The Role Of Prohibitin 2-mediated Mitophagy In ANG? Induced Renal Tubular Epithelial Cells Injury

Objective:Renal tubular epithelial cell injury is a key pathological hallmark for renal fibrosis and end-stage renal disease.Current studies suggest that activation of mitochondrial ROS/Nlrp3 inflammasome axis can directly mediate tubular epithelial cell damage.Mitophagy is important to mitochondrial quality control and is widely involved in the regulation of various kidney diseases.However,its specific mechanism remains unclear.Prohibitin 2?PHB2?is a mitochondrial inner membrane protein which is closely related to the maintenance of mitochondrial morphology and function.Recent studies found that PHB2 could bind to autophagy-associated protein LC3 and selectively removed the damaged mitochondria via mitophagy.This study investigated the role of mitophagy in ANG II-induced renal tubular epithelial cell injury,and explored the regulation of PHB2 on mitochondrial dysfunction/Nlrp3 inflammasome,in order to provide new therapeutic targets for chronic kidney disease.Methods:Part I:HK-2 cells were treated with ANG? of different concentrations(10^-8,10^-7,10^-66 and 10^-55 mol/L)for 12 hours.The levels of ATG7,LC3II were analyzed by western blot to indicate autophagy flux.Then,chloroquine,a common inhibitor of lysosome,was pretreated to further confirm the activation of autophagy.Next,we used 3-MA,a widely used inhibitor of autophagy,as well as rapamycin,an autophagy inducer,to determine whether autophagy played a protective role in HK-2 cells.Tunel staining and the CCK-8 assay were applied to reveal apoptosis and cytotoxicity in HK-2 cells treated with ANG?.Part II:PHB2 was deleted in renal proximal tubular cells via transfection of shRNA against PHB2,and its efficiency was confirmed by western blotting analysis.MitoTracker and lysoTracker were used to assess mitophagy.HK-2 cells were transfected with the PHB2 overexpression?OE?plasmid 1 day before the ANG? intervention?10^-66 mol/L?.The influence on mitophagy and apoptosis were determined as above.Part III:To further elucidate the regulatory role of PHB2 in mitophagy and inflammation,DCFDA,mitoSOX,JC-1 staining and RT-PCR analysis were conducted to assess cellular/mitochondrial ROS,membrane potential,ATP and mtDNA respectively.We also used western blot analysis to detect the expression of the components of the inflammasome,including NLRP3,ASC,caspase 1,and the downstream cytokines IL-1?and IL-18.Results:Our results showed that autophagy was stimulated by ANG? in a dose-dependent way.After incubation with 3-MA,the degree of autophagy induced by ANG??10^-66 mol/L?was significantly reduced.Consistent with the results of previous studies,pre-treatment with rapamycin remarkably increased autophagy flux.Additionally,amelioration of apoptosis and improvement in cell viability were observed in HK-2 cells in the presence of rapamycin.Strikingly,PHB2 knockdown reduced the level of mitophagy and augmented cell death,while overexpression of PHB2 provided protection against NLRP3-induced inflammatory pathways through amelioration of mitochondrial dysfunction.Conclusion:In summary,we found that autophagy was stimulated in ANG?-induced renal tubular epithelial malfunction,and that it protected HK-2cells against apoptosis.PHB2 is involved in this progression as a mitophagy receptor.Overexpression of PHB2 ameliorates tubular epithelial cells injury,improves mitochondrial dysfunction and reduces NLRP3 inflammasome activation by promoting mitophagy.