, L. A., Godley, L. A., Aldaz, C. M., Pyle, R., Shi, Y. P., Pinkel, D., et al. (1995). Deficiency of p53 accelerates mammary tumorigenesis in Wnt-1 transgenic mice and promotes chromosomal instability. Genes and Development, 9(7), 882–95. Donovan, M. J., Natoli, T. A., Sainio, K., Amstutz, A., Jaenisch, R., Sariola, H., et al. (1999). Initial differentiation of the metanephric mesenchyme is independent of WT1 and the ureteric bud. Developmental Genetics, 24(3–4), 252–62.

Renal development

81

Dressler, G. R., Deutsch, U., Chowdhury, K., Nornes, H. O., and Gruss, P. (1990). PAX2 a new murine paired-box-containing gene and its expression in the developing excretory system. Development, 109, 787–95. Dressler, G. R. and Douglas, E. C. (1992). Pax-2 is a DNA-binding protein expressed in embryonic kidney and Wilms tumor. Proceedings of the National Academy of Sciences, USA, 89, 1179–83. Dressler, G. R., Wilkinson, J. E., Rothenpieler, U. W., Patterson, L. T., Williams-Simons, L., and Westphal, H. (1993). Deregulation of Pax-2 expression in transgenic mice generates severe kidney abnormalities. Nature, 362, 65–7. Dudley, A. T., Lyons, K. M., and Robertson, E. J. (1995). A requirement for bone morphogenetic protein-7 during development of the mammalian kidney and eye. Genes and Development, 9(22), 2795–807. Duke, V. M., Winyard, P. J. D., Thorogood, P., Soothill, P., Bouloux, P. M., and Woolf, A. S. (1995). KAL, a gene mutated in Kallmann’s syndrome, is expressed in the first trimester of human development. Molecular and Cellular Endocrinology, 110(1–2), 73–9. Durbeej, M., Larsson, E., Ibraghimov-Beskrovnaya, O., Roberds, S. L., Campbell, K. P., and Ekblom, P. (1995). Non-muscle a-dystroglycan is involved in epithelial development. Journal of Cell Biology, 130, 79–91. Ekblom, P. (1981). Formation of basement membranes in the embryonic kidney: an immunohistological study. Journal of Cell Biology, 91(1), 1–10. Ekblom, P. (1996). Extracellular matrix and cell adhesion molecules in nephrogenesis. Experimental Nephrology, 4(2), 92–6. Ekblom, P., Ekblom, M., Fecker, L., Klein, G., Zhang, H.-Y., Kadoya, Y., et al. (1994). Role of mesenchymal nidogen for epithelial morphogenesis in vitro. Development, 120, 2003–14. Ekblom, P., Lehtonen, E., Saxen, L., and Timpl, R. (1981). Shift in collagen type as an early response to induction of the metanephric mesenchyme. Journal of Cell Biology, 89(2), 276–83. Elenius, K., Maatta, A., Salmivirta, M., and Jalkanen, M. (1992). Growth factors induce 3T3 cells to express bFGF-binding syndecan. Journal of Biology Chemistry, 267(9), 6435–41. Falk, M., Salmivirta, K., Durbeej, M., Larsson, E., Ekblom, M., Vestweber, D., et al. (1996). Integrin alpha 6B beta 1 is involved in kidney tubulogenesis in vitro. Journal of Cell Science, 109(Pt 12), 2801–10. Favor, J., Sandulache, R., Neuhauser-Klaus, A., Pretsch, W., Chatterjee, B., Senft, E., et al. (1996). The mouse PAX2 (1Neu) mutation is identical to a human PAX2 mutation in a family with renalcoloboma syndrome and results in developmental defects of the brain, ear, eye, and kidney. Proceedings of the National Academy of Sciences, USA, 93(24), 13870–5. Gage, J. C. and Sulik, K. K. (1991). Pathogenesis of ethanol-induced hydronephrosis and hydroureter as demonstrated following in vivo exposure of mouse embryos. Teratology, 44, 299–312. Gilbert, S. F. (1997). Developmental biology 5th edn. Sinauer Associates Inc, Sunderland, Massachusetts, USA. Gnarra, J. R. and Dressler, G. R. (1995). Expression of Pax-2 in human renal cell carcinoma and growth inhibition by antisense oligonucleotides. Cancer Research, 55, 4092–8. Godley, L. A., Kopp, J. B., Eckhaus, M., Paglino, J. J., Owens, J., and Vermus, H. E. (1996). Wild-type p53 transgenic mice exhibit altered differentiation of the ureteric bud and possess small kidneys. Genes and Development, 10, 863–50. Gomez, R. A., Lynch, K. R., Chevalier, R. L., Wilfong, N., Everett, A., Carey, R. M., et al. (1988). Renin and angiotensinogen gene expression in maturing rat kidney. American Journal of Physiology, 254(4, Pt 2), F582–F587. Grobstein, C. (1955). Inductive interaction in the development of the mouse metanephros. Journal of Experimental Zoology, 130, 319–40.

82

Paul J. D. Winyard

Guay-Woodford, L. M. (1998). Bartter syndrome: unraveling the pathophysiologic enigma. American Journal of Medicine, 105(2), 151–61. Halder, G., Callaerts, P., and Gehring, W. J. (1995). Induction of ectopic eyes by targeted expression of the eyeless gene in Drosophila [see comments]. Science, 267(5205), 1788–92. Hatini, V., Huh, S. O., Herzlinger, D., Soares, V. C., and Lai, E. (1996). Essential role of stromal mesenchyme in kidney morphogenesis revealed by targeted disruption of Winged Helix transcription factor BF-2. Genes and Development 10(12), 1467–78. Herzlinger, D., Abramson, R., and Cohen, D. (1993). Phenotypic conversions in renal development. Journal of Cell Science, 106, 61–4. Herzlinger, D., Qiao, J., Cohen, D., Ramakrishna, N., and Brown, A. M. (1994). Induction of kidney epithelial morphogenesis by cells expressing Wnt-1. Developmental Biology, 166(2), 815–18. Hsieh-Li, H. M., Witte, D. P., Weinstein, M., Branford, W., Li, H., Small, K., and Potter, S. S. (1995). Hoxa 11 structure, extensive antisense transcription, and function in male and female fertility. Development, 121(5), 1373–85. Hughes, R. C. (1994). Mac 2. A versatile galactose binding protein of mammalian tissues. Glycobiology, 4(1), 5–12. Hyink, D. P., Tucker, D. C., St John, P. L., Leardkamolkarn, V., Accavitti, M. A., Abrass, C. K., et al. (1996). Endogenous origin of glomerular endothelial and mesangial cells in grafts of embryonic kidneys. American Journal of Physiology, 270(5, Pt 2), F886–F899. Kakuchi, J., Ichiki, T., Kiyama, S., Hogan, B. L., Fogo, A., Inagami, T., et al. (1995). Developmental expression of renal angiotensin II receptor genes in the mouse. Kidney International, 47(1), 140–7. Kampmeier, O. F. (1926). The metanephros or so-called permanent kidney in part provisional and vestigial. Anatomical Record, 33, 115–20. Kawaida, K., Matsumoto, K., Shimazu, H., and Nakamura, T. (1994). Hepatocyte growth factor prevents acute renal failure and accelerates renal regeneration in mice. Proceedings of the National Academy of Sciences, USA, 91(10), 4357–61. Kiefer, M. C., Stephans, J. C., Crawford, K., Okino, K., and Barr, P. J. (1990). Ligand-affinity cloning and structure of a cell surface heparan sulfate proteoglycan that binds basic fibroblast growth factor. Proceedings of the National Academy of Sciences, USA, 87(18), 6985–9. Kirk, J. M. W., Grant, D. B., Besser, G. M., Shalet, S., Smith, C. S., White, M., et al. (1994). Unilateral renal aplasia in X-linked Kallmann’s Syndrome. Clinical Genetics, 46(3), 260–2. Kispert, A., Vainio, S., and McMahon, A. P. (1998). Wnt-4 is a mesenchymal signal for epithelial transformation of metanephric mesenchyme in the developing kidney. Development, 125(21), 4225–34. Klamt, B., Koziell, A., Poulat, F., Wieacker, P., Scambler, P., Berta, P., et al. (1998). Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 /KTS splice isoforms. Human Molecular Genetics, 7(4), 709–14. Klein, G., Langegger, M., Garidis, C., and Ekblom, P. (1988a). Neural cell adhesion molecules during embryonic induction and development of the kidney. Development, 102, 749–61. Klein, G., Langegger, M., Timpl, R., and Ekblom, P. (1988b). Role of laminin A chain in the development of epithelial cell polarity. Cell, 55, 331–41. Knudson, C. M. and Korsmeyer, S. J. (1997). Bcl-2 and Bax function independently to regulate cell death. Nature Genetics, 16(4), 358–63. Kolatsi-Joannou, M., Moore, R., Winyard, P. J., and Woolf, A. S. (1997). Expression of hepatocyte growth factor/scatter factor and its receptor, MET, suggests roles in human embryonic organogenesis. Pediatric Research, 41(5), 657–65. Korhonen, M., Ylanne, J., Laitinen, L., and Virtanen, I. (1990b). Distribution of beta 1 and beta 3 integrins in human foetal and adult kidney. Laboratory Investigations, 62(5), 616–25. Korhonen, M., Ylanne, J., Laitinen, L., and Virtanen, I. (1990a). The alpha 1-alpha 6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron. Journal of Cell Biology, 111(3), 1245–54.

Renal development

83

Koseki, C., Herzlinger, D., and Al-Awqati, Q. (1992). Apoptosis in metanephric development. Journal of Cell Biology, 119(5), 1327–33. Kreidberg, J. A., Donovan, M. J., Goldstein, S. L., Rennke, H., Shepherd, K., Jones, R. C., et al. (1996). Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis. Development, 122(11), 3537–47. Kreidberg, J. A., Sariola, H., Loring, J. M., Maeda, M., Pelletier, J., Housman, D., et al. (1993). WT-1 is required for early kidney development. Cell, 74, 679–91. Kriz, W. (1981). Structural organization of the renal medulla: comparative and functional aspects. American Journal of Physiology, 241(1), R3–R16. Lai, E., Clark, K. L., Burley, S. K., and Darnell, J. E. J. (1993). Hepatocyte nuclear factor 3/fork head or winged helix proteins: a family of transcription factors of diverse biologic function. Proceeding of the National Academy of Sciences, USA, 90(22), 10421–3. Lako, M., Strachan, T., Bullen, P., Wilson, D. I., Robson, S. C., and Lindsay, S. (1998). Isolation, characterisation and embryonic expression of WNT11, a gene which maps to 11q13.5 and has possible roles in the development of skeleton, kidney and lung. Gene, 219(1–2), 101–10. Larsen, W. J. (1993). Human embryology 1st edn. Churchill Livingstone Inc., New York, USA. LeBrun, D. P., Warnke, R. A., and Cleary, M. L. (1993). Expression of BCL-2 in fetal tissues suggests a role in morphogenesis. American Journal of Pathology, 142, 743–53. Lehnert, S. A. and Akhurst, R. J. (1988). Embryonic expression pattern of TGF beta type-1 RNA suggests both paracrine and autocrine mechanisms of action. Development, 104(2), 263–73. Letterio, J. J., Geiser, A. G., Kulkarni, A. B., Roche, N. S., Sporn, M. B., and Roberts, A. B. (1994). Maternal rescue of transforming growth factor-beta 1 null mice. Science, 264(5167), 1936–8. Leveen, P., Pekny, M., Gebre-Medhin, S., Swolin, B., Larsson, E., and Betsholtz, C. (1994). Mice deficient for PDGF- show renal, cardiovascular, and hematological abnormalities. Genes and Development, 8(16), 1875–87. Little, M. and Wells, C. (1997). A clinical overview of WT1 gene mutations. Human Mutation, 9(3), 209–25. Liu, S., Cieslinski, D. A., Funke, A. J., and Humes, H. D. (1997). Transforming growth factor-beta 1 regulates the expression of Pax-2, a developmental control gene, in renal tubule cells. Experimental Nephrology, 5(4), 295–300. Loughna, S., Hardman, P., Landels, E., Jussila, L., Alitalo, K., and Woolf, A. S. (1997). A molecular and genetic analysis of renal glomerular kidney development. Angiogenesis, 1, 84–101. Loughna, S., Yuan, H. T., and Woolf, A. S. (1998). Effects of oxygen on vascular patterning in Tie1/LacZ metanephric kidneys in vitro. Biochemical and Biophysical Research Communications, 247(2), 361–6. Luo, G., Hofmann, C., Bronckers, A. L., Sohocki, M., Bradley, A., and Karsenty, G. (1995). BMP-7 is an inducer of nephrogenesis, and is also required for eye development and skeletal patterning. Genes and Development, 9(22), 2808–20. Maas, R., Elfering, S., Glaser, T., and Jepeal, L. (1994). Deficient outgrowth of the ureteric bud underlies the renal agenesis phenotype in mice manifesting the limb deformity (ld) mutation. Developmental Dynamics, 199, 214–18. Mansouri, A., Chowdhury, K., and Gruss, P. (1998). Follicular cells of the thyroid gland require Pax8 gene function. Nature Genetics, 19(1), 87–90. Mariano, J. M., Montuenga, L. M., Prentice, M. A., Cuttitta, F., and Jakowlew, S. B. (1998). Concurrent and distinct transcription and translation of transforming growth factor-beta type I and type II receptors in rodent embryogenesis. International Journal of Developmental Biology, 42(8), 1125–36. Maulbecker, C. C. and Gruss, P. (1993). The oncogenic potential of Pax genes. EMBO Journal, 12(6), 2361–7.

