Proteomic analyses of kidney glomerular extracellular matrix in health and disease A thesis submitted to the University of Manchester for the degree of Doctor of Philosophy in the Faculty of Medical and Human Sciences 2014 Michael Randles School of Medicine Institute of Human Development 2 Contents List of figures .................................................................................................................................................... 7 List of tables ..................................................................................................................................................... 9 Abstract ........................................................................................................................................................... 10 Declaration ..................................................................................................................................................... 11 Copyright statement ...................................................................................................................................... 11 Acknowledgments ......................................................................................................................................... 12 Rationale for an alternative thesis format .................................................................................................. 14 Key Abbreviations ......................................................................................................................................... 15 1 General introduction .............................................................................................................................. 16 1.1 Overview of the thesis ................................................................................................................. 16 1.2 The kidney .................................................................................................................................... 17 1.3 Glomerular filtration ..................................................................................................................... 19 1.3.1 The glomerular filtration barrier ............................................................................................. 19 1.3.2 Size and charge selective glomerular filtration ................................................................... 22 1.3.3 The albumin retrieval hypothesis .......................................................................................... 22 1.3.4 The electrokinetic model ........................................................................................................ 23 1.4 Microalbuminuria .......................................................................................................................... 24 1.5 Cellular components of the glomerulus .................................................................................... 25 1.5.1 The glomerular endothelium .................................................................................................. 25 1.5.2 Podocytes .................................................................................................................................26 1.5.3 Mesangial cells ........................................................................................................................ 28 1.5.4 Parietal epithelial cells ............................................................................................................ 28 1.6 Glomerular extracellular matrix .................................................................................................. 31 3 1.6.1 The glomerular basement membrane .................................................................................. 31 1.6.2 The mesangial extracellular matrix and Bowman's capsule ............................................. 36 1.6.3 Regulation of the extracellular matrix by transforming growth factor beta ...................... 36 1.6.4 The Matrisome ......................................................................................................................... 38 1.7 Podocyte adhesion ...................................................................................................................... 38 1.7.1 Integrin biology ......................................................................................................................... 39 1.7.2 Integrin α3β1............................................................................................................................ 41 1.7.3 CD151 ....................................................................................................................................... 41 1.7.4 Syndecans ................................................................................................................................ 42 1.7.5 Dystroglycan ............................................................................................................................. 42 1.7.6 Integrin mediated adhesion complexes ................................................................................ 43 1.7.7 The adhesome ......................................................................................................................... 46 1.7.8 Slit diaphragm .......................................................................................................................... 48 1.8 Mass spectrometry-based proteomics ...................................................................................... 53 1.9 Thesis aims ................................................................................................................................... 55 2 Defining the glomerular matrisome ..................................................................................................... 58 2.1 Introduction ................................................................................................................................... 58 2.2 Statements .................................................................................................................................... 59 2.3 Global analysis reveals the complexity of the human glomerular extracellular matrix ...... 60 2.3.1 Abstract ..................................................................................................................................... 61 2.3.2 Introduction ............................................................................................................................... 62 2.3.3 Results ...................................................................................................................................... 64 2.3.4 Materials and Methods ........................................................................................................... 76 2.3.5 Acknowledgments ................................................................................................................... 81 2.3.6 Statement of competing financial interests .......................................................................... 81 2.3.7 Supplementary data ................................................................................................................ 82 4 3 Glomerular cell-derived matrices ........................................................................................................ 91 3.1 Introduction ................................................................................................................................... 91 3.2 Statements .................................................................................................................................... 92 3.3 Glomerular cell crosstalk influences the composition and assembly of extracellular matrix93 3.3.1 Abstract ..................................................................................................................................... 94 3.3.2 Introduction ............................................................................................................................... 95 3.3.3 Results ...................................................................................................................................... 96 3.3.4 Discussion ............................................................................................................................. 110 3.3.5 Methods ................................................................................................................................. 112 3.3.6 Acknowledgments ................................................................................................................ 119 3.3.7 Statement of competing financial interests ....................................................................... 120 3.3.8 Supplementary data ............................................................................................................. 121 4 Genetic background alters glomerular matrix ................................................................................ 141 4.1 Introduction ................................................................................................................................ 141 4.2 Statements ................................................................................................................................. 142 4.3 Genetic background is a key determinant of glomerular extracellular matrix composition and organisation ............................................................................................................................................