Effect of autophagy on the hypoxia-induced human renal proximal tubular cell apoptosis
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Effect of autophagy on the hypoxia-induced human renal proximal tubular cell apoptosis
Journal of Chongqing Medical UniversityVol. 43, Issue 3, Pages: 431(2018)
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重庆医科大学附属第一医院急诊科,重庆,400042
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Published:2018,
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Yang Shan, Li Guansheng, Chen Xuemei. Effect of autophagy on the hypoxia-induced human renal proximal tubular cell apoptosis. [J]. Journal of Chongqing Medical University 43(3):431(2018)
DOI:
Yang Shan, Li Guansheng, Chen Xuemei. Effect of autophagy on the hypoxia-induced human renal proximal tubular cell apoptosis. [J]. Journal of Chongqing Medical University 43(3):431(2018)DOI:
Effect of autophagy on the hypoxia-induced human renal proximal tubular cell apoptosis
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.
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