84

Paul J. D. Winyard

McConnell, M. J., Cunliffe, H. E., Chua, L. J., Ward, T. A., and Eccles, M. R. (1997). Differential regulation of the human Wilms tumour suppressor gene (WT1) promoter by two isoforms of PAX2. Oncogene, 14(22), 2689–700. Medjebeur, A. A., Bussieres, L., Gasser, B., Gimonet, V., and Laborde, K. (1997). Experimental bilateral urinary obstruction in foetal sheep: transforming growth factor-beta 1 expression. American Journal of Physiology, 273(3, Pt 2), F372–F379. Mendelsohn, C., Lohnes, D., Decimo, D., Lufkin, T., LeMeur, M., Chambon, P., et al. (1994). Function of the retinoic acid receptors (RAR) during development. Development, 120, 2749–71. Milbrandt, J., de Sauvage, F. J., Fahrner, T. J., Baloh, R. H., Leitner, M. L., Tansey, M. G., et al. (1998). Persephin, a novel neurotrophic factor related to GDNF and neurturin. Neuron, 20(2), 245–53. Miyamoto, N., Yoshida, M., Kuratani, S., Matsuo, I., and Aizawa, S. (1997). Defects of urogenital development in mice lacking Emx2. Development, 124(9), 1653–64. Miyazaki, Y., Oshima, K., Kume, T., Dunn, R., Fogo, A., Hogan, B. L. M., et al. (1999). Ontogeny of the congenital anomalies of the kidney and urinary tract (CAKUT): Incomplete elongation of the branching ureter leads to hypo-dysplastic kidney in heterozygous Bmp4 knockouts (Bmp4/). American Journal of Nephrology, 10(408A), (Abstract). Montesano, R., Schaller, G., and Orci, L. (1991). Induction of epithelial tubular morphogenesis in vitro by fibroblast-derived soluble factors. Cell, 66, 697–711. Moore, K. L. and Persaud, T. V. N. (1998). The developing human. Clinically oriented embryology. WB Saunders, Philadelphia, Pennsylvania. Morham, S. G., Lanhenbach, R., Loftin, C. D., Tiano, H. F., Vouloumanos, N., Jennette, J. C., et al. (1995). Prostaglandin synthase 2 gene disruption cuases severe renal pathology in the mouse. Cell, 83, 473–82. Mugrauer, G. and Ekblom, P. (1991). Contrasting expression patterns of three members of the myc family of protooncogenes in the developing and adult mouse kidney. Journal of Cell Biology, 112(1), 13–25. Muller, U., Wang, D., Denda, S., Meneses, J. J., Pedersen, R. A., and Reichardt, L. F. (1997). Integrin alpha8beta1 is critically important for epithelial-mesenchymal interactions during kidney morphogenesis. Cell, 88(5), 603–13. Obara-Ishihara, T., Kuhlman, J., Niswander, L., and Herzlinger, D. (1999). The surface ectoderm is essential for nephric duct formation in intermediate mesoderm. Development, 126(6), 1103–8. Okada, H., Strutz, F., Danoff, T. M., and Neilson, E. G. (1996). Possible pathogenesis of renal fibrosis. Kidney International Supplement, 54, S37–S38. Patek, C. E., Little, M. H., Fleming, S., Miles, C., Charlieu, J. P., Clarke, A. R., et al. (1999). A zinc finger truncation of murine WT1 results in the characteristic urogenital abnormalities of Denys–Drash syndrome. Proceedings of the National Academy of Sciences, USA, 96(6), 2931–6. Pelletier, J., Bruening, W., Kashtan, C. E., Mauer, S. M., Manivel, J. C., Striegel, J. E., et al. (1991). Germline mutations in the Wilms’ tumor suppressor gene are associated with abnormal urogenital development in Denys–Drash syndrome. Cell, 67(2), 437–47. Perantoni, A. O., Dove, L. F., and Karavanova, I. (1995). Basic fibroblast growth factor can mediate the early inductive events in renal development. Proceedings of the National Academy of Sciences, USA, 92(10), 4696–700. Pichel, J. G., Shen, L., Sheng, H. Z., Granholm, A. C., Drago, J., Grinberg, A., et al. (1996). GDNF is required for kidney development and enteric innervation. Cold Spring Harbor Symposia on Quantitative Biology, 61, 445–57. Pope, J. C., Brock, J. W., III, Adams, M. C., Stephens, F. D., and Ichikawa, I. (1999). How they begin and how they end: classic and new theories for the development and deterioration of congenital anomalies of the kidney and urinary tract, CAKUT. Journal of the American Society of Nephrology, 10(9), 2018–28.

Renal development

85

Potter, E. L. (1972). Normal and abnormal development of the kidney, Year Book. Medical Publishers Inc., Chicago, IL. Pritchard-Jones, K., Fleming, S., Davidson, D., Bickmore, W., Porteous, D., Gosden, C., et al. (1990). The candidate Wilms’ tumour gene is involved in genitourinary development. Nature, 346(6280), 194–7. Pritchard-Jones, K. and Hawkins, M. M. (1997). Biology of Wilms’ tumour. Lancet, 349(9053), 663–4. Qiao, J., Cohen, D., and Herzlinger, D. (1995). The metanephric blastema differentiates into collecting system and nephron epithelia in vitro. Development, 121, 3207–14. Qiao, J., Cohen, J., and Herzlinger, D. (1996). FGF-7 uncouples the mitogenic and nephron inducing activities of the ureteric bud. Journal of the American Society of Nephrology, 7(1604A), (Abstract). Qiao, J., Sakurai, H., and Nigam, S. K. (1999). Branching morphogenesis independent of mesenchymalepithelial contact in the developing kidney. Proceedings of the National Academy of Sciences, USA, 96(13), 7330–5. Quaggin, S. E., Vanden Heuvel, G. B., and Igarash, P. (1998). Pod-1, a mesoderm-specific basichelix-loop-helix protein expressed in mesenchymal and glomerular epithelial cells in the developing kidney. Mechanisms of Development, 71(1–2), 37–48. Rackley, R. R., Kessler, P. M., Campbell, C., and Williams, B. R. (1995). In situ expression of the early growth response gene-1 during murine nephrogenesis. Journal of Urology, 154(2, Pt 2), 700–5. Rogers, S. A., Ryan, G., and Hammerman, M. R. (1991). Insulin-like growth factors I and II are produced in the metanephros and are required for growth and development in vitro. Journal of Cell Biology, 113, 1447–53. Rogers, S. A., Ryan, G., and Hammerman, M. R. (1992). Metanephric transforming growth factor- is required for renal organogenesis in vitro. American Journal of Physiology, 262(4, Pt 2), F533–F539. Rogers, S. A., Ryan, G., Purchio, A. F., and Hammerman, M. R. (1993). Metanephric transforming growth factor-1 regulates nephrogenesis in vitro. American Journal Physiology, 264(6, Pt 2), F996–F1002. Rossant, J. and Nagy, A. (1995). Genome engineering: the new mouse genetics. Nature Medicine, 1(6), 592–4. Rothenpieler, U. W. and Dressler, G. R. (1993). Pax-2 is required for mesenchymal-to-epithelium conversion during kidney development. Development, 119, 711–20. Ruoslahti, E. and Yamaguchi, Y. (1991). Proteoglycans as modulators of growth factor activities. Cell, 64, 867–9. Rupprecht, H. D., Drummond, I. A., Madden, S. L., Rauscher, F. J., and Sukhatme, V. P. (1994). The Wilms’ tumor suppressor gene WT1 is negatively autoregulated. Journal of Biological Chemistry, 269(8), 6198–206. Ryan, G., Steele-Perkins, S. V., Morris, J. F., Rausher, F. J. 3., and Dressler, G. R. (1995). Repression of Pax-2 by WT1 during normal kidney development. Development, 121(3), 867–75. Saelman, E. U., Keely, P. J., and Santoro, S. A. (1995). Loss of MDCK cell alpha 2 beta 1 integrin expression results in reduced cyst formation, failure of hepatocyte growth factor/scatter factor-induced branching morphogenesis, and increased apoptosis. Journal of Cell Science, 108(Pt 11), 3531–40. Sainio, K., Suvanto, P., Davies, J., Wartiovaara, J., Wartiovaara, K., Saarma, M., et al. (1997). Glial-cellline-derived neurotrophic factor is required for bud initiation from ureteric epithelium. Development, 124(20), 4077–87. Sakurai, H., Barros, E. J., Tsukamoto, T., Barasch, J., and Nigam, S. K. (1997). An in vitro tubulogenesis system using cell lines derived from the embryonic kidney shows dependence on multiple soluble growth factors. Proceedings of the National Academy of Sciences, USA, 94(12), 6279–84. Sanford, L. P., Ormsby, I., Gittenberger-de Groot, A. C., Sariola, H., Friedman, R., Boivin, G. P., et al. (1997). TGFbeta2 knockout mice have multiple developmental defects that are non-overlapping with other TGFbeta knockout phenotypes. Development, 124(13), 2659–70.

