Expression of apoptosis-related genes in chronic cyclosporine nephrotoxicity in mice

To define the mechanism of cyclosporine (CsA)-induced apoptosis, we investigated the expression of apoptosis-related genes in experimental chronic CsA nephrotoxicity. Mice on a low-salt (0.01%) diet were given vehicle (VH, olive oil, 1 mg/kg/day), or CsA (30mg/kg/day), and sacrificed at 1 and 4weeks. Apoptosis was detected with deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) stain, and the expressions of apoptosis-related genes were evaluated by reverse transcription-polymerase chain reaction, immunoblot or immunohistochemistry. The activity of caspase 1 and 3 was also evaluated. The CsA group showed increases in apoptotic cells compared with the VH group (54±41 vs. 3±3, p <0.05), and the number of apoptotic cells correlated well with interstitial fibrosis scores (r = 0.83, p <0.01). The CsA group showed a significant increase in Fas-ligand mRNA (0.20 vs. 0.02amol/pg total RNA, p <0.05) and Fas protein expression (146% vs. 95%, p <0.05), compared with the VH group. The CsA group showed significant increases in ICE mRNA (0.21 vs. 0.03amol/μg total RNA at 4weeks, p <0.05) and CPP32 mRNA (0.18 vs. 0.03amol/μg total RNA at 4weeks, p <0.05), compared with the VH group. The enzymatic activity of ICE (16.6 vs. 7.9 pmol/μg/h, p <0.05) and CPP32 protease (15.6 vs. 2.7pmol/μg/h, p 0.<05) proteases were increased in the CsA group, compared with the VH group. The ratio between bax and bcl-2 protein increased significantly in the CsA group (5.3-fold), compared with the VH group. Levels of p53 protein also increased in the CsA group. Immunohistochemical detection of Fas, Fas-ligand, ICE and CPP32 revealed strong immunoreactivity in renal tubular cells in areas of structural injury. These findings suggest that local activation of the apoptosis-related genes is associated with CsA-induced apoptotic cell death.