Effect of autophagy on oxalate-induced toxicity of human proximal renal tubular epithelial cell.

Abstract

To investigate the role of autophagy in oxalate-induced toxicity of human proximal renal tubular epithelial cell (HK-2). HK-2 cells were exposed to oxalate (1 mmol/L) for 2 h and 3-methyladenine (3-MA) was used to inhibit autophagy. Then Western blotting was used to measure the expression of autophagy-related protein LC3II. Cell viability and cell apoptosis were measured by MTT assay and flow cytometry assay, respectively. Cytoplasmic vacuolization was observed in HK-2 cells after treating with oxalate for 2 h. However, 3-MA showed no effects on the formation of cytoplasmic vacuolization regardless of the dose at 1 or 5 mmol/L. The expression of LC3II protein was significantly increased in the HK-2 cells in the presence of oxalate (0.62±0.03 vs 0.35±0.02, P<0.05). The expression of LC3II protein in HK-2 cells was downregulated by 3-MA at both 1 and 5 mmol/L compared with the blank control (0.17±0.03 vs 0.35±0.02, 0.16±0.03 vs 0.35±0.02, both P<0.05). Oxalate-induced upregulation of LC3II was reversed by 3-MA only at the concentration of 5 mmol/L (0.47±0.04 vs 0.62±0.03, P<0.05) rather than 1 mmol/L (0.61±0.04 vs 0.62±0.03, P>0.05). Oxalate attenuated viability [(77.32±2.69)% vs 100%, P<0.05] and increased the apoptosis [(8.32±1.05)% vs (2.36±0.29)%, P<0.05] in HK-2 cells, and these effects were reversed by 3-MA only at the concentration of 5 mmol/L [(91.91±3.36)% vs (77.32±2.69)%, (3.45±0.21)% vs (8.32±1.05)%, respectively, both P<0.05] rather than 1 mmol/L [(80.48±3.41)% vs (77.32±2.69)%, (7.81±0.47)% vs (8.32±1.05)%, both P>0.05, respectively]. Autophagy of HK-2 cells is enhanced by oxalate at the concentration of 1 mmol/L. Inhibition of 3-MA-induced autophagy protects HK-2 cells from the oxalate-induced cytotoxicity.