The Glomerular Basement Membrane and Nephritis. Thesis submitted for the Degree of Doctor of Philosophy in the University of Adelaide. Andrew Wootton Department of Pathology. September 1985. Âuaeuso 2ç FeÅr.f ¡gE6 Table of Contents. Table of Contents. Abstract vii Statement of Originality viii Acknowledgements ix Chapter 1 Background and lntroduction 1 lmmunopathology of Glomerulonephritis 3 Circulating lmmune Complex Disease... 3 Disease Caused by Antibodies to Structural Antigens.. 4 "ln Situ" lmmune Complex Formation. 6 Summary of Pathogenic Mechanisms of Glomerulonephritis 7 Anti-Glomerular Basement Membrane-Antibody Disease 8 Anti-GBM-Antibody Disease in Humans I Experimental Models of Anti-GBM-Antibody Disease I Diagnosis I Antigen. 10 lnduction of Antibody Formation....... 10 Pathogenic Significance of Antibody 11 Mediation of Damage. 11 Therapy ..12 Chapter 2 The GBM; Binding prope-rties 13 lntroduction 15 Structure and Function of the GBM.. 15 Antibody binding to the G8M........ 15 Binding of other compounds to the GBM. 16 Factors affecting localisation of antigens within the kidney 16 Present study............... 17 Materials and Methods 18 GBM preparation.............. 18 Reagents 18 Cationisation of lgG. 19 lsoelectric focusing.. 19 Radiolabelling........... 19 Neuraminidase treatment of lgG 19 ln vitro binding assay 20 ln vivo binding of modified lgG 20 Elution studies. 21 ill Table of Contents Results 22 ln vitro binding. 22 ln vivo binding. 22 Elution studies. 23 Discussion 28 Chapter 3 Anti-GBM-Antibody; Measurement and Properties 30 il 8ta 31 Introduction 32 Background. 32 lmmunofluorescence 32 lmmunoassay 32 Present study........ 33 Materials and Methods 35 lsolation of G8M........ 35 Collagenase purification 35 Collagenase digestion............... 35 lodination. 35 Solid phase RlA.. 35 Assay optimisation............... 36 Patients 36 Results 37 Glomerular preparation............. .tl Purification of the antigen 37 Assay optimisation 37 Assay results......... 41 Discussion 43 38 Aviditv 46 lntroduction 47 Significance of Circulating Antibodies in Human Anti-GBM-Disease.. 47 Steblay model of anti-GBM disease 47 Antibody avidity....... 48 Materials and Methods 50 Antigen Preparation 50 Selection of serum.. 50 Elution of glomerular immunoglobulin.. 51 Radioimmunoassay 51 Avidity estimation 51 Results 53 Modification of RlA.... 53 tv Table of Contents. Inhibition of binding of serum antibody..... 53 Eluate binding studies. 54 Discussion 58 Chapter 4 Experimental Anti-GBM-Antibody lnduced Disease; Analysis of Treatment 60 Background 61 4A PE and CT 62 Introduction 63 Materials and Methods 64 1mmunogen............... 64 lmmunisation 64 Plasma Exchange Procedure 64 Chemotherapy... 65 Renal Clearance 67 lmmunofluorescence 67 Elution 68 Results 69 Disease Development. 69 Survival.... 69 RenalClearance. 69 Anti-human GBM-antibody levels 76 Anti-sheep G BM-antibody levels 76 Eluted antibody 79 Discussion 81 48 PGEr 85 lntroduction 86 Effect of Prostaglandin.s on Cell Mediated-lmmune Response 86 Effect of Prostaglandins on PMNs. 86 Effect of Prostaglandins on Vasopermeability.... 87 Effects of Prostaglandins on the Humoral Anti body Response 87 Summary of the Effects of Prostaglandin on Experimental lmmune Disease 88 Nephrotoxic Nephritis 88 Materials and Methods 90 Animals. 90 15(s)-1S-methyl PGEl 90 Nephrotoxic Serum.. 90 Experi mental P rotocol - Heterologous Phase............. 91 v Table of Contents. Experimental Protocol - Autologous Phase 92 Microscopic Methods., 92 Biochemical Methods. 93 Results 95 lmmunofluorescence 95 Mesangial Enlargement 95 PMNs/g lomerulus....... 98 Urinary Protein..... 98 Serum Urea. 100 Serum Creatinine 100 Serum Rat anti-rabbit antibody... 100 Discussion 101 Chapter 5 Conclusions and lmplications 104 Circulating lmmune Complex Disease - importance in glomerulonephritis................ 106 Relevance of the In Situ model of glomerulonephritis 107 Experimental models of anti-GBM disease - general considerations and specificity of antibodies... 109 Anti-GBM antibody assay...... 110 Avidity and relevance of circulating antibody.......... 110 Treatment of anti-GBM disease.............. 112 Final remarks... 