The Effects Of Down-regulation Of Augmenter Of Liver Regeneration On The Autophagy Level Of Human Renal Tubular Epithelial Cells After Ischemia-reperfusion Treatment

Part1: Construction of human renal tubular epithelialcell line with stable down-regulation of 23 k D augmenter ofliver regenerationObjective: To construct human renal tubular epithelial cell line with stable down-regulation of 23 k D augmenter of liver regeneration(ALR)by lentivirus infection and purine screening.Methods: A effective RNAi lentiviral vectro harboring RNAi sequence targeting human ALR gene were construct.Then it was transformed into HK-2 cells.HK-2 cells were transfected by sh RNA/control as control group.The screening concentrations of puromycin were determined by treating HK-2 cells with different concentrations of puromycin.The expression of 23 k D ALR in HK-2 cells was detected by real-time fluorescence quantitative PCR and immunoblotting.MTS was used to detect HK-2 cell proliferation.Results: Sequence alignment confirmed that the lentivirus oligonucleotide sequence was inserted correctly and the purine screening concentration was confirmed to be 3μg/ml.Real-time fluorescent quantitative PCR and Western blot analysis showed that the expression of endogenous 23 k D ALR m RNA and protein in the sh RNA/ALR group was significantly lower than that in the normal group and the sh RNA/control group(P <0.05).MTS results showed that down-regulation of 23 k D ALR had no significant effect on HK-2 cell proliferation(P> 0.05).Conclusion: HK-2 cell line with stable down-regulation of 23 k D ALR were successfully constructed.Part2: Effects of I/R treatment on the expression of 23 k DALR and the level of autophagy in HK-2 cellsObjective: To investigate the change of the expression of 23 k D ALR and autophagy-related proteins in HK-2 cells after ischemia/reperfusion(I/R)treatment.Methods: HK-2 cells were treated with serum-free and phosphate-free phosphate medium combined with hypoxic and reoxygenation treatment.The expression of 23 k D ALR and autophagy-related proteins LC3 II and p62 in HK-2 cells were detected by immunoblotting.Immunofluorescence was used to observe the expression of LC3 fluorescent spots in HK-2 cells.Results: After I/R treatment,the expression of 23 k D ALR in HK-2 cells increased first and returned to normal gradually.The expression of LC3 II was significantly increased at 3,6,12 and 24 hours after I/R treatment.The expression of p62 decreased at each time point.Immunofluorescence also indicated the LC3 fluorescence was significantly enhanced in HK-2 cells after I/R treatment.These results suggest that the level of autophagy in HK-2 cells elevated after I/R treatment.Conclusion: The autophagy level of HK-2 cells increased first and then decreased gradually after I/R treatment.The variation tendency was consistent with the change process of autophagy induced by I/R model in vivo.The expression of ALR in HK-2 cells increased during I/R treatment.Part 3: The effect of 23 k D ALR down-regulation on the level of autophagy induced by ischemia-reperfusion in HK-2 cellsObjective: To investigate the effect of ALR down-regulation on the autophagy level induced by ischemia-reperfusion in HK-2 cells.Methods: The autophagy level in HK-2 cells was upregulated after treated with ischemia-reperfusion as previous described.The expression of ALR in HK-2 cells was downregulated after transfected with lentivirus sh RNA/ALR.The expression of ALR and autophagy-related proteins such as LC3 II,p62,p-AMPK,AMPK,p-m TOR and m TOR were detected by immunoblotting.The expression of caspase-3 also was detected with immunobloting.The LC3 fluorescent spots in HK-2 cells was detected by laser confocal microscopy.The levels of reactive oxygen species(ROS)were measured by flow cytometry.Electron microscopy was used to detect the formation of autophagosome in HK-2 cells.Flow cytometry was used to analyze the level of apoptosis in HK-2 cells.Results: The results showed that the level of autophagy in sh RNA/ALR group was significantly higher than that in the sh RNA/control group.The number of autophagosome in the sh RNA/control group increased significantly compared with the sh RNA/ALR group after I/R treatment.Compared with the sh RNA/control group,Western blots showed p-AMPK level increased and p-m TOR level decreased in the sh RNA/ALR group.After treatment with Compound C,the level of autophagy was significantly decreased and the level of apoptosis was significantlyincreased in the sh RNA/control group and the sh RNA/ALR group.Western blots showed the expression of caspase-3 upregulated while autophagy was downregulated,especially in the sh RNA/ALR group.Conclusion: 23 k D ALR is involved in the autophagy process induced by ischemia-reperfusion.The down-regulation of 23 k D ALR expression increased the level of autophagy in HK-2 cells.The AMPK/m TOR signaling pathway is hyperactive in the sh RNA/ALR group.This effect may be associated with the level of ROS increased after down-regulation of 23 k D ALR in the sh RNA/ALR group.Autophagy inhibit apoptosis and play a protective role under conditions of oxidative stress.