The Role And Mechanism Of Autophagy-associated Protein FIP-200 In Regulating Renal Tubular Epithelial Cell Apoptosis In Renal Ischemia-Reperfusion Injury

Objective:With increase of its incidence and mortality,acute kidney injury(AKI)can easily make progress to chronic kidney diseases(CKD)and end stage renal diseases(ESRD).It brings great burden to society and economy the worldwide.Renal ischemia reperfusion injury(RIRI)is an important cause of AKI,and it is also one of the main factors affecting the early recovery and long-term survival of the replaceable kidney after renal transplantation.The pathogenesis of renal IRI is complex,and its pathological characteristic mainlyinvolves withinjury and deathof renal tubular epithelial cells.Apoptosis is one of the main mechanisms for renal tubular epithelial cell death induced by hypoxia.Autophagy is involved in regulating apoptosis induced by oxidative stress in IRI.Autophagy also plays a key regulatory role in the development of renal IRI.A comprehensive and intensive study of the interaction mechanism between autophagy and apoptosis in tubular epithelial cells upon renal IRI will make a breakthrough in the recognition and prevention of diseases such as AKI caused by renal IRI.This study will focus on the role of the autophagy-associated protein focal adhesion kinase family interacting protein FIP-200(fak-family interacting protein of 200 kDa)in renal IRI and its relationship between autophagy and apoptosis signaling pathways.Current study of autophagy and apoptosis signaling pathway in IRI provides a new mechanism to reveal the effects of FIP-200 on prevention and treatment of AKI caused by IRI.Andit also provides novel ideas and targets for the prevention and treatment of AKI in the future.Methods:1.Establish a time point model of in vitro human renal tubular epithelial cells(HK2)by Bilupus hypoxia and reoxgenation(H/R)modular system.Transfect GFP-LC3 plasmid into HK2 cells using lipofectamine 3000.And observe the cells with a laser confocal fluorescence microscope to determine optimal hypoxia time by measurement the formation of GFP-LC3 autophagy puncta.2.After respectively overexpression or knockdown of FIP-200 in human renal tubular epithelial cell line HK2 cells,Quantitative real-time polymerase chain reaction(qRT-PCR)and Western blot analysis were used to detect the expression of FIP200,and western blot was used to detect the expression of autophagy Anti-microtubule-associated protein 1 light chain 3(LC3),beclinl and apoptosis-associated proteins caspase3,cleaved caspase3,Bcl-2,etc.Lipofectamine 3000 was used to respectively co-transfect GFP-RFP-LC3 tandem plasmid with FIP-200 overexpression plasmid or FIP-200 siRNA into HK2 cells.Confocal was used to observe the autophagy flows inHK2cells.3.In wild-type mice,the renal artery was clamped for 15 min,30min,45min,1h,2h,4h,and 6h to establish an IRI model.Based on results of the previous in vivo time point model,for the wild-type mice and renal tubular epithelial cells,a 45-minute clamping time point was used to establish the IRI model upon renal specific conditional knockout FIP-200mice(FKO mice).And there were four groups,sham group,IRI group,FKO sham group,FKO IRI group.After sacrificed the mice,measured the level of serum creatinine and urea nitrogen,then observed renal tubular injury by conventional pathological staining such as HE and PAS staining.Detected the expressionand distribution of FIP-200and HMGB1 by immunohistochemistry(IHC)staining.Western blot measured the expression of FIP-200,LC3,Beclin 1,caspase3,cleaved caspase3,Bcl-2 and HMGB1.4.Use both HMGB1 antibody and FIP-200 antibody to perform immunoprecipitation to verify the interaction between FIP-200 and HMGB1.Results:1.In the H/R study,with the increase of hypoxia time,the activation of both autophagy and apoptosis increased.At 45min of hypoxia,both autophagy and apoptosis were significantly enhanced.2.In the RIRI model,with the increase of ischemic time,both autophagy and apoptosis enhanced,and renal tubules appeared damages.At 45 minutes of ischemia,the renal tubules were significantly damaged,and IHC showed a significant increase in FIP-200 expression.3.In the H/R model and IRI model,the expressions of HMGB 1 and FIP-200 were significantly increased.4.Compared with wild-type mice,FKO mice had no significant differences in tubules,creatinine and urea nitrogen.After the establishment of the IRI model,the renal tubular epithelial cells of FKO mice were significantly damaged and the levels of serum creatinine and urea nitrogen were significantly increased after IRI.Electron microscopy showed that the number of autophagosomes in renal tubular epithelial cells was significantly reduced,compared that of wild-type mice.The results suggest that autophagy is inhibited.The TUNEL and Western blot results showed that the apoptosis of renal tubular cells in FKO mice was significantly activated after IRI.5.In HK2 cells,transfected with FIP-200 overexpression plasmid,FIP-200 expression was significantly increased compared with the normal group.Autophagy was enhancedand and HMGB1 expression was significantly increased.However,transfected with FIP-200 siRNA,the FIP-200 expression was significantly reduced compared to the normal group.Following knockdown of FIP-200,autophagy was inhibited and HMGB1 expressionwas reduced.6.Immunoprecipitation showed that FIP-200 interacted with HMGB1 in renal tubular epithelial cells.Conclusions:1.In the H/R model upon HK2 cells,FIP-200 knockdown inhibited autophagy and reduced expression of HMGB1 and then enhanced apoptosis,whereas overexpression of FIP-200 enhanced autophagy and increased expression of HMGB1,then inhibited apoptosis.2.In FKO mice,tubule damage was significantly aggravated after IRI.And HMGB1 expression of FKO mice was significantly reduced.3.FIP-200 interacted with HMGB1 in renal tubular epithelial cells,which might be a potential target for prevention and treatment of AKI caused by IRI.