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Santos, O. F. P., Barros, E. J. G., Yang, X.-M., Matsumoto, K., Nakamura, T., Park, M., et al. (1994). Involvement of hepatocyte growth factor in kidney development. Developmental Biology, 163, 525–9. Sanyanusin, P., Schimmentl, L. A., McNoe, L. A., Ward, T. A., Pierpoint, M. E. M., Sullivan, M. J., et al. (1995). Mutations of the PAX2 gene in a family with optic nerve colobomas, renal anomalies and vesicoureteral reflux. Nature Genetics, 9, 358–64. Sariola, H. (1996). Does the kidney express redundant or important molecules during nephrogenesis? Experimental Nephrology, 4(2), 70–6. Sariola, H., Ekblom, P., Lehtonen, E., and Saxen, L. (1983). Differentiation and vascularisation of the metanephric kidney grafted on the chorioallantoic membrane. Developmental Biology, 96, 427–35. Sato, S. and Hughes, R. C. (1992). Binding specificity of a baby hamster lectin for H type I and II chains of polylactosamine glycans and ppropriately glycosylated forms of laminin and fibronectin. Journal of Biological Chemistry, 267, 6983–90. Schmid, P., Cox, D., Bilbe, G., Maier, R., and McMaster, G. K. (1991). Differential expression of TGF beta 1, beta 2 and beta 3 genes during mouse embryogenesis. Development, 111(1), 117–30. Schmid, P., Schulz, W. A., and Hameister, H. (1989). Dynamic expression pattern of the myc protooncogene in midgestation mouse embryos. Science, 243(4888), 226–9. Schmidt, C., Bladt, F., Goedecke, S., Brinkmann, V., Zschiesche, W., Sharpe, M., et al. (1995). Scatter factor/hepatocyte growth factor is essential for liver development. Nature, 373(6516), 699–702. Schuchardt, A., D’Agati, V., Larsson-Blomberg, L., Costantini, F., and Pachnis, V. (1994). Defects in the kidney and enteric nervous system of mice lacking the tyrosine kinase receptor Ret [see comments]. Nature, 367(6461), 380–3. Shawlot, W. and Behringer, R. R. (1995). Requirement for Lim1 in head-organizer function. Nature, 374(6521), 425–30. Sonnenberg, E., Meyer, D., Weidner, K. M., and Birchmeier, C. (1993). Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development. Journal of Cell Biology, 123, 223–35. Sorenson, C. M., Padanilam, B. J., and Hammerman, M. R. (1996). Abnormal postpartum renal development and cystogenesis in the bcl-2 (/) mouse. American Journal of Physiology, 271(1, Pt 2), F184–F193. Sorenson, C. M., Rogers, S. A., Korsmeyer, S. J., and Hammerman, M. R. (1995). Fulminant metanephric apoptosis and abnormal kidney development in bcl-2 deficient mice. American Journal of Physiology, 268, F73–F81. Sorokin, L., Sonnenberg, A., Aumailley, M., Timpl, R., and Ekblom, P. (1990). Recognition of the laminin E8 cell-binding site by an integrin possessing the alpha 6 subunit is essential for epithelial polarization in developing kidney tubules. Journal of Cell Biology, 111, 1265–73. Soussi-Yanicostas, N., Hardelin, J. P., Arroyo-Jimenez, M. M., Ardouin, O., Legouis, R., Levilliers, J., et al. (1996). Initial characterization of anosmin-1, a putative extracellular matrix protein synthesized by definite neuronal cell populations in the central nervous system. Journal of Cell Science, 109(Pt 7), 1749–57. Stanton, B. R. and Parada, L. F. (1992). The N-myc proto-oncogene: developmental expression and in vivo site-directed mutagenesis. Brain Pathology, 2, 71–83. Stark, K., Vainio, S., Vassileva, G., and McMahon, A. P. (1994). Epithelial transformation of metanephric mesenchyme in the developing kidney regulated by Wnt-4. Nature, 372(6507), 679–83. Takayama, H., LaRochelle, W. J., Sabnis, S. G., Otsuka, T., and Merlino, G. (1997). Renal tubular hyperplasia, polycystic disease, and glomerulosclerosis in transgenic mice overexpressing hepatocyte growth factor/scatter factor. Laboratory Investment, 77(2), 131–8.