112 Appendix 1 Published Work 113 Aooendix 2 Abbreviations 117 Bibliooraohv 119 vl Abstract ln this study, the binding of antibody and other compounds to the glomerular basement membrane (GBM) was investigated, together with the immunopathology and treatment of anti-GBM-antibody disease. The binding to GBM of a variety of compounds which are associated with membranous nephropathy was investigated using an in vifro binding assay. These included gold, mercury, captopril and cationic lgG with DNA, BSA and C1q used as controls. Cationised lgG and mercury were found to bind to GBM whilst gold and captopril did not. However study of the pl of glomerular lgG from membranous nephropathy patients failed to demonstrate increased amounts of cationic lgG indicating that this mechanism is unlikely to be responsible for the immunopathology of this condition. A solid phase radioimmunoassay for anti-GBM antibodies was established and used to study the circulating antibody in the diagnosis and monitoring of disease. Although the assay distinguished antibody positive patients from normal controls, several patients with SLE were also positive in the assay and the significance of this is discussed with reference to the antigens of the basement membrane. Further studies of the anti-GBM antibody were performed with the determination of the avidity of the serum antibody showing that the circulating antibody is of low avidity. This ímplies that the circulating antibody may be of limited relevance to on-going renal disease. Plasma exchange and chemotherapy are normally performed together in the treatment of anti-GBM disease. The separate use of these therapies was investigated in the Steblay model of anti-GBM nephritis in sheep and they were shown to be effective by different mechanisms. Plasma exchange is probably efficacious by removing non-antibody inflammatory agents from the circulation whilst chemotherapy reduces antibody production. Further investigations of treatment were performed using the prostaglandin PGEl since this has been found to be of therapeutic use in an number of different forms of nephritis. PGEI was shown to be protective in experimental anti-GBM disease. The most likely effect appeared to be upon inflammatory cells and their products since antibody deposition was not affected. vil Statement of Originality This thesis contains no material that has been accepted for the award of any other degree or diploma in any University with the exception of some of the data included in Chapter 4A. This study was a joint project with members of The Centre for Biomedical Engineering, University of New South Wales (approximate share 50%) and some data from this project have been detailed in the MSc thesis by Allan Pollack in the University of NSW. These data are presented here with the consent of my co-investigators. To the best of my knowledge and belief, this thesis contains no material previously published or written by another person, except where due reference has been made. I consent to this thesis being made available for photocopying and loan if accepted for the award of the degree. 3' l tt' vilt Acknowledgements Many people have assisted with this project. Without their generous cooperation it could not have been possible. In particular I wish to acknowledge with gratitude the role of the following people: Prof B. Vernon-Robeils, the supervisor of my candidature; Dr R. G. Edwards of the lnstitute of Medical and Veterinary Science for his role in enabling the project; Klaus Schindhelm and Allan Pollack who performed much of the experimental work described in Chapter 4A; Aileen Thompson and Gordon Howarth for their meticulous technique and assistance with immunofluorescence ; Mark FitzGerald and Silvia Schottmann for photography and Sonia Priadko for valuable technical assistance; and especially the support and encouragement of Howard Morris and Alison Carroll, without which this thesis would undoubtedly never have been written. Most importantly, I thank Andrew Woodroffe. lt was he who conceived the project and provided ideas, laboratory space and motivation, and of course many a Wednesday night of fruitful discussion. Financial support for this project was provided partially with an L. C. Hughes Fellowship from the Royal Adelaide Hospital and partly by the Institute of Medical and Veterinary Science. This thesis has been printed using an þple LaserWriteilu. I am gratefulto Random Access Ry Ltd, Adelaide for providing this facility and in particular to Tim Kleemann and John Gilbert foi generously giving the¡r time and advice in its use. ix Chapter 1. Background and Introduction. 1 Chapter 1. Background and lntroduction. 2 lmmunopathology of Glomerulonephritis 3 Circulating lmmune Complex Disease. 3 Disease Caused by Antibodies to Structural Antigens.. 4 "ln Situ" lmmune Complex Formation 6 Summary of Pathogenic Mechanisms of Glomerulonephritis 7 Anti-Glomerular Basement Membrane-Antibody Disease I Anti-GBM-Antibody Disease in Humans I Experimental Models of Anti-GBM-Antibody Disease I Diagnosis. I Antigen..... ... 10 lnduction of Antibody Formation 10 Pathogenic Significance of Antibody 11 Mediation of Damage .................. 11 Therapy. 12 Chapter 1. Background and lntroduction. 3 lmmunopathology of Glomerulonephritis lmmune damage to the glomerulus has tradítionally been thought to arise by