Table 1.
Average ages of mice used for analysis.
Table 2.
Primer sequences used for RT-qPCR validations.
Fig 1.
Histology of wild type (WT) and Cd2ap+/-, Fyn-/- three allele mutant (3A) glomeruli.
H&E staining, top panels, show partial blockage of mutant capillaries and regions of scarring, particularly pronounced on the left extreme. (3A was 14 months, Ctrl was 10 months). Bottom panels show Jone’s Silver stain, with increased staining of glomerular basement membranes in 3A glomerulus. (3A was 15 months, Ctrl was 10 months).
Fig 2.
Scanning electron microscopy of wild type (WT) and three allele mutant (3A) glomeruli.
Note uniform capillary coverage by wild type podocytes, with evenly spaced foot processes and slit diaphragms. In contrast the mutant podocytes are disorganized, partially detached, with foot processes sometimes fused (arrowhead). In addition, the mutant glomerulus showed abundant fibrils (arrow). (Both 3A and Ctrl were 14 months).
Fig 3.
Transmission electron microscopy of glomeruli of wild type (WT), left panels, and mutant (3A), middle and right panels. Upper panels are lower magnification and lower panels are higher magnification (see size bars). Note evenly spaced foot processes and slit diaphragms in the WT, with the mutant showing fused foot processes (black arrow) and expanded glomerular basement membrane (white arrow). (Both 3A and Ctrl were 14 months).
Fig 4.
Heatmap showing gene expression changes in control (ctrl) versus Cd2ap+/-, Fyn-/- three allele mutant (3A) podocytes.
There were three samples of each, with red indicating stronger expression and blue indicating weaker expression. Genes with greater than 2 fold change are shown. An expandable version including gene names is included in supplementary data (S1 Fig). Lists of genes with fold change are included in S1 Table.
Fig 5.
Cytoscape showing some of the genes upregulated in Cd2ap+/-, Fyn-/- mutant podocytes and their associated functionalities.
Red hexagons represent upregulated genes in mutant podocytes. Molecular functions (Protein kinase activity), Biological processes (response to wounding, regulation of cell adhesion, regulation of MAPK cascade, macrophage cytokine production), Cellular components (extracellular matrix), Pathways (AGE-RAGE signaling pathway in diabetic complications), Computational (neighborhood of STAT6) and Co-expression (genes defining epithelial-mesenchymal transition), as determined using ToppGene (Toppgene.cchmc.org).
Fig 6.
RT-QPCR validation of gene expression changes.
Using RNA from isolated glomeruli the RNA-seq predicted changes in expression of Endou, Parm1, Serpine1, Spon2, and Rspo1 in mutant podocytes, and Cpe, Aldh1a2, Tnc, Cpne7, Frzb, and Thbs1 in mutant mesangial cells were validated.