Renal development

87

Threadgill, D. W., Dlugosz, A. A., Hansen, L. A., Tennenbaum, T., Lichti, U., Yee, D., et al. (1995). Targeted disruption of mouse EGF receptor. Effect of genetic background on mutant phenotype. Science, 269, 230–4. Torres, M., Gomex-Pardo, E., Dressler, G. R., and Gruss, P. (1995). Pax-2 controls multiple steps of urogenital development. Development, 121, 4057–65. Towers, P. R., Woolf, A. S., and Hardman, P. (1998). Glial cell line-derived neurotrophic factor stimulates ureteric bud outgrowth and enhances survival of ureteric bud cells in vitro. Experimental Nephrology, 6(4), 337–51. Tufro, A., Norwood, V. F., Carey, R. M., and Gomez, R. A. (1999). Vascular endothelial growth factor induces nephrogenesis and vasculogenesis. Journal of the American Society of Nephrology, 10(10), 2125–34. Vainio, S., Jalkanen, M., Bernfield, M., and Saxen, L. (1989a). Transient expression of syndecan in mesenchymal cell aggregates of the embryonic kidney. Developmental Biology, 152, 221–32. Vainio, S., Lehtonen, E., Jalkanen, M., Bernfield, M., and Saxen, L. (1989b), Epithelial-mesenchymal interactions regulate the stage-specific expression of a cell surface proteoglycan, syndecan, in the developing kidney. Developmental Biology, 134(2), 382–91. Vega, Q. C., Worby, C. A., Lechner, M. S., Dixon, J. E., and Dressler, G. R. (1996). Glial cell line-derived neurotrophic factor activates the receptor tyrosine kinase RET and promotes kidney morphogenesis. Proceeding of the National Academy of Sciences, USA, 93(20), 10657–61. Veis, D. J., Sorenson, C. M., Shutter, J. R., and Korsmeyer, S. J. (1994). Bcl-2 deficient mice demonstrate fulminant lymphoid apoptosis, polycystic kidneys and hypopigmented hair. Cell, 75, 229–40. Ventatachalam, M. A. and Kriz, W. (1998). Anatomy. In Heptinstall’s pathology of the kidney, 5th edn, (ed. J. C. Jennette et al.), vol. 1, pp. 3–66. Lippincott-Raven Publishers, Philadelphia, PA. Vestweber, D. and Kemler, R. (1985). Identification of a putative cell adhesion domain of uvomorulin. EMBO Journal, 4, 3393–8. Wallner, E. I., Yang, Q., Peterson, D. R., Wada, J., and Kanwar, Y. S. (1998). Relevance of extracellular matrix, its receptors, and cell adhesion molecules in mammalian nephrogenesis. American Journal of Physiology, 275(4, Pt 2), F467–F477. Winyard, P. J., Bao, Q., Hughes, R. C., and Woolf, A. S. (1997). Epithelial galectin-3 during human nephrogenesis and childhood cystic diseases. Journal of the American Society of Nephrology, 8(11), 1647–57. Winyard, P. J., Nauta, J., Lirenman, D. S., Hardman, P., Sams, V. R., Risdon, R. A., et al. (1996a). Deregulation of cell survival in cystic and dysplastic renal development. Kidney International, 49(1), 135–46. Winyard, P. J. D., Risdon, R. A., Sams, V. R., Dressler, G., and Woolf, A. S. (1996b), The PAX2 transcription factor is expressed in cystic and hyperproliferative dysplastic epithelia in human kidney malformations. Journal of Clinical Investigation, 98, 451–9. Woolf, A. S. (1999). Embryology. In Pediatric nephrology, 4th edn (ed. T. M. Barratt and E. D. Avner) pp 1–19. Lippincott Williams and Wilkins, Philadelphia, PA. Woolf, A. S., Kolatsi-Joannou, M., Hardman, P., Andermacher, E., Moorby, C., Fine, L., et al. (1995). Roles of hepatocyte growth factor/scatter factor and the met receptor in the early development of the metanephros. Journal of Cell Biology, 128, 171–84. Woolf, A. S. and Loughna, S. (1998). Origin of glomerular capillaries: is the verdict in? Experimental Nephrology, 6(1), 17–21. Woolf, A. S. and Winyard, P. J. (1998). Advances in the cell biology and genetics of human kidney malformations. Journal of the American Society of Nephrology, 9(6), 1114–25. Woolf, A. S. and Winyard, P. J. D. (2000). Gene expression and cell turnover in human renal dysplasia. Histology and Histopathology, 15(1), 159–66.

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Wrana, J. L. (1998). TGF-beta receptors and signalling mechanisms. Minerals and Electrolyte Metabolism, 24(2–3), 120–30. Xu, P. X., Adams, J., Peters, H., Brown, M. C., Heaney, S., and Maas, R. (1999). Eya1-deficient mice lack ears and kidneys and show abnormal apoptosis of organ primordia. Nature Genetics, 23(1), 113–17. Yang, S. P., Woolf, A. S., Yuan, H. T., Scott, R. J., Risdon, R. A., O’Hare, M. J., et al. (2000). Biological role of transforming growth factor beta 1 in human congenital kidney malformations. Submitted. American Journal of Pathology. Zhang, P., Liegeois, N. J., Wong, C., Finegold, M., Hou, H., Thompson, J. C., et al. (1997). Altered cell differentiation and proliferation in mice lacking p57KIP2 indicates a role in Beckwith–Wiedemann syndrome. Nature, 387(6629), 151–8.

4 Kidney and lower urinary tract malformations Adrian S. Woolf

Introduction Congenital malformations of the kidney and lower urinary tract are common causes of chronic renal failure in infants and young children. In animal experiments, the main causes of such malformations are mutations, chemical and pharmaceutical teratogens, physical obstruction of fetal urinary flow and alterations of maternal nutrition. The focus of this chapter is to consider how mutations alter gene expression during development to cause human urinary tract malformations. Some of these disorders are associated with congenital anomalies in multiple organ systems and two such syndromes are considered in detail: first, the renalcoloboma syndrome where mutations of the PAX2 transcription factor cause partial failure of urinary tract growth; second, Kallmann syndrome where mutations of the KAL gene coding for a cell signaling molecule called anosmin-1 are associated with absence of the urinary tract. In most patients, however, kidney and lower urinary tract malformations occur in isolation and in some of these individuals a genetic pathogenesis is strongly suggested by a positive family history supported by positive linkage studies: one common example is primary vesicoureteric reflux which is said to affect 1–2 per cent of very young children. Apparently sporadic kidney and lower urinary tract malformations have recently been shown to be associated with polymorphisms of genes expressed during development. In the long-term, an understanding of the genetic aspects of human urinary tract malformations could help to unravel the pathogenesis of these disorders and may facilitate the design of genetic screening tests with a view to early diagnosis. Normal development of the kidney and lower urinary tract The mammalian kidney derives from two tissue compartments of the embryonic metanephros: the ureteric bud, an epithelium which branches recurrently to form the collecting ducts, and the renal mesenchyme, which undergoes an epithelial transformation to form nephron components including glomerular and proximal tubule epithelia (Woolf 1999a; Risdon and Woolf 1998). The human metanephros appears at 5 weeks gestation and the first layer of glomeruli form by 9 weeks. Branching and nephrogenesis continue to occur in the outer rim of the kidney, the nephrogenic cortex, until 34 weeks whereas further maturation, in the form of growth and differentiation, continues postnatally. Recent histological studies using detailed measurements suggest that about two-thirds of nephrons are generated in the last third of human gestation, and that the range of nephrons found in a healthy kidney is

