The Study Of The Role Of Autophagy In Proteinuria-Induced Renal Interstitial Fibrosis

Objective:Proteinuria is not only a common feature of chronic kidney diseases(CKD),but also an independent risk factor promoting CKD progression to end-stage renal failure(ESRD).It is the result of glomerular damage and the primary cause of secondary renal tubular injury.It plays a pivotal role in the progression of chronic kidney disease,in glomerular disease to end stage renal disease(ESRD).It plays a pivotal role in the progression of glomerular disease to end stage renaldisease(ESRD).Therefore,it is of great significance to protect the renal tubule from being damaged by large amount of urinary protein and progress to the stage of fibrosis,which is one of the key treatment strategies to prevent or delay the progression of CKD to ESRD.Autophagy,which is a highly conserved metabolic process unique to cells in eukaryotes,is inseparable from the homeostasis of the intracellular environment.It is essential for a variety of physiological and pathophysiological processes.In mammalian systems baseline autophagy occurs under normal conditions,but it can be further stimulated by starvation or by various pathologic conditions,including ischemic,toxic,immunological and oxidative insults.And autophagy can protect cells from eliminating the misfolded proteins.In recent years,many studies have shown that autophagy is involved in the progression of kidney disease.Albumin,the major protein in proteinuria,can induce autophagy in renal tubular epithelial cells,but its underlying role remains uncertain.In conclusion,our study is focused on the role of autophagy in proteinuria-induced tubulointerstitial fibrosis,providing new therapeutic strategies and targets for reversing the progression of CKD to ESRD.Methods:Part I : Retrospective review of 30 cases of IMN,diagnosed by renal biopsy at Shengjing Hospital affiliated to China Medical University from June 2015 to December 2016,as the experimental group.And all patients must conform to the inclusion criteria and exclusion criteria.The number of autophagosomes in renal tubular epithelial cells was observed by transmission electron microscopy.The expression of autophagy-related proteins Beclin-1 and LC3Ⅱ in renal tubular epithelial cells was detected by immunohistochemistry.The correlation of the expression of autophagy and 24 hour urinary protein quantification,renal interstitial fibrosis degree,the clinical indications of renal function were analysed.PartⅡ:(1)HK-2 cells,selected as experimental subjects,treated with different concentrations of BSA(0,1,5,10 mg/ml)for 72 hours,and EMT related indicators,including the protein of e-cadherin and α-SMA,were detected by Western blot.(2)HK-2 cells were treated with 10 mg/ml BSA for 0,24,48 and 72 h respectively.And the expression of the e-cadherin protein and α-SMA protein was detected by Western blot.(3)HK-2 cells were treated with different concentrations of BSA(0,1,5,10mg/ml)for 72 h,and the expression of autophagy-related proteins LC3 II and Beclin1 were detected by Western blot.(4)Autophagy inhibitors(3-MA)whose final concentration is 5 m M,were administered 1 h before treatment of HK-2 cells with10 mg/ml BSA,and then cotreated with BSA for 72 h,divided into 4 groups: control(C group),control+3-MA(C+3-MA group),10mg/ml BSA(BSA group),10mg/ml BSA+3-MA(BSA+3-MA group),the e-cadherin protein 、 α-SMA protein,the autophagy-related proteins LC3 II and Beclin 1 were detected by Western blot.(5)Autophagy agonist(Rapa)whose final concentration is 5 μM,were administered 1 h before treatment of HK-2 cells with 10 mg/ml BSA,and then cotreated with BSA for72 h,divided into 4 groups: control(C group),control+Rapa(C+Rapa group),10mg/ml BSA(BSA group),10 mg/ml BSA+Rapa(BSA+Rapa group),the e-cadherin protein 、α-SMA protein,the autophagy-related proteins LC3 II and Beclin1 were detected by Western blot.Results:Part Ⅰ:Compared with normal renal tissues,the number of autophagosomes and the expression of autophagy-related proteins Beclin-1 and LC3 were significantly increased in IMN group;In the IMN group,there was a significant increase in the number of autophagosomes and the expression of Beclin1 and LC3Ⅱ protein in the fibrotic group,compared with the nonfibrotic group.(2)In the IMN group,the number of autophagosomes and the expression of Beclin1 and LC3Ⅱ protein were positively correlated with the degree of renal interstitial fibrosis;LC3 Ⅱ was negatively correlated with 24-hour urine protein quantitation,Beclin 1 and serum urea nitrogen(BUN)was positively correlated,and there was a correlation between the number of autophagosomes and the sex of the patients.Part Ⅱ:(1)Treatment of HK-2 cells with different concentrations of BSA for 72 h,revealed that BSA could induce EMT in a concentration-dependent manner,that is of a decreasing trend in the epithelial marker e-cadherin protein and increasing trend in the mesenchymal marker α-SMA protein with the increase of BSA concentration.(2)Treatment of HK-2 cells with the same concentration(10mg/ml)BSA for 0,24,48,and 72 h respectively,which were found that BSA can induce EMT in a time-dependent manner,that is of a decreasing trend in the epithelial marker e-cadherin protein and an increasing trend in the mesenchymal marker α-SMA protein with the gradual increase of BSA induction time at same concentration of BSA.(3)After pretreatment of 3-MA,it was found that the expression of autophagy-related proteins LC3 II and Beclin1 were decreased,meanwhile the progress of EMT was further aggravated,including the downregulation of the epithelial marker e-cadherin protein and the upregulation of the expression of the mesenchymal marker α-SMA protein in BSA+3-MA group,compared with BSA group.(4)After pretreatment of Rapa,it was found that the expression of autophagy-related proteins LC3 II and Beclin 1 were increased,meanwhile the BSA induced-EMT was reversed to restore the epithelial marker e-cadherin protein and reduce the expression of the mesenchymal marker α-SMA protein.Conclusions:1.Autophagy in renal tubular epithelial cells is involved in the progression of renal interstitial fibrosis in IMN.2.Albumin can induce EMT in renal tubular epithelial cells in a concentration-and time-dependent manner.3.Autophagy can be induced by albumin in renal tubular epithelial cells in a concentration-dependent manner,which is gradually increased with the aggravation of EMT.4.Autophagy may reverse albumin-induced EMT of HK-2.