Figure 3
Effect of diabetes and cinaciguat on renal fibrosis. (a) Immunostaining for collagen IV was performed on paraffin embedded kidney sections. Representative photomicrographs (200x magnification) show moderate tubulointerstitial and glomerular staining in controls, strong tubulointerstitial and thickened glomerular expression in DM but only mild collagen IV expression in DM-Cin groups (scale bar represents 50 μm). (b,c) Compared to controls, the renal mRNA and protein expression analysis of profibrotic TGF-ß as well as CTGF mRNA expression showed a marked overexpression in DM kidneys, which was significantly attenuated in DM-Cin group. TGF-ß protein expression of each sample was normalized for tubulin expression and given as fold change relative to a calibrator. The mRNA expression were normalized for GAPDH expression using the formula 2 −ΔΔCt. (d) DM kidneys showed significantly increased phosphorylation of the p44/p42 MAPK (ERK1/2) as compared to non-diabetic controls, which was inhibited by cinaciguat treatment, as seen in DM-Cin kidneys. Representative immunoblots are shown. Phospho-ERK1/2 expression was normalized for total ERK1/2 expression of each sample and given as fold change. Data are presented as mean ± SD (n = 8–10/group). **p < 0.01, ***p < 0.001 (two-way ANOVA with Sidak’s multiple comparison test).
