Apoptosis inducing factor mediates cisplatin-induced apoptosis of renal tubular epithelial cells

Abstract

To investigate the involvement of apoptosis inducing factor (AIF) in caspase-independent pathway mediating apoptosis of cultured renal tubular epithelial cells induced by cisplatin (CP). Western Blot analysis and real-time PCR were performed to detect cytosol AIF (cAIF), nuclear AIF (nAIF) and AIF mRNA expression in cultured renal epithelial cells (HK-2) treated with cisplatin (CP) at various concentrations (0 - 200 micromol/L) and time courses (0 - 12 h). Immunofluorescence analysis was used to detect the AIF protein distribution in HK-2 cells. Pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA treatment, TUNEL and flow cytometer were used to measure the suppression of apoptosis induced by CP in HK-2 cells. The expressions of cAIF, nAIF protein and AIF mRNA were all increased to some extent in HK-2 cells treated with CP at various concentrations and time points. cAIF expression was 2.3-fold (P < 0.05) increased after 25 micromol/L CP treatment for 12 h and 1.7-fold (P < 0.01) increased after 50 micromol/L CP treatment for 3 h, compared with that of control groups, and showed a concentration- and time-dependent increment. The nAIF expression reached a peak (4.3-fold increase) (P < 0.005) after 150 micromol/L CP treatment for 12 h and 3.7-fold incease (P < 0.05) after 50 micromol/L CP treatment for 9 h, compared with that of the 25 micromol/L group and 3 h group, respectively. The expression of nAIF was approximately consistent with cleaved-PARP expressive pattern. Real-time PCR showed that AIF mRNA increased gradually with prolonged treatment with 50 micromol/L CP and reached a peak at 9 h. Immunofluorescence assay showed AIF translocation from cytosol to nuclei in some cultured HK-2 cells treated with CP. Applying pan-caspase inhibitor (Z-VAD-FMK) and AIF-siRNA to CP-treated HK-2 cells, the apoptotic rates were decreased by 60.1% and 39.2%, respectively. The inhibitory effect on HK-2 cell apoptosis was even more significant with combination of both Z-VAD-FMK and AIF-siRNA. The AIF activation and translocation to nuclei with the increment of its mRNA expression mediates CP-induced apoptosis of renal tubular epithelial cells in vitro. It may provide a new therapeutic target for protecting from nephrotoxciity of cisplatin.