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rather large, about 0.5  106–1.5  106 (Hinchliffe et al. 1991, 1992b, 1993). The ureteric bud also gives rise to the epithelium of the renal pelvis, the ureter and the bladder trigone, also called the urothelium. The remainder of the bladder epithelium is derived from endoderm and both ureter and bladder become enveloped in mesodermal-derived smooth muscle. A more detailed description of these events is provided in Chapter 3. During the second half of human gestation fetal urine is the main component of amniotic fluid and is thought to be important for fetal lung growth. However, the production of urine before birth is not essential for fetal survival since the placenta effectively performs both hemodialysis and gas exchange during gestation. The spectrum of human kidney and urinary tract malformations As might be expected for such a complex pattern of development, these processes are often perturbed (Bernstein et al. 1997; Risdon and Woolf 1998), making kidney and lower urinary tract malformations relatively common (Table 4.1). Furthermore, since the development of the ureteric bud and renal mesenchyme depend on mutually inductive interactions (Woolf 1999a; Vainio and Muller 1997), it is not surprising that a primary defect of either component will affect the development of its partner. This provides one explanation for the clinical observation that an abnormal insertion of the ureter into the bladder is associated with congenital renal parenchymal anomalies (Vermillion and Heale 1973; Schwartz et al. 1981). Human urinary tract malformations are phenotypically diverse but have in common a failure of normal development before birth (Table 4.1). For instance, in agenesis the kidney and ureter are absent, whereas dysplastic kidneys contain undifferentiated tissues and are often Table 4.1 The spectrum of kidney and lower urinary tract malformations Upper urinary tract Renal agenesis: kidney is absent Renal dysplasia: kidney contains undifferentiated tissues and may be tiny (aplasia) or distended by cysts (multicystic and cystic dysplastic kidneys) Renal hypoplasia: kidney contains formed nephrons but significantly fewer than normal—when nephrons are large the condition is called oligomeganephronia Duplex kidney: the organ is separated into an upper part, which is often dysplastic and attached to an obstructed ureter, and a lower part, which is attached to a refluxing ureter Horseshoe kidney: both kidneys have become fused during development and may include dysplastic components Lower urinary tract Agenesis: the ureter and bladder trigone is absent Hydronephrosis: the renal pelvis is distended and the parenchyma may be hypoplastic or dysplastic—the ureter may be refluxing or obstructed at the level of the pelvi-ureteric or uretero-vesical junction Duplication of the ureter: may occur in association with a duplex kidney Vesicoureteric reflux: urine flows retrogradely from the bladder into the ureter, pelvis and medullary collecting ducts of the kidney Posterior urethral valves: the outflow to the urinary bladder is anatomically obstructed Some forms of upper and lower urinary tract malformations. The following incidences have been quoted: duplex ureter, 1/100; vesicoureteric reflux, 1/100; horseshoe kidney, 1/200; unilateral renal agenesis, 1/500–1/1000; unilateral multicystic kidney, 1/5000. Bilateral agenesis/dysplasia occurs in 1/5000–1/10,000 births.

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attached to structurally abnormal lower urinary tracts. Other variations are listed in Table 4.1. Any bilateral renal malformation can be associated with oligohydramnios, oligoanuria and lung hypoplasia, and this is sometimes called the ‘Potter sequence’ after the fetal pathologist, Edith Potter. The definition of malformation can be extended to microscopic abnormalities such as ‘tubular dysgenesis’ where proximal tubules form abnormally. Although some types of polycystic kidney diseases can affect infants and children (Bernstein et al. 1997; Risdon and Woolf 1998), cysts in these disorders generally arise after the period of nephron formation and these diseases are considered in full elsewhere in this book. The clinical impact of human kidney and lower urinary tract malformations With the increasing use of fetal ultrasound scanning, more individuals with congenital urinary tract malformation are being diagnosed (Anderson et al. 1997; Hiraoka et al. 1997; Yeung et al. 1997; Jaswon et al. 1999). In fact, these disorders account for about 30 per cent of all prenatally diagnosed anomalies (Noia et al. 1996). Although many appear to have minimal clinical significance, for the more serious anomalies such as bilateral renal agenesis or dysplasia, early diagnosis allows consideration of termination or active therapeutic intervention. The latter includes fetal decompression of obstructed urinary tracts as well as planning for the need for dialysis soon after birth (Noia et al. 1996). In these severe cases, accompanying lung hypoplasia can also be life threatening after birth. Severe kidney and urinary tract malformations are the main cause of chronic renal failure in young children (Ehrich et al. 1992; Drozdz et al. 1998). For example, in our own clinical service at Great Ormond Street Hospital for Children in London, UK, malformations account for over 80 per cent of children under 5 years of age with end-stage renal failure. With advances in technology, babies with minimal renal function can be dialysed from birth and infants can receive kidney transplants from the age of 1 year. These strategies, together with general improvements in the care of children with chronic renal failure will ultimately mean an increase in long-term survival into the adult period for these individuals. Clues from animal studies of kidney development The explosion of knowledge regarding the mechanisms of murine kidney development is relevant to our understanding of human malformations (Vainio and Muller 1997; Woolf and Winyard 1998). Clifford Grobstein (1967) was the first to demonstrate that mouse metanephric mesenchyme and ureteric bud failed to differentiate when cultured separately but they formed nephrons and collecting ducts when recombined. Using genetically engineered mice and organ culture, it is now established that nephrogenesis is controlled by diverse molecules, some of which may act as inductive signals postulated by Grobstein. These molecules include transcription factors, growth factors, and adhesion molecules which are essential for control of cell survival, proliferation, differentiation, and morphogenesis during normal mammalian development. ●

Transcription factors include BF2 (Hatini et al. 1996), EMX2 (Miyamoto et al. 1997), EYA1 (Johnson et al. 1999; Xu et al. 1999), hepatocyte nuclear factors (HNFs) (Lazzaro et al. 1992), HOXa11 and HOXd11 (Davis et al. 1995), LIM1 (Shawlot and Behringer 1995), LMX1B (Chen et al. 1998), N-MYC (Stanton et al. 1992), PAX2 (Torres et al. 1995), POD-1 (Quaggin et al. 1998) and WT1 (Kreidberg et al. 1993).

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Growth factors with positive effects include epidermal growth factor, bone morphogenetic protein 7 (Dudley et al. 1995), fibroblast growth factors (FGFs) 2 (Perantoni et al. 1995) and FGF 7 (Qiao et al. 1996), glial cell line-derived neurotrophic factor (Schuchardt et al. 1994; Sanchez et al. 1996; Towers et al. 1998), hepatocyte growth factor (Woolf et al. 1995), insulin-like growth factors (IGFs) I and II (Rogers et al. 1991), platelet-derived growth factor  (Leveen et al. 1994), transforming growth factor  (TGF) (Rogers et al. 1992), vascular endothelial growth factor (Loughna et al. 1997) and WNT4 (Stark et al. 1994). Inhibitory ‘growth’ factors include activin (Rivtos et al. 1995), leukaemia inhibition factor (Bard and Ross 1991), tumor necrosis factor  (Cale et al. 1998) and TGF (Rogers et al. 1993). Cell adhesion molecules include laminins (Klein et al. 1988; Noakes et al. 1995) as well as integrins (Muller et al. 1997; Kreidberg et al. 1996). Diverse other molecules include survival factors (e.g. BCL2) (Veis et al. 1994; Sorenson et al. 1995), retinoic acid receptors (RARs) (Mendelsohn et al. 1994), enzymes (e.g. COX2) (Morham et al. 1995; Dinchuk et al. 1995) and molecules with unknown specific functions (e.g. formins; Maas et al. 1994).

