Role of 3-methyladenine in uric acid-induced apoptosis of renal tubular epithelial cells / 中华肾脏病杂志

ABSTRACT

Objective:To investigate the effect of autophagy inhibitor 3-methyladenine (3-MA) on uric acid (UA)-induced apoptosis of renal tubular epithelial cells.Methods:(1) Totally 24 SD rats were randomly divided into 4 groups control group, treatment with 3-MA group, hyperuricemic nephropathy (HN) group, and HN+3-MA group, with 6 rats in each group. According to the body weight of the rats, adenine (100 mg/kg) and potassium oxonate (1 500 mg/kg) were mixed with distilled water to make a suspension, and the rats were given intragastrically once daily for consecutive 21 days to establish HN rat model. The control group and the 3-MA treatment group were fed an equivalent amount of distilled water. At the same time, the 3-MA treatment group and the HN+3-MA group were intraperitoneally injected with 3-MA (15 mg/kg), and the control group and HN group were intraperitoneally injected with an equal volume of saline once daily for 21 consecutive days. Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling assay (TUNEL) was used to observe renal cell apoptosis. Western blotting was used to detect the expression levels of cleaved caspase-3 and Bax, and immunofluorescence staining was used to detect the expression and localization of cleaved caspase-3 in renal tissue. (2) Human renal tubular epithelial cells (HK-2) were stimulated with UA (800 μmol/L), and cells were administrated with different concentrations of 3-MA or Beclin-1 small interfering RNA (siRNA). The apoptosis of renal tubular epithelial cells was detected by Western blotting and immunofluorescence staining.Results:Compared with the normal rats, the apoptosis of renal tubular epithelial cells in the HN group was significantly increased ( P<0.01), and the expression levels of cleaved caspase-3 and Bax were significantly up-regulated (both P<0.05). Compared with the HN group, the apoptosis of renal tubular epithelial cells in the HN+3-MA group was significantly decreased ( P<0.01). In addition, high level of uric acid could significantly increase the levels of apoptosis associated proteins in HK-2 cells (all P<0.05), and using different concentrations of 3-MA or transfecting with Beclin-1 siRNA could significantly reduce the expression of cleaved caspase-3 and Bax (all P<0.05). Conclusion:Autophagy plays an important role in uric acid-induced apoptosis of renal tubular epithelial cells. Inhibiting the excessive activation of autophagy may be a new strategy to prevent the progression of HN.