Changes of p53 and Caspase-3 in aristolochic acid-induced tubular epithelial cell apoptosis
AIM: To investigate the features of p53 expression and Caspase-3 activity assay in aristolochic acid (AA) induced renal tubular epithelial cell apoptosis. METHODS: The direct toxic effect of AA on HK-2 cells was evaluated by Lactate dehydrogenase(LDH) releasing essay, Apoptosis was observed under fluorescence microscope after the cells were stained by Hoechst 33258. The apoptosis rate of HK-2 cells induced by AA was estimated with flow cytometric analysis and the expression of wildtype p53 mRNA was measured by RT-PCR. Caspase-3 activity assay was performed by spectrophotometric method. RESULTS: The LDH release rate significantly increased after stimulation with AA at the concentrations of 80 and 160 mg/L(compared with norreal control, P＜0.01). A.A(at the concentrations of 20 and 40 rag/L) inhibited HK-2 cells growth and induced cell apoptesis. The apoptotic index in AA groups markedly increased compared with that in normal control(P＜0.01). The expressions of wildtype p53 mRNA in HK-2 cells after AA treatment(at the concentrations of 10, 20 and 40 mg/L) were similar to these in control group. The activity of Caspase-3 markedly increased after AA treatment(at the concentrations of 20 and 40 mg/L) compared with that in normal control(P＜0.01 ). CONCLUSION: AA is cytotoxic to renal tubular epithelial cells by inhibiting renal tubular epithelial cells growth and inducing apeptosis. Our results demonstrate that AA may induce apoptosis not through p53-independent pathway in renal tubular epithelial cells. Activation of Caspases-3 occurs in AA-induced apoptosis pathway.
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