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Global Analysis of Kidney Glomerular Extracellular Matrix in Mice of Different Strains and Genders
[Thesis]. Manchester, UK: The University of Manchester; 2013.
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Abstract
Research to date has shown that human populations of different racial backgrounds and sexes have differing levels of albuminuria, suggesting that the genetics of an individual influences the function and integrity of their glomerular filtration barrier (GFB). In addition, different strains and sexes of mice are known to have variable patterns of albuminuria comparable with those seen amongst human races and sexes.The aim of this research project was to use mass spectrometry (MS)-based proteomics to characterise the effect of sex and strain on the protein composition of glomerular ECMs from male and female mice from the C57BL/6J (C57) and FVB inbred strains as well as identify any specific glomerular ECM components that vary in these mice and relate to their degree of albuminuria. Therefore, the central hypothesis for this research project stated that global proteomic analysis of the glomerular extracellular matrix (ECM) would produce novel data regarding sex and strain-related differences in protein composition in the murine GBM and mesangial matrix.This report presents the investigations in four related areas: 1: whether techniques relying on either sieving or magnetic Dynabeads are more effective for the isolation of murine glomeruli; 2: the enrichment of the murine glomerular ECMs using buffered washes; 3: the analysis of ECM components via liquid chromatography–tandem mass spectrometry; and 4: a comparison of analogous data sets relating to RNA expression and protein abundance in the murine glomerulus.The results have shown that the Dynabead-based glomerular isolation method was superior because it produced an isolate containing nearly five times as many glomeruli compared with its sieving based alternative. A sample suitable for analysis by MS was produced through the successful enrichment of glomerular ECM proteins as assessed by Western blotting. Analysis of the samples by MS provided a list of 120 ECM proteins that make up the glomerular matrisome, or the ensemble of ECM proteins and associated factors, in the four mouse groups studied. The different patterns of abundance observed in these proteins across the mouse groups have shown that the glomerular ECMs of male and female mice from the C57 and FVB strains are compositionally different. Additionally, the presence or absence of certain proteins can be associated with increased levels of albuminuria. Comparative analysis of RNA array and MS datasets has revealed previously unreported patterns of gene expression relating to the mouse groups studied.This work provides a basis for an MS analysis of glomerular ECMs and for future investigations aimed at determining the roles of specific components and mechanisms that control sex and strain-associated changes in the structure and function of the glomerular ECM.