Effect of pirfenidone on apoptosis-regulatory genes in chronic cyclosporine nephrotoxicity

Background. Apoptosis was shown to play a role in the progression of fibrosis in a chronic cyclosporine A (CsA) nephrotoxicity animal model. In addition, the antifibrotic molecule pirfenidone (PFD) was shown to ameliorate fibrosis in this model. We evaluated the role of PFD on the expression of apoptosis regulatory genes in the kidneys of CsA-treated rats. Methods. Rats were administered CsA 7.5 mg/kg per day, CsA+PFD (250 mg/kg/day), vehicle (VH), or VH+PFD, and sacrificed at 28 days. Physiologic and histologic changes were studied, and apoptosis was detected by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling stain. The mRNA expression of pro-apoptotic genes p53 and Fas-ligand was evaluated by quantitative polymerase chain reaction, and that of Bcl-xL, an anti-apoptotic gene, was evaluated by Northern blot analysis. In addition to mRNA expression, immimohistochemical studies of caspase 3 were performed. Result. PFD administration to CsA-treated rats significantly ameliorated nephrotoxicity. Apoptosis-positive cells were increased by CsA but significantly reduced by PFD treatment (68 +/- 19 vs. 3 +/- 1, P<0.01). In addition, PFD downregulated the mRNA expression of CsA-induced p53 and Fas-ligand (P<0.0 1) and increased that of Bcl-xL, previously reduced by CsA (P<0.01). Finally, PFD significantly down-regulated caspase 3 expression, present mostly on renal tubular epithelial cells. None of these changes were observed in VH-treated rats. Conclusion. Whereas CsA favored the expression of pro-apoptotic genes, that effect was ameliorated by PFD. Because apoptosis can partly explain the loss of cells associated with fibrosis, the influence of PFD on apoptosis-regulatory genes in a manner that reduces apoptosis may explain sonic of its antifibrotic properties.