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Global Analysis of Kidney Glomerular Extracellular Matrix in Mice of Different Strains and Genders

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Global Analysis of Kidney Glomerular Extracellular Matrix in Mice of Different Strains and Genders Global Analysis of Kidney Glomerular Extracellular Matrix in Mice of Different Strains and Genders A thesis submitted to the University of Manchester for the degree of MPhil in the Institute of Human Development, Faculty of Medical and Human Sciences September 2013 Thomas Robert Denny School of Medicine 2 Table of Contents Table of Contents .................................................................................................. 2 List of Figures ........................................................................................................ 6 List of Tables ......................................................................................................... 8 Abstract ..................................................................................................................... 9 Details and Declaration ........................................................................................ 10 Copyright ............................................................................................................. 11 Acknowledgements .............................................................................................. 12 Part 1: General Introduction .................................................................................... 13 The glomerulus .................................................................................................... 13 Albuminuria and glomerular pathologies .............................................................. 14 Different urinary albumin excretion rates in humans of different genetic backgrounds and sexes ....................................................................................... 15 Urinary albumin excretion rates vary in inbred mouse strains .............................. 16 Part 2: Glomerular Filtration Barrier Structure and Function .................................... 20 Podocytes ............................................................................................................ 22 Fenestrated Endothelial Cells .............................................................................. 22 Glomerular Mesangial Cells ................................................................................. 22 Glomerular extracellular matrix ............................................................................ 23 Laminins .............................................................................................................. 25 Collagens ............................................................................................................ 25 3 Nidogens ............................................................................................................. 26 Heparan Sulphate Proteoglycans ........................................................................ 26 Part 3: Methodologies ............................................................................................. 28 Proteomics .......................................................................................................... 28 Mass Spectrometry .............................................................................................. 29 Simplification of glomerular ECM Sample ............................................................ 30 RNA Array ........................................................................................................... 33 Bioinformatic Analysis .......................................................................................... 34 Aims and Hypotheses .......................................................................................... 36 Aims .................................................................................................................... 36 Hypotheses.......................................................................................................... 37 Methods .................................................................................................................. 38 Antibodies ............................................................................................................ 38 Isolation of mouse glomeruli by sieving ................................................................ 39 Isolation of mouse glomeruli by magnetic dynabeads .......................................... 39 Quantification of glomerular isolation method effectiveness ................................. 40 Enrichment of glomerular ECM proteins .............................................................. 40 Western blotting ................................................................................................... 41 In-gel proteolytic digestion ................................................................................... 42 Liquid chromatography–tandem mass spectrometry analysis .............................. 43 4 Bioinformatic Analysis .......................................................................................... 44 RNA Microarray analysis of isolated glomeruli ..................................................... 46 Analysis of RNA Microarray results ...................................................................... 46 Statistical analysis of RNA Microarray data.......................................................... 47 Comparison of Mass Spectrometric and RNA Microarray data sets ..................... 48 Results Part 1: Comparison of glomerular isolation methodologies ......................... 50 Discussion ........................................................................................................... 54 Results Part 2: Enrichment of Isolated Glomerular Extracellular Matrix Proteins ..... 57 Discussion ........................................................................................................... 59 Results Part 3: Analysis of Mass Spectrometric Data .............................................. 61 Discussion ........................................................................................................... 80 Results Part 4: Comparison of Mass Spectrometric and RNA Array Data Sets ....... 82 Discussion ........................................................................................................... 90 Overall Discussion .................................................................................................. 92 Genes of interest ................................................................................................. 93 Genes coding for proteins expressed in the female gender only .......................... 94 Umod ................................................................................................................... 94 Fras1 ................................................................................................................... 95 Genes coding for proteins expressed in the FVB strain only ................................ 96 Fgf2 ..................................................................................................................... 96 Serpina3k and Agrin ............................................................................................ 97 5 Comparison of Mass Spectrometric and RNA data .............................................. 98 Main Conclusions .................................................................................................... 99 Future Directions of Investigation ....................................................................... 100 References ............................................................................................................... 102 Zhang B. Zhou K. K. and Ma J. (2010) Inhibition of Connective Tissue Growth Factor Overexpression in Diabetic Retinopathy by SERPINA3K via Blocking the WNT/β- Catenin Pathway, Diabetes, 59: 1809–1816. ............................................................ 113 Appendices ............................................................................................................... 115 Appendix 1: Tables of glomerular extracellular matrix proteins detected by mass spectrometry in male FVB, female FVB, male C57 and female C57 mice .......... 115 Appendix 2: Visual comparison of data (MS NSC/RNA RFU) ............................ 124 Words: 27,079 6 List of Figures Figure 1 Graphs showing glomerular number per kidney (A) and urinary 18 albumin excretion rate (B) in male (M) and female (F) mice from the C57 and FVB strains. Figure 2 The glomerulus 21 Figure 3 Coomassie stained gel and Western blots showing enrichment 32 of glomerular ECM proteins Figure 4 Light microscope image of a relatively pure glomerular isolate 51 Figure 5 Mean numbers of whole glomeruli from sieving and Dynabead 53 based isolation methods Figure 6 A Workflow of sieving-based glomerular isolation method 55 Figure 6 B Workflow of Dynabead-based glomerular isolation method 56 Figure 7 Coomassie stained gel and Western blots showing enrichment 58 of glomerular ECM proteins Figure 8 Venn diagram of MS analysis of enriched glomerular isolates 62 from male and female mice from the C57 and FVB strains Figure 9 Bar charts summarizing MS data values for protein products of 68 selected genes characteristic of one sex or one strain Figure 10 Total numbers of glomerular ECM proteins that are unique to 70 each group and classes of glomerular ECM protein that are unique to each mouse group Figure 11 Bar Relative sizes of the four protein classes in the common 73 7 matrisome Figure 12 Percentages that each of the protein classes contribute to the 75 glomerular matrisomes Figure 13 Mean
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