Effect Of Autophagy On The Hypoxia-induced Human Renal Proximal Tubular Cell Apoptosis

Background: Acute kidney injury is the most common acute illness in intensive care patients with a high risk of death.Ischemic kidney injury is a common cause of acute kidney injury.Various factors such as sepsis,trauma,gastrointestinal hemorrhage and other factors could lead to a rapid decline in the body's effective circulating blood volume and renal hypoperfusion,which would cause renal cell ischemia.Hypoxia caused tubular cell damage and increased tubular cell apoptosis.Under the pressure of energy deficiency or hypoxia,autophagy initiates the maintenance of cell homeostasis.However,it was unclear whether autophagy is involved in apoptosis of renal tubular epithelial cells in ischemic acute renal injury.In this study,we aim to discover the changes and interactions of autophagy and apoptosis in renal tubular epithelial cells during hypoxia.Methods: Human renal tubular epithelial cell line(HK2 cells)was used to construct the hypoxia model in this study.Western Blot was used to detect autophagy-related proteins Beclin1,LC3II/LC3 I,P62,Lamp2,TFEB,mTORC1 and apoptosis related protein cleaved-caspase3 expression.Flow cytometry was used to detect the apoptosis of HK2 cells.qPCR was used to detect the change of P62 mRNA level,and P62 protein levels induced by hypoxia were observed when ubiquitin degradation pathway inhibitor MG132 was presented.The spots of GFP-LC3 and P62 were observed under a fluorescence microscope after the GFP-LC3 stably expressing HK2 cells were induced by hypoxia for 18 h.Changes in autophagosomes were observed under electron microscope.HK2 was further treated with hypoxia combined autophagy inducer rapamycin(RaPa)or autophagy inhibitor bafilomycin A1(BafA1),then the expression of cleaved-caspase3 was detected by Western Blot,and apoptosis was detected by flow cytometry.Results: Compared with the control group,the hypoxia group shown: a significantly increased expression of cleaved-caspase3 and the apoptosis;and higher expression of autophagy-related proteins Beclin1,LC3II/LC3 I,TFEB and more autophagosomes,lower expression of p-mTORC1?Lamp2;also increased in transcription level but decreased in protein level P62,which were not influenced by the proteasomal degradation pathway inhibitor MG132.After blocking autophagic degradation by BafA1,the expression of autophagy-related proteins LC3II/LC3 I,P62,and Lamp2 increased in HK2 cells,and the expression of cleaved-caspase3 protein and apoptosis were further increased.Then enhance autophagy by Rapa,the expression of autophagy-regulating proteins Beclin1 and LC3II/LC3 I was up-regulated,and the expression of p-mTORC1 was further decreased,while the expression of cleaved-caspase3 and apoptosis were significantly reduced.Conclusion: Hypoxia promotes apoptosis and activation of autophagy in HK2 cells.Autophagy plays a protective role in hypoxia-induced apoptosis in HK2 cells.