Effect of autophagy on the hypoxia-induced human renal proximal tubular cell apoptosis

Abstract:

Objective：To investigate the effect of autophagy on the hypoxia-induced apoptosis in human renal proximal tubular cells（HK-2）. Methods：Human proximal tubular epithelial cells cultured in vitro were divided into the control group（21%O2） and hypoxia group（1%O2），based on the oxygen concentration. HK-2 cells cultured in hypoxia condition were then divided into 6 h hypoxia sub－group，12 h hypoxia subgroup，18 h hypoxia subgroup and 24 h hypoxia subgroup. The expression of autophagy-related proteins，Beclin1，LC3Ⅱ/LC3Ⅰ，P62 and apoptosis-related protein，activated cleaved-caspase 3 were detected by Western blot analysis，and the apoptosis was analyzed by flow cytometry assay. HK-2 cells stably expressing GFP-LC3 were induced by hypoxia for 18 hours，and the alteration of LC3 autophagosomes was examined by immunofluorescence（IF） assay. Then HK-2 cells were co-treated with hypoxia and autophagy inducer rapamycin or autophagy inhibitor Bafi-lomycin A1，and they were divided into normoxia control group，nor－moxia plus rapamycin/ Bafilomycin A1 group，hypoxia control group，hypoxia plus rapamycin/Bafilomycin A1 group. The expression of Beclin1，LC3Ⅱ/LC3Ⅰ，P62 and cleaved-caspase3 were detected by Western blot analysis，while the apoptosis was analyzed by flow cytometry assay. Results：Compared with the control group（0.161±0.043），the cleaved-caspase3 expression in 12 h[（0.315±0.060），P=0.019]，18 h[（0.586±0.063），P=0.000] and 24 h hypoxia subgroup[（0.698±0.085），P=0.000] increased significantly. The apopto－sis of HK-2 cells in 12 h[（23.633±5.346）%]，18 h[（25.200±4.782）%] and 24 h hypoxia subgroup[（38.567±8.046）%] increased significantly as compared with the control group[（11.800±3.966）%]（P=0.036，P=0.021，P=0.000）. The Beclin1 expression in 18 h[（0.557±0.071），P=0.001] and 24 h hypoxia subgroup[（0.897±0.074），P=0.000] were higher than that in the control group（0.249±0.04）. The LC3Ⅱ/LC3Ⅰ expression in 6 h[（1.795±0.143），P=0.036]，12 h[（2.873±0.078），P=0.000]，18 h[（3.029±0.089），P=0.000] and 24 h hypoxia subgroup[（3.735±0.059），P=0.000] were higher than that in the control group（1.322±0.191）. The P62 ex－pression in 6 h[（0.776±0.061），P=0.028]，12 h[（0.602±0.119），P=0.001]，18 h[（0.419± 0.117），P=0.000] and 24 h hypoxia subgroup[（0.384±0.068），P=0.000] were lower than that in the control group（0.957±0.035）. The counting value of GFP-LC3 puncta in HK-2 cells in hypoxia group（40.667±6.028） was obviously more than that in the control group（18.667±5.508）（P=0.010）. We also found that the treatment of the autophagy inducer rapamycin significantly reduced the hypoxia-induced apoptosis[（26.433±3.402），P=0.012] and cleaved-caspase3 expression[（0.265±0.066），P=0.000] of HK-2 cells compared with that in the hypoxia control group[（36.133±5.856），（0.358±0.060）]，while the treatment of the autophagy inhibitor Bafilomycin A1 significantly increased the hypox－ia-induced apoptosis[（49.233±3.412），P=0.000] and cleaved-caspase3 expression[（1.242±0.110），P=0.000] of HK-2 cells as com－pared to the hypoxia control group[（25.933±3.650），（0.659±0.060）]. Conclusion：Autophagy protects against the hypoxia- induced apoptosis of HK-2 cells.