Figure 1.
Pirfenidone improved general parameters in SNx rats.
Urinary protein excretion (A), NAG activity (B), blood urea nitrogen (C) and serum creatinine in sham-operated, SNx and PFD-treated rats at the 12th week post-surgery. NAG, N-acetyl-β-D-glycosaminidase; BUN, blood urea nitrogen; Scr, serum creatinine; SNx, 5/6 nephrectomy; PFD, pirfenidone. Values are represented as the means±SEM (n=10). * P<0.05, SNx vs. sham; # P<0.05, SNx+PFD vs. SNx rats.
Figure 2.
Pirfenidone protected renal proximal tubular cell mitochondrial structure.
(A) Representative images of mitochondria in renal proximal tubular cells in rats using transmission electron microscopy. Bars=500nm. (B) Representative images of HK2 cells using transmission electron microscopy. Arrows indicate the mitochondria in different groups. Bars=0.2μm. (C) Flameng grading of injury of renal proximal tubular mitochondria in SNx rats or HK2 cells. SNx, 5/6 nephrectomy; PFD, pirfenidone.
Figure 3.
Pirfenidone stabilized the mitochondrial membrane potential (MMP) and decreased intracellular ROS in renal proximal tubular cells.
(A) Quantitative analysis of MMP in the kidney cortex of rats. Values are represented as the means±SEM (n=10). (B) MMP in HK2 cells was measured using flow cytometry after JC-1 staining; Quantitative analysis of MMP deterioration in HK2 cells. Values are represented as the means±SEM of three replicates from three different experiments. CCCP was used as a positive control. (C) Dichlorofluorescein (DCF) fluorescence was determined using a fluorescence multimode microplate reader. Quantitative analysis of DCF in the kidney cortex of rats or HK2 cells. Rosup (50mg/ml) was used as a positive control in HK2 cells. (D) H2O2 release by HK2 cells was measured in 50μl cell culture media preincubated with Amplex Red reagent for 30 min at room temperature. Values are represented as the means±SEM (n=10); Quantitative analysis of DCF in HK2 cells. Values are represented as the means±SEM of three replicates from three different experiments. SNx, 5/6 nephrectomy; PFD, pirfenidone. * P<0.05, SNx rats vs. sham or rotenone/CCCP/ H2O2 incubation vs. control; # P<0.05, SNx+PFD vs. SNx rats or PFD+rotenone incubation vs. rotenone incubation alone.
Figure 4.
Pirfenidone increased the ATP content and stabilized the mitochondrial DNA (mtDNA) copy number in renal proximal tubular cells.
(A) Quantitative analysis of ATP in kidney cortexes of rats. Values are represented as the means±SEM (n=10); Quantitative analysis of ATP in HK2 cells. Values are represented as the means±SEM of three replicates from three different experiments. (B) quantitative analysis of mtDNA in kidney cortexes of rats. Values are represented as the means±SEM (n=10); Quantitative analysis of mtDNA in HK2 cells. Values are represented as the means±SEM of three replicates from three different experiments. SNx, 5/6 nephrectomy; PFD, pirfenidone. rRNA, ribosomal RNA. * P<0.05, SNx rats vs. sham or rotenone incubation vs. control; # P<0.05, SNx+PFD vs. SNx rats or PFD+rotenone incubation vs. rotenone incubation alone.
Figure 5.
Pirfenidone ameliorated renal injuries including renal tubulointerstitial fibrosis and proximal tubular cell apoptosis in SNx rats.
Representative light micrographs of renal tissues obtained from sham-operated, SNx and PFD-treated rats 12 weeks after renal ablation. A (200× magnification) is representative of periodic acid-schiff and masson trichrome staining (200× magnification) and TdT-mediated dUTP nick end labeling (TUNEL) (400× magnification). Renal tubulointerstitial fibrosis score (B) and proximal tubular cell apoptosis indexes (C) were calculated. Correlations between tubulointerstitial fibrosis and the apoptosis index were analyzed in SNx (D) and PFD-treated rats (E). SNx, 5/6 nephrectomy; PFD, pirfenidone; TIF, tubulointerstitial fibrosis. PAS, periodic acid-schiff. Values are represented as the means±SEM (n=10). * P<0.05, SNx vs. sham; # P<0.05, SNx+PFD vs. SNx rats.
Figure 6.
Pirfenidone sustained HK2 cells viability and inhibited their apoptosis after mitochondrial injury.
Rotenone induced cell injury, and pirfenidone was used at 100, 500 and 1000 μg/ml (A). HK2 cell apoptosis was determined using annexin ν/PI staining and flow cytometry analysis (B). PFD, pirfenidone. Values are represented as the means±SEM of three replicates from three different experiments. * P<0.05, rotenone incubation vs. control; # P<0.05, PFD+rotenone incubation vs. rotenone incubation alone.
Figure 7.
Effect of pirfenidone on cytochrome C, prohibitin and cleaved caspase-3/9 in kidney cortexes of rats.
(A) Western blotting analysis for cyto C, prohibitin, cleaved caspase-3 and cleaved caspase-9. (B) Quantification of cyto C and prohibitin. (C) Quantification of cleaved caspase-3 and cleaved caspase-9. SNx, 5/6 nephrectomy; PFD, pirfenidone. Values were represented as the means±SEM, n=6. * P<0.05, SNx rats vs. sham; # P<0.05, SNx+PFD vs. SNx rats.
Figure 8.
Effect of pirfenidone on cytochrome C, prohibitin and cleaved caspase-3/9 in HK2 cells.
(A) Western blotting analysis for cyto C and prohibitin; Quantification of cyto C and prohibitin. (B) Western blotting analysis for cleaved caspase 3 and cleaved caspase 9; Quantification of cleaved caspase 3/9. PFD, pirfenidone; Values are represented as the means±SEM of three replicates from three different experiments. * P<0.05, rotenone incubation vs. control; # P<0.05, PFD+rotenone incubation vs. rotenone incubation alone.
Figure 9.
Evaluation of totoal SOD (TSOD), Mn-SOD, MDA and GSH activity by pirfenidone intervention.
(A) Activities of TSOD, Mn-SOD, MDA and GSH in kidney cortexes of rats were displayed. Values were represented as the means±SEM, n=10; (B) Quantification of TSOD, Mn-SOD, MDA and GSH in HK2 cells. Values are represented as the means±SEM of three replicates from three different experiments. SNx, 5/6 nephrectomy; PFD, pirfenidone; SOD, superoxide dismutase. Mn-SOD, Manganese superoxide dismutase. MDA, malondialdehyde; GSH, glutathione. * P<0.05, SNx rats vs. sham or rotenone incubation vs. control; # P<0.05, SNx+PFD vs. SNx rats or PFD+rotenone incubation vs. rotenone incubation alone.
Figure 10.
Pirfenidone’s protection of mitochondria in renal proximal tubular cells.