Figure 5

Evaluation of podocyte damage, glomerular cell proliferation and apoptosis. (a) Immunostaining for desmin, as marker of podocyte damage, was performed on paraffin embedded kidney sections. Representative photomicrographs (400x magnification) show no staining in non-diabetic controls, but several positive podocytes (arrows) in DM with significantly reduced staining intensity in DM-Cin kidneys (scale bar represents 50 μm). (b) Compared to controls, the mRNA expression of nephrin and podocin was reduced by at least 40% in DM kidneys, showing diabetic podocyte damage. Both nephrin and podocin expression was significantly ameliorated in DM-Cin kidneys. The mRNA expression was normalized for GAPDH expression using the formula 2 ^−ΔΔCt. (c) Representative photomicrographs for Ki-67 are shown (400x magnification), as a marker of cell proliferation. We detected significantly more Ki-67 positive proliferating glomerular cells in DM rats than in non-diabetic controls, which difference was abolished by cinaciguat treatment. (d) TUNEL assay demonstrated a marked increase of apoptotic glomerular and tubular cell count in DM rats vs. controls, which was significantly reduced in DM-Cin kidneys. Scale bar represents 50 μm. Data are presented as mean ± SD (n = 8–10/group). *p < 0.05, **p < 0.01, ***p < 0.001 (two-way ANOVA with Sidak’s multiple comparison test).