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, immunohistochemical studies of caspase 3 were performed.


RESULTS
: 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 down-regulated the mRNA expression of CsA-induced p53 and Fas-ligand (P<0.01) 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.


CONCLUSIONS
: 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 some of its antifibrotic properties.
Shihab
Fuad S
FS
Division of Nephrology, University of Utah School of Medicine, 30 N. 1900 E., Salt Lake City, UT 84132, USA.
Bennett
William M
WM
Yi
Hong
H
Andoh
Takeshi F
TF
D000818:Animals; D017209:Apoptosis; D053148:Caspase 3; D020169:Caspases / Q000235:genetics; D016572:Cyclosporine / Q000633:toxicity*; D053222:Fas Ligand Protein; D005786:Gene Expression Regulation / Q000187:drug effects*; D016158:Genes, p53; D020287:In Situ Nick-End Labeling; D007668:Kidney / Q000187:drug effects* / Q000473:pathology; D008297:Male; D008562:Membrane Glycoproteins / Q000235:genetics; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D011728:Pyridones / Q000494:pharmacology*; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D051020:bcl-X Protein
C494813:Bcl2l1 protein, rat; D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D019253:Proto-Oncogene Proteins c-bcl-2; D011728:Pyridones; D012333:RNA, Messenger; D051020:bcl-X Protein; D016572:Cyclosporine; C093844:pirfenidone; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases