| PART Ⅰ THE EFFECTS OF ISCHEMIA-REPERFUSION TREATMENT ON THE EXPRESSION OF AUGMENTER OF LIVER REGENERATION AND THE LEVEL OF MITOPHAGY IN HUMAN KIDNEY PROXIMAL TUBULAR CELLSObjective: To test the augmenter of liver regeneration(ALR)protein expression and the level of mitophagy in human kidney proximal tubular(HK-2)cells induced by ischemia-reperfusion(I/R)in vitro.Methods: HK-2 cells were treated by hypoxic/reoxygenation and phosphate/serum-free medium in vitro.The time point of I/R treatment was set as follows: reoxygenation for 3 hours,6 hours,12 hours and 24 hours after hypoxia for 6 hours(I6R3,I6R6,I6R12 and I6R24).The expression of LC3 II,TOMM20,TIMM23 and 23 kDa ALR in HK-2 cells were detected by immunoblotting.transmission electron microscopy(TEM)was used to observe the morphologic alterations of mitochondria and mitophagosomes in HK-2 cells.Results: In response to I/R treatment,the protein expression level of 23 kDa ALR and LC3-Ⅱ increased gradually,and increased to the highest at the time point of I6R12,and then decreased;while the protein level of TOMM20 and TIMM23 decreased gradually,and decreased to the lowest at the time point of I6R12,and then increased.After I/R treatment,mitochondrial swelling,loss of mitochondrial cristae,mitophagosomes and autophagosomes were apparent in HK-2 cells.Conclusion:1.The results of western blot and TEM showed that I/R treatment successfully induced mitochondrial damage and mitophagy.After I/R treatment,the level of mitophagy increased and was most obvious in I6R12,which was the observation point of subsequent experiments.2.23 kDa ALR was involved in the process of I/R-induced mitophagy,and its protein expression level was consistent with that of LC3-Ⅱ,which gradually increased with time,reached the highest level at 12 h,and then gradually decreased.PART Ⅱ THE EFFECT OF KNOCKDOWN OF ALR ON THE LEVEL OF MITOPHAGY CAUSED BY ISCHEMIA-REPERFUSION IN HK-2 CELLSObjective: To construct HK-2 cell line which stably down-regulating ALR.To investigate the role of down-regulation of ALR in the mitophagy induced by I/R.Methods: HK-2 cells were transduced with LV-ALR shRNA and LV-shRNA according to the manufacturer’s operations.After 72 h,the successfully transduced cells were selected with puromycin.The experimental groups were as follows: IR+shRNA/ALR group,IR+shRNA/control group,IR group and normal group.The ALR m RNA level was examined by quantitative real-time PCR(q PCR),and the ALR protein expression was tested by immunoblotting.The lactate dehydrogenase(LDH)cytotoxicity assay and the cell number count were used to test cell injury caused by I/R.The colocalization of LC3 and mitochondria in HK-2 cells was examined by laser scanning confocal microscopy.The protein level of ALR,LC3-Ⅱ,P62,TOMM20,TIMM23 and cleaved Caspase-3 were detected by Western blot.The levels of mitochondrial reactive oxygen species(ROS)were measured by flow cytometry.Mitochondrial membrane potential(MMP)was assessed using flow cytometry and confocal microscopy.The intracellular ATP levels of HK-2 cells was assessed using an ATP assay kit.The rate of cell apoptosis was tested by flow cytometry.Results: The results of immunoblotting analysis and q PCR showed that the ALR protein expression and ALR m RNA level in the shRNA/ALR group were significantly lower than that in the shRNA/control group and the normal group(P<0.05).the release of LDH in the shRNA/ALR group was clearly increased after I/R treatment.In addition,the number of cells in the shRNA/ALR group was significantly decreased after I/R compared with the shRNA/control(P<0.05).The colocalization of LC3 and mitochondria was decreased in the IR+shRNA /ALR group.The protein expression of ALR and LC3-Ⅱ in the IR+shRNA/ALR group were significantly lower than that of the IR+shRNA/control group,while the protein expression of P62,TOMM20,TIMM23 and cleaved Caspase-3 in the IR+shRNA/ALR group were significantly higher than that of the IR+shRNA/control group(p<0.05).The level of MMP and mitochondrial ROS in the IR+shRNA/ALR group decreased compared with IR+shRNA/control group,while the level of ROS and cell apoptosis were increased in the IR+shRNA/ALR group compared with IR+shRNA/control group(P<0.05).Conclusion: 1.HK-2 cell line which stably down-regulating 23 kDa ALR is successfully established.2.Knockdown of 23 kDa ALR in HK-2 cells can reduce the level of mitophagy,which leads to the aggravation of mitochondrial damage,the increase of ROS released by mitochondria,and finally the increase of cell apoptosis.Therefore,the protective effect of ALR on HK-2 cells is related to the enhancement of mitophagy.PART Ⅲ THE EFFECT OF KNOCKDOWN OF ALR ON THE LEVEL OF PINK1/PARKIN PATHWAYObjective: To investigate the role of PINK1/Parkin in I/R-induced mitophagy and the effect of down-regulating ALR expression on PINK1/Parkin pathway.Methods: The methods of group dividing and model making were the same as the second part.After I/R treatment,the colocalization of PINK1 and Parkin with mitochondria was detected by confocal microscopy.The mitochondria fraction was isolated by mitochondrial protein extraction kit,and the protein expression of PINK1 and Parkin in each experimental group were detected by Western blot.Results: After I/R treatment,immunoblot analysis showed that the expression of PINK1 and parkin extracted from mitochondrial protein increased(P<0.05),and the confocal microscopy results also showed that the fluorescence signals of PINK1 and parkin obviously overlapped with mitochondria.In response to I/R,HK-2 cells with ALR shRNA had a decrease in PINK1 and Parkin protein expression,as well as colocalization with mitochondria in immunofluorescence staining compared with cells with control shRNA(P<0.05).Conclusion: 1.PINK1/Parkin pathway is activated in I/R-induced mitophagy.2.Silencing of 23 kDa ALR suppressed the activation of PINK1/Parkin pathway via double immunofluorescent staining and Western blot.These results suggested that 23 kDa ALR may regulate mitophagy via the PINK1/Parkin pathway. |