Table 4.2 Molecules involved in nephrogenesis in null mutant mice Transcription factors BF2: small, fused and undifferentiated kidneys (Hatini et al. 1996) EYA11: absent kidneys (Johnson et al. 1999; Xu et al. 1999) EMX2: absent kidneys (Miyamoto et al. 1997) HOXa11 / HOXd112: small or absent kidneys (Davis et al. 1995) LIM1: absent kidneys (Shawlot and Behringer 1995) LMX1B1: malformed glomeruli (Chen et al. 1998) N-MYC: poorly developed mesonephric kidneys (Stanton et al. 1992) PAX21,3: small or absent kidneys (Torres et al. 1995) WT11: absent kidneys (Kreidberg et al. 1993) Growth factors and receptors EGF receptor: cystic collecting ducts (Threadgill et al. 1995) BMP7: undifferentiated kidneys (Dudley et al. 1995) GDNF3 and its receptor, RET: small or absent kidneys (Schuchardt et al. 1994; Sanchez et al. 1996) PDGF B: absent mesangial cells (Leveen et al. 1994) WNT4: undifferentiated kidneys (Stark et al. 1994) Adhesion molecules and receptors 3 integrin: decreased collecting duct branching (Kreidberg et al. 1996) 8 integrin: impaired ureteric bud branching and nephron formation (Muller et al. 1997) s-laminin/laminin 2: nephrotic syndrome (Noakes et al. 1995) Miscellaneous molecules BCL2: small kidneys (Veis et al. 1994; Sorenson et al. 1995) COX2: small kidneys (Dinchuk et al. 1995; Morham et al. 1995) Formin: absent kidneys (Maas et al. 1994) Neuronal nitric oxide synthase: bladder outflow obstruction (Burnett et al. 1997) RAR/22: small or absent kidneys (Mendelsohn et al. 1994) Unless otherwise stated, malformations only occur in homozygous null mutants. 1Mutations of these genes have also been implicated in malformations of the human kidney or lower urinary tract (see Table 4.4). 2Null mutation of two homologous genes is required to cause malformation. 3Heterozygous mutations also produce kidney or lower urinary tract malformations.

Table 4.2 summarizes some of the genes known to affect nephrogenesis based on analyses of null mutant mice. As with human disease, the resulting malformations are agenesis, dysplasia, and hypoplasia. These respective phenotypes represent defects in regulation of metanephric formation, epithelial morphogenesis, and nephron numbers. In addition, overexpression of

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either PAX2 (Dressler et al. 1993) or FGF7 (Nguyen et al. 1997) leads to cyst formation suggesting a role for these genes in epithelial overgrowth. These experiments show that mouse embryonic kidneys have a limited range of responses to gene inactivation or overactivity. Mutant mice also demonstrate that: ii(i) the urinary tract malformation can result from mutation of different genes (e.g. null mutation of either WTI or PAX2 transcription factor genes causes renal agenesis) (Kreidberg et al. 1993; Torres et al. 1995); i(ii) the presence and severity of the urinary tract malformation is often dependent on the genetic background of the animal (Threadgill et al. 1995); (iii) in some instances, more than one related gene must be mutated before development is perturbed (Mendelsohn et al. 1994; Davis et al. 1995). (iv) mouse ‘nephrogenesis genes’ are often also expressed in other organs where they are also critical for development for example PAX2 mutants have renal and ocular defects while WT1 / mice have a syndrome of kidney, gonadal, and cardiothoracic anomalies. Teratogenic and physical causes of kidney and lower urinary tract malformations Based in animal experiments, the major influences which perturb kidney and lower urinary tract development comprise mutations, chemical and pharmacological teratogens, urinary tract obstruction, and maternal undernutrition. The focus of this chapter is on how mutations alter gene activity and the normal program of development to generate disease. The reader is referred to recent reviews, which cover in detail the biology of the other areas (MerletBenichou et al. 1997; Chevalier 1999). Several teratogens implicated in the pathogenesis of kidney and lower urinary tract malformations are listed in Table 4.3. It is important to realize that teratogens may often act by altering the expression of genes during development (Brown 1997), as demonstrated by these three examples. 1. The use of angiotensin converting enzyme inhibitors to treat hypertension during pregnancy can cause neonatal renal failure which may have a hemodynamic basis or be associated with a lesion which resembles renal tubular dysgenesis: this can be associated with a skull malformation called hypocalvaria (Pryde et al. 1993; Barr 1994; Sedman et al. 1995). Of note, a strikingly similar disorder occurs in the absence of teratogen exposure (McFadden et al. 1997; Milunsku et al. 1997), and perhaps these individuals harbor mutations which affect angiotensin action. Certainly, animal experiments point to the importance of angiotensin action during normal kidney and urinary tract development (see below). 2. Evidence of teratogens affecting gene expression is provided by the increased incidence of kidney and lower urinary tract malformations in the offspring of mothers with diabetes (Novak and Robinson 1994; Lynch and Wright 1997). There is experimental evidence that exposure of murine embryonic kidneys to glucose alters the expression of a basement membrane component called s-laminin (Abrass et al. 1997). 3. Vitamin A and its retinoic acid derivatives provide another example. In humans and animals, high doses have been asscoaited with severe malformations such as renal agenesis,

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Adrian S. Woolf sometimes associated with ‘caudal regression’, the reduction of distal structures such as the sacrum and hindlimbs (Von Lennep et al. 1985; Rothman et al. 1995; Padmanabhan 1998). It has been reported that retinoic acid, a vitamin A derivative, can induce renal malformations in mice accompanied by fulminant apoptosis of renal mesenchyme and a downregulation of WT1 transcription factor gene expression (Tse 1998). The vitamin A story is further complicated because there is convincing animal evidence that too little may also result in failure of fetal kidney growth, perhaps mediated by impaired ureteric bud branching and downregulation of glial cell line derived neurotrophic factor receptor expression (Vilar et al. 1996; Lelievre-Pegorier et al. 1998).

Although it is unusual to elicit a history of teratogen exposure, it remains possible that occult or low-grade exposure is important. For example, the incidence of diverse malformations increases with a daily intake of vitamin A above levels as low as 10,000 IU; this includes a weak association with urogenital defects (Rothman et al. 1995). Another example of a relatively subtle environmental change which can have significant effects on development of the kidney is the effect of a low protein diet during pregnancy which in animals can lead to reduced numbers of nephrons (renal hypoplasia) and hypertension postnatally (Gilbert et al. 1990; Lucas et al. 1997). Similar effects have been postulated to occur in humans (Hinchliffe et al. 1992b; Rossing et al. 1995). In fact, it is interesting to speculate that dietary influences may explain part of the normal variation of nephron numbers found in healthy kidneys (Hinchliffe et al. 1991, 1992b, 1993). A significant minority of kidney malformations in girls, and about half of malformations in boys, are associated with obstructed urinary tracts (Peters 1997). Kidneys associated with obstruction in early gestation are usually dysplastic whereas obstruction in the last third of gestation is associated with hydronephrosis and subcortical cysts. Below, we describe how fetal urinary tract obstruction in humans and experimental animals can alter cell proliferation in developing kidney epithelia accompanied by alteration of the expression of a transcription factor called PAX2. Human malformation syndromes affecting the kidney and lower urinary tract In some cases, the kidney and lower urinary tract disorder occurs in association with a multi-organ malformation syndrome that commonly affects the central nervous, cardiovascular and skeletal systems. A brief list is shown in Table 4.3 and reader is also referred to other Table 4.3 Some teratogens of the kidney and lower urinary tract Angiotensin converting enzyme inhibitors (Fogo et al. 19902; Pryde et al. 19931; Barr 19941; Sedman et al. 19951; Tufro-McReddie et al. 19952; McCausland et al. 19972) Cocaine (Battin et al. 19951) Corticosteroids (Hulton and Kaplan 19951; Celsi et al. 19982) Ethanol (Gage and Sulik 19912; Taylor et al. 19941; Boehm et al. 19972; Moore et al. 19971) Gentamicin (Gilbert et al. 19912; Hulton and Kaplan 19951) Glucose (Novak and Robinson 19941; Abrass et al. 19972; Lynch and Wright 19971) Non-steriodal anti-inflammatory drugs (Voyer et al. 19941) Vitamin A and its derivatives (Von Lennep et al. 19851; Rothman et al. 19951; Padmanabhan 19982) 1Teratogen 2Teratogen

demonstrated in humans. demonstrated in animal experiments.

