Browsing by Author "Humphries, Jonathan D."

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    Genetic Background is a Key Determinant of Glomerular Extracellular Matrix Composition and Organization
    (Wolters Kluwer, 2015-04-20) Randles, Michael J.; Woolf, Adrian S.; Huang, Jennifer L.; Byron, Adam; Humphries, Jonathan D.; Price, Karen L.; Kolatsi-Joannou, Maria; Collinson, Sophie; Denny, Thomas; Knight, David; Mironov, Aleksandr; Starborg, Toby; Korstanje, Ron; Humphries, Martin J.; Long, David A.; Lennon, Rachel
    Glomerular disease often features altered histologic patterns of extracellular matrix (ECM). Despite this, the potential complexities of the glomerular ECM in both health and disease are poorly understood. To explore whether genetic background and sex determine glomerular ECM composition, we investigated two mouse strains, FVB and B6, using RNA microarrays of isolated glomeruli combined with proteomic glomerular ECM analyses. These studies, undertaken in healthy young adult animals, revealed unique strain- and sexdependent glomerular ECM signatures, which correlated with variations in levels of albuminuria and known predisposition to progressive nephropathy. Among the variation, we observed changes in netrin 4, fibroblast growth factor 2, tenascin C, collagen 1, meprin 1-a, and meprin 1-b. Differences in protein abundance were validated by quantitative immunohistochemistry and Western blot analysis, and the collective differences were not explained by mutations in known ECM or glomerular disease genes. Within the distinct signatures, we discovered a core set of structural ECM proteins that form multiple protein–protein interactions and are conserved from mouse to man. Furthermore, we found striking ultrastructural changes in glomerular basement membranes in FVB mice. Pathway analysis of merged transcriptomic and proteomic datasets identified potentialECMregulatory pathways involving inhibitionofmatrixmetalloproteases, liverXreceptor/retinoidX receptor, nuclear factor erythroid 2-related factor 2, notch, and cyclin-dependent kinase 5. These pathways may therefore alter ECM and confer susceptibility to disease.
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    Glomerular Cell Cross-Talk Influences Composition and Assembly of Extracellular Matrix
    (American Society of Nephrology, 2014-01-16) Randles, Michael J.; Byron, Adam; Humphries, Jonathan D.; Mironov, Aleksandr; Hamidi, Hellyeh; Harris, Shelley; Mathieson, Peter W.; Saleem, Moin A.; Satchell, Simon C.; Zent, Roy; Humphries, Martin J.; Lennon, Rachel
    The glomerular basement membrane (GBM) is a specialized extracellular matrix (ECM) compartment within the glomerulus that contains tissue-restricted isoforms of collagen IV and laminin. It is integral to the capillary wall and therefore, functionally linked to glomerular filtration. Although the composition of the GBM has been investigated with global and candidate-based approaches, the relative contributions of glomerular cell types to the production of ECM are not well understood. To characterize specific cellular contributions to the GBM, we used mass spectrometry–based proteomics to analyze ECM isolated from podocytes and glomerular endothelial cells in vitro. These analyses identified cell type–specificdifferences in ECM composition, indicating distinct contributions to glomerular ECM assembly. Coculture of podocytes and endothelial cells resulted in an altered composition and organization of ECM compared with monoculture ECMs, and electron microscopy revealed basement membrane–like ECM deposition between cocultured cells, suggesting the involvement of cell–cell cross-talk in the production of glomerular ECM. Notably, compared with monoculture ECM proteomes, the coculture ECM proteome better resembled a tissue-derived glomerular ECM dataset, indicating its relevance to GBM in vivo. Protein network analyses revealed a common core of 35 highly connected structural ECM proteins that may be important for glomerular ECM assembly. Overall, these findings show the complexity of the glomerular ECM and suggest that both ECM composition and organization are context-dependent.