Figure 3.

CIN85 exon2 deletion ameliorates proteinuria and glomerular matrix accumulation in diabetic mice. A: Immunofluorescence staining of mouse glomeruli using antibodies against nephrin (green) and ubiquitin (red). Ubiquitin partially colocalized with nephrin (yellow) in diabetic C57BL/6J mice. Ubiquitin-positive podocytes were increased in 16-week-old diabetic wild-type mice, whereas nephrin expression was downregulated. Nephrin expression was preserved in CIN85^Δex2 mice, and substantially fewer ubiquitin-positive podocytes were detected under diabetic conditions compared with C57BL/6J diabetic mice. Original magnification ×60. B: Albuminuria in C57BL/6J mice was determined by analysis of spot urine samples of mice 16 weeks after low-dose STZ injection (n = 5 in first three conditions, n = 12 in CIN85^Δex2 diabetic mice). The error bars show the mean ± SEM. ***P ≤ 0.001 by unpaired t test. C: Ubiquitination assay using immunoprecipitation (IP) with antibody against nephrin and ubiquitin showed the ubiquitinated nephrin content increased 1 h after high glucose stimulation. D: Representative images of glomeruli stained by immunofluorescence using anti-collagen IV. Collagen IV staining indicated increased matrix accumulation caused by diabetes. Original magnification ×60. E: Glomerular collagen IV expression in glomeruli from the indicated mice was semiquantitatively scored in a blinded fashion: score 1, very mild; score 2, mild; score 3, moderate; score 4, intense. The error bars show the mean ± SEM (n = 5 per condition). *P ≤ 0.05; n.s., not statistically significant by unpaired t test.