WT (A–H) or Ripk3^–/– mice (C–H) were subjected to sham operation or UUO, and kidneys were harvested at 7 days after surgery. (A) Ripk3 mRNA expression was analyzed by qRT-PCR. Gapdh was the standard (n = 7, WT Sham; n = 8, WT UUO). *P < 0.05 by Mann-Whitney test. (B) RIPK3 expression was determined by Western blot analysis. GAPDH was the standard (n = 3, WT Sham; n = 7, WT UUO). Dot plots represent densitometric analysis of Western blot data (mean ± SEM, 3 independent experiments). *P < 0.05 by Mann-Whitney test. (C) Kidney tissue lysates were subjected to Western blot analysis for α–smooth muscle actin (αSMA), type-I collagen (Col-I), and fibronectin (FN). GAPDH was the standard. Dot plots represent densitometric analysis of Western blot data (mean ± SEM, 3 independent experiments). *P < 0.05 compared with WT UUO mice by ANOVA with Newman-Keuls post-hoc test. (D) Kidney tissues were analyzed by qRT-PCR for Col1a1 and Acta2 mRNA expression. Gapdh was the standard. *P < 0.05 compared with WT UUO mice by ANOVA with Newman-Keuls post-hoc test (mean ± SEM, 3 independent experiments). (E and F) Kidney tissues were stained with (E) Masson’s trichrome or (F) immunostained for RIPK3. Representative images are shown. Tubular epithelial cells (arrowheads) and interstitial cells (arrows) are indicated. Scale bars: 100 μm. (G) Immunofluorescence staining for RIPK3 (red), vimentin (green), and Hoechst (blue). Scale bars: 10 μm. C–G; n = 6 for UUO; n = 3 for Sham. (H) Quantitative analysis of TUNEL-positive cells. The number of TUNEL-positive cells was counted in 5 random high-power fields (HPF) of the left kidney of the WT mice and Ripk3^–/– mice 7 days after UUO or sham surgery. The dot represents the average of the number of TUNEL-positive cells per HPF for each mouse. WT Sham (n = 4), WT UUO (n = 7), Ripk3^–/– Sham (n = 3), Ripk3^–/– UUO (n = 6). *P < 0.05 compared with WT UUO mice by ANOVA with Newman-Keuls post-hoc test. All values represent mean ± SEM from 3 independent experiments.