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references (McKusick 1992; Woolf and Winyard 1998) and to the constantly updated version of Online Mendelian Inheritance in Man (http:www4.ncbi.nlm.nih.gov/Omim/). Although malformation syndromes are individually rare they collectively account for considerable morbidity. Some are associated with gross chromosomal anomalies such as trisomies, 4p-syndrome and dup (10p)/del (10q) (Egli and Stalder 1973; Kulharya et al. 1993; Benjamin and al-Harbi 1997; Gilbert-Barness 1998), but cytogenetic aberrations are absent in most cases of urinary tract malformation syndromes. Other renal malformation syndromes are inherited in Mendelian patterns and in several, of them disease loci and mutations have been defined (Table 4.4). Table 4.4 Genes and human kidney and lower urinary tract malformation syndromes Apert syndrome (FGFR2 mutation—growth factor receptor) hydronephrosis and duplicated renal pelvis (Cohen and Kreiborg 1993; Wilkie et al. 1996) Bardet–Biedl syndrome1 (at least 5 loci: 11q13, 16q22, 3p13, 15q21 and 2q31): renal dysplasia and calyceal malformation (Harnett et al. 1988; Beales et al. 1999) Beckwith–Wiedemann syndrome1 (in a minority of patients, 557KIP2 mutation—cell cycle gene) renal overgrowth, cysts and dyplasia) (Choyke et al. 1999; Lam et al. 1999) Branchio-oto-renal syndrome (EYA12 mutation—transcription factor: renal agenesis and dysplasia (Konig et al. 1994; Abdelhak et al. 1997) Campomelic dysplasia (SOX9 mutation—transcription factor): diverse renal malformations (Houston et al. 1983; Wagner et al. 1994) Carnitine palmitoyltransferase II deficiency3: renal dysplasia (North et al. 1995) CHARGE association3: diverse urinary tract anomalies (Regan et al. 1999) Denys–Drash syndrome (WT12 mutation—transcription/splicing factor): calyceal defects and mesangial cell sclerosis (Jadresic et al. 1990; Coppes et al. 1993) Di George syndrome1 (locus at 22q11): renal agenesis, dysplasia and vesicoureteric reflux (Budarf et al. 1995; Czarnecki et al. 1998; Stewart et al. 1999) Glutaric aciduria type II3: cystic and dysplastic disease (Wilson et al. 1989) Fanconi anaemia (FAA mutation—DNA repair): renal agenesis, ectopic/horseshoe kidney (Lo Ten Foe et al. 1996) Kallmann Syndrome (KAL mutation—cell signaling molecule): renal agenesis (Franco et al. 1991; Legouis et al. 1991; Duke et al. 1998) Maturity onset diabetes of the young/renal malformation syndrome (HNF1 mutation—transcription factor): renal dysplasia and cysts (Bingham et al. 2000) Meckel syndrome1 (locus at 17q21–q24): cystic renal dysplasia (Salonen 1984; Paavola et al. 1995) Nail-patella syndrome (LMX1B2 mutation—transcription factor): malformation of the glomerulus and renal agenesis (Gubler and Levy 1993; Haga et al. 1997; Dreyer et al. 1998) Renal-coloboma syndrome (PAX22 mutation—transcription factor): renal hypoplasia and vesicoureteric reflux (Sanyanusin et al. 1995a,b) Simpson–Golabi–Behmel syndrome (GPC3 mutation—proteoglycan): renal overgrowth, cysts and dysplasia (Hughes-Benzie et al. 1992; Pilia et al. 1996) Smith–Lemli–Opitz syndrome1 (locus at 7q32—defect in cholesterol biosynthesis): cystic dysplastic kidneys (Akl et al. 1977; Wallace et al. 1994) VACTERL/VATER association3 (one patient with mitochondial gene mutation): diverse urinary tract anomalies (Damian et al. 1996) Zellweger syndrome (peroxisomal protein mutation): cystic dysplastic kidneys (Powers and Moser 1998; Shimozawa et al. 1992) 1Locus

known but gene undefined. of these genes have also been implicated in malformations of the murine kidney or lower urinary tract (see Table 4.4). 3Putative genetic basis in some cases. 2Mutations

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In the remainder of this chapter we firstly detail two malformation syndromes which act as striking examples where advances in genetics have illuminated the pathogenesis of abnormal urinary tract development. Secondly, we consider whether non-syndromic human urinary tract anomalies may have a genetic basis.

The renal coloboma syndrome—mutation of the PAX2 transcription factor gene As renal mesenchymal cells begin to differentiate into nephron tubules, they aggregate into condensates and undergo a burst of proliferation with increased expression of PAX2 transcription factor (Eccles et al. 1992; Winyard et al. 1996a). At the same time, the termini of the adjacent ureteric bud branches express the same gene and are highly proliferative. Later in nephrogenesis both proliferation and PAX2 expression are downregulated. Urinary tract growth is impaired in mice with inactivating PAX2 mutations (Keller et al. 1994; Torres et al. 1995; Favor et al. 1996). Null-mutants lack kidneys and ureters because the ureteric bud fails to branch from the mesonephric duct, while an associated absence of Fallopian tubes in female embryos is explained by the expression of PAX2 in Mullerian duct development. Haploinsufficiency (a partial lack of functional protein) in heterozygous PAX2 / mice is associated with renal hypoplasia and, in some cases, hydronephrosis and hydroureter, consistent with the presence of vesicoureteric reflux. The human renal-coloboma syndrome has a striking phenotypic similarity to PAX2 / mice. It comprises blindness due to an optic nerve malformation, the coloboma, with vesicoureteric reflux and hypoplastic kidneys. Sanyanusin et al. (1995a) described heterozygous mutations of PAX2 (chromosome 10q24–q25) in one kindred, and reports of other affected individuals followed (Sanyanusin et al. 1995a,b; Narahara et al. 1997; Schimmenti et al. 1997). The eye disease is explained by PAX2 expression during ocular development and it is notable that PAX2 / mutant mice also have eye disease (Keller et al. 1994; Favor et al. 1996). The first-reported human mutation was found to have arisen de novo with subsequent inheritance in a dominant manner (Sanyanusin et al. 1995a). It most likely caused haploinsufficiency because the mutated gene coded for a protein with an intact DNA-binding paired box, an interrupted octapepide domain, and a novel, truncated, carboxy terminus which was postulated to perturb the transactivation of target genes. Homozygous PAX2 mutations have yet to be described in humans, although kindred