Study On The Mechanism Of Mitochondrial Autophagy In Renal Tubular Epithelial Cell Injury

Acute kidney injury(AKI)refers to the rapid decline in renal function caused byvarious reasons with the emergence of clinical syndrome.A large sample from multi center epidemiological investigation in our country in 2013 showed that the incidence of AKI was 11.6%.The community AKI morbidity was 2.5%and the hospitalized period AKI morbidity was 9.1%.The mortality of patients with AKI in hospitalwas 8.8%,which has already become a hazard to human health and is the important public issues[1].Although thenephrologistsare paying more and more attention to AKI,but there is still no specific treatmentin recent years.The pathophysiology of AKI is complex,including apoptosis,necrosis,production of reactive oxygen species(ROS),microvascular injury caused by ischemia,endothelial cell dysfunction and the release of inflammatory factors.The injury and necrosis of renal tubular cells is the main pathological process of AKI.So,the key to block the progression of AKIis protecting the renal tubular cells from damage.Therefore,how to prevent and treat the injury of human proximaltubular epithelial cell line(HK-2)is the main problems in the treatment of AKI.Autophagy can clear the damaged and aging organelles and no longer needed biological macromolecules,which playimportant rolesin cell growth of terminally differentiated cells.At the same time of digestion,autophagy also provide materials for construction of organelles.Berkenstam isolated phagocytic vesicles from rat kidney cortex in 1983[2].Previous research has confirmed that autophagy plays a protective role in HK-2 cell injury[3,4].The body can remove damaged or aging organelles and macromolecules through autophagy pathway,including mitophagy,pexophagy,lipophagy,aggrephagy,ER-phagy,ribophagy,xenophagy,ciliophagy,in order to maintain the normal physiological function[5-7].Mitophagy has attracted much attention in nervous system disease and tumor.The ubiquitin proteasome system(UPS)identify the damaged mitochondria by the decrease of mitochondrial membrane potential and the change of mitochondrial morphology.ATP and mitochondrial reactive oxygen species production can regulate mitophagy[8].Therefore,it is necessary to study the relationship between mitophagy and mitochondrial function in kidney disease.We envision mitophagy can clear damaged mitochondria,further improve the mitochondrial function,so as to reduce the cell damage.To verify the hypothesis,we studythe activation and inhibition of autophagy and mitophagy from two aspects in renal tubular epithelial cell injury modelinduced bycisplatin.Researchersfound that the expressionof blood Pink-Parkin mRNAwassignificantly lower in diabetic nephropathy patients and nephropathy patientswith no diabetes than that in healthy control.WhetherPinkl-Parkin is involved in cisplatin induced injury of HK-2 cells and the ralationship between mitochondrial function in HK-2 cells have not been reported.We carried on the preliminary discussion by siRNA interferenceand the overexpression plasmid in this study.Part ? Mitophagy in renal tubular epithelial cell injury induced by cisplatinObjective:To investigate mitophagy and mitochondrial function in renal tubular epithelial cell injury induced by cisplatin.Methods:Human renal proximal tubular epithelial cells(HK-2)were cultured in vitro,with different cisplatin concentrations(0,5,10,20umol/L)to stimulate cells for12h.Western blot was used to detecte autophagy related protein Beclin,LC3.Electron microscopy was used to observe the morphology of autophagy.Rapamycin(100nmol/L)or 3-methyladenine(3-MA,5mmol/L)stimulatedHK-2 cells 30min inadvance,then cisplatin(10umol/L)treated HK?2 cells for 12h.Western blot was used to detected autophagy related protein Beclin andLC3expression.Pre-transfection of adenovirus(mRFP-GFP-LC3)labeled autophagy for12h,We used confocal microscopy observed the expression of autophagy.Mitotracker andlysotracker costaining was used to observe the expression of mitophagy.Mitochondrial reactive oxygen species,mitochondrial membrane potential and the content of ATP were also detected.CCK?8 detected the cell viabilityand apoptosis was detected by flow cytometry.Results:(1)With the increase of the concentration of cisplatin,the expression of Beclinand LC3?/LC3? increased gradually.We observed the morphology of autophagy by electron microscopy.(2)Beclin and LC3?/LC3?decreased in 3-MA+cisplatin groupcompared with cisplatin.The number of autophagosomes transfectedby mRFP-GFP-LC3 was decreased.The number of mitotracker and lysotracker colocalization was alsodecreased.Mitosox fluorescencewas enhanced.JC-1 red/green fluorescence and ATP content was reduced.Cell apoptosis was increased.Cell viability determined by CCK-8 was decreased(P<0.05).(3)Beclin and LC3?/LC31?increased in Rapa+cisplatin groupcompared with cisplatin.The number of autophagosomes transfectedby mRFP-GFP-LC3 was increased.The number of lysotracker and mitotracker colocalization was also increased.Electron microscopy saw increased mitophagy numbers.MitoSOX fluorescence was reduced.JC-1 red/green fluorescence and ATP contentenhanced.Cell apoptosis was decreased.The cell viability wasincreased(P<0.05).Conclusions:Cisplatin can induceautophagy and mitophagyin renal tubular epithelial cell.Promoting mitophagy can protect mitochondrial function and reduce the renal tubular epithelial cell injuryinduced by cisplatin.While,inhibition ofmitophagy damage mitochondrial function and further aggravate renal tubular epithelial cell injuryinduced bycisplatin.Part ? Pinkl-Parkin regulate mitophagyin HK-2 cell injury induced by cisplatinObjective:To investigate the protective effects of Pinkl-Parkinin HK-2 cells injuryand dysfunction of mitochondria induced by cisplatin·Methods:HK-2 cells were cultured in vitro withdifferent doses of cisplatin(0,5,10,20umol/L).Western blot was used to detectePinkl and Parkin protein expression.HK-2 cells were transfected with PinklsiRNAor Parkin siRNA and transfected with Pinkl or Parkin overexpression plasmid,then cisplatin stimulated HK-2 cells.Western blot was used to detecteBeclin,LC3,Pinkl,Parkin protein expression.Mitochondrial function detected by intracellular ROS,mitochondrial membrane potential(JC-1)and content of ATP.CCK-8 was used to detecte cellviability.Cell apoptosis was detected by flow cytometry.Results:(1)With the increase ofcisplatin concentration,the expression of Pinkl and Parkin increased gradually.(2)HK-2 cells transiently transfected with Pinkl siRNA or Parkin siRNA,then HK-2 cells was stimulated by cisplatin.Compared with cisplatin group,the expression of Beclin,LC3?/LC3?,Pinkl and Parkin were reduced.Expression of JC-1 and the content of ATP were reduced.Apoptosis was increased andcellviability determined by CCK-8 was decreased(P<0.05).(3)Pinklor Parkin overexpression plasmid treated HK-2 cells,then HK-2 cells was stimulated by cisplatin.Compared with cisplatin group,the expression of Beclin,LC3?/LC3?,Pinkl and Parkinwereenhanced.Expression of JC-1 and the content of ATP were enhanced.Apoptosis was reduced andcellviabilitydetermined by CCK-8increased(P<0.05).Conclusion:Pinkl and parkin expressionwere increased in HK-2 cellsstimulated by cisplatin.The protective effect of Pinkl/Parkin regulates mitophagyand mitochondrial function in HK-2 cells injury induced by cisplatin.