Genetic Background is a Key Determinant of Glomerular Extracellular Matrix Composition and Organization

Abstract

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 potential ECM regulatory pathways involving inhibition of matrix metalloproteases, liver X receptor/retinoid X 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.

ECM Characterization Reveals a Massive Activation of Acute Phase Response during FSGS

“…Manual curation revealed 111 bona fide ECM-associated proteins according to the matrisome resource database [15] plus additional proteins not primarily associated with ECM (e.g., members of the complement pathway, see below) (Table S2). Our findings are in line with a comparable study identifying 115 ECM-associated proteins in two different mouse backgrounds [7]. We found strong and significant expression changes for 18 up-and two downregulated proteins in ECM isolates in miR-193a FSGS mice, four proteins were even exclusively detected in FSGS (Table 1.)…”

“…By adapting a protocol established by Rachel Lennon and coworkers [7,11], we isolated the ECM of healthy control mice and mice over-expressing miR-193a for 8 weeks [14] (Figure 1a-d). The ECM of four mice per group was characterized by MS analysis.…”

“…The glomerular ECM provides a supporting scaffold for podocytes and endothelial cells and integrates signals between cells and their environment, including survival and growth factor signaling [1][2][3]. Dysregulation of ECM deposition and formation of excessive connective tissue histologically manifests as sclerosis and can cause organ dysfunction or failure [4], especially in glomerular diseases [5][6][7].…”

“…Recently, the composition of the glomerular ECM of humans and mice have been established [11][12][13]. Furthermore, comparison of the glomerular ECM of mice with different genetic backgrounds and basic levels of proteinuria revealed changes in certain proteins, which might enhance disease susceptibility [7]. To gain deeper insight into the changes during FSGS development and progression, we characterized the ECM proteome during FSGS in the murine miR-193a transgenic model by mass spectrometry (MS) analysis.…”

Comparative proteomic assessment of matrisome enrichment methodologies

“…In an elaboration of this method, Hill et al [13] performed a further chemical digestion step to aid in the solubilisation of the urea-insoluble fraction prior to trypsin digestion, improving the recovery of matrisome components identified in rat lungs. Treatment by alkaline detergent has been used to enrich matrisomal components from murine glomeruli [21,22]. The alkaline detergent improves the solubilisation of cellular components and disrupts cell–ECM interactions.…”