The Effects Of Integrin β6 And Triptolide On Diabetic Kidney Disease

Part Ⅰ The Effects of Integrin β6 on Epithelial-MesenchymalTransdifferentiation in Diabetic Kidney Disease ObjectiveAs one of the common complications of diabetes mellitus(DM),diabetes kidney disease(DKD)has been affecting life quality of more and more patients.Nowadays there is not sufficiently effective methods for DKD.The main pathological changes in DKD are fibrosis and epithelial-mesenchymal transition(EMT).Integrin(ITG)is a family of trans-membrane proteins that mediate communication between cells and extra-cellular matrix(ECM).Its main function is regulating cell adhesion and migration.Integrin β6 is exclusively located in epithelial cells and only binds with integrin αv,reacting with fibronectin(FN)in ECM.This experiment aimed at the role of integrin β6 in the occurrence and development of DKD,and explored possible mechanisms for potential therapeutic targets.MethodsThe 16-week-old db/db and littermate db/m mice were employed,with blood glucose and urinary protein detected.Degree of fibrosis was evaluated by Masson and immunohistochemical(IHC)/FN staining.Renal tissue samples were subjected to RNA sequencing(RNA-Seq)and bio-informatics analysis to seek differently expressed genes.Western blot(WB)and IHC experiments were conducted to assess degrees of integrin β6 and EMT.Phosphorylation levels of focal adhesion kinase(Fak),Src and Yes-associated protein(YAP)were detected by WB,as well as Notch1.The human renal tubular epithelial cells(HK-2)were intervened by poly-l-lysine(PLL)and FN for 48 h.Expressions of integrin β6 and E-cadherin(E-Ca)were assessed by WB and immunofluorescence(IF).The levels of phosphorylated focal adhesion kinase(Fak),Src(sarcoma gene)and Yes-associated protein(YAP)were detected by WB,as well as that of Notch1.Urine was collected from negative controls(NC),DM patients(without DKD)and DKD patients,followed by urine protein profile detection.Enzyme linked immunosorbent assay(ELISA)was operated for urinary integrin β6 evaluation.Results1.Db/db mice showed obviously higher blood glucose and urinary protein levels.Collagen and FN were accumulated in db/db mice,indicating elevated fibrosis.2.Cell-adhesion genes were significantly changed in db/db mice.Integrin β6were obviously increased in db/db mice,closely related to other cell-adhesion genes.3.Integrin β6 expression was significantly increased in db/db kidneys.EMT was obviously elevated in db/db mice.Phosphorylations of Fak,Src and YAP were increased,as well as Notch1.4.Expressions of integrin β6 were significantly increased in FN intervened cells.Levels of E-Ca was decreased,indicating elevated EMT.Phosphorylations of Fak,Src and YAP were increased in FN group,as well as Notch1.5.Phosphorylation levels of Fak,Src and YAP were significantly inhibited,but levels of Notch1 and EMT were reduced in ITGβ6-si RNA transfected HK-2 cells.EMT was obviously alleviated in Notch1-si RNA transfected cells.6.Urinary marker proteins of renal tubule function and glomerular function were significantly increased in DKD patients.ELISA results showed that the expressions of urinary integrin β6 were significantly increased in DKD patients.ConclusionIntegrin β6 was significantly increased in db/db mice,renal tubular epithelial cells treated by FN,and urine of DKD patients respectively.FN is accumulated in DKD,which acts on integrin β6 on cell membrane,activates Fak/Src pathway,promotes phosphorylation of YAP,increases expression of Notch1,and then promotes EMT in epithelial cells.Urinary integrin β6 may be used as a potential bio-marker for DKD.And inhibition of integrin β6 in renal tubular epithelial cells may be a new target for clinical prevention and treatment for DKD.Part ⅡThe Effects of Triptolide on Autophagy and Fibrosis in Diabetic Kidney DiseaseObjectiveAs one of the common complications of DM,DKD is a serious threat to human health.Autophagy is a physiological process by which the body degrades organelle components and proteins to maintain normal metabolism.Autophagy is significantly inhibited and fibrosis elevated in DKD models.Tripterygium wilfordii Hook F(TWHF)is a medicine extracted from traditional Chinese herbal medicine Tripterygium wilfordii.Adverse reactions of TWHF can not be ignored,such as liver injury.Triptolide(TP)is one of TWHF’s main active ingredients.It is not clear whether TP maintains therapeutic effect of TWHF while avoiding adverse reactions.This experiment aimed at the effect and mechanism of TP on DKD to provide new ideas for clinical treatment and prevention for DKD.MethodsMale SD rats were randomly divided into 3 groups,standard diet feeding group(NC),high-fat diet(HFD)combined with single streptozocin(STZ)intraperitoneal injection group(DKD),and HFD/STZ models treated with 200 g/kg/d TP gavage(DKD + TP).Blood glucose and urine micro-albumin(UMA)levels were detected.IHC and WB of collagen Ⅳ(Col Ⅳ),FN,P62 and LC3 were operated to evaluate the degree of fibrosis and autophagy.Human renal mesangial cells(HRMCs)were intervened by 5.5mmol/L glucose(NG),5.5mmol/L glucose with 19.5mmol/L mannitol(MA),25mmol/L glucose(HG),25mmol/L glucose with 10μmol/L TP(HG + TP)for 48 h.A specific inhibitor of autophagy,3-Methyladenine(3-MA),and autophagy-related gene(Atg)5-si RNA were transfected to evaluate relationship between autophagy and fibrosis.Mi RNA chip analysis was performed to seek for differently expressed mi RNA.Target genes were predicted by Target Scan database and confirmed by luciferase reporter gene experiment.Changes of autophagy and fibrosis were detected after transfection of phosphatase and tensin homologue(PTEN)si RNA.Expressions of PTEN and protein kinase B(Akt)/mammalian target of rapamycin(m TOR)were detected after transfection of mi R-141-3p mimics and inhibitor respectively.Results1.DKD rats showed significantly increased blood glucose and UMA.But UMA levels were significantly decreased in DKD+TP group.There were no statistical differences in liver function and blood routine.The levels of fibrosis were elevated but those of autophagy were repressed in DKD rats,but restored in DKD+TP group.2.Expressions of Col Ⅳ,FN and P62 were significantly increased,and LC3 Ⅱ/Ⅰratio was decreased,indicating inhibited autophagy and elevated fibrosis in HG group.After TP intervention,autophagy was increased and fibrosis was alleviated obviously.3.After blocking the initial of autophagy by 3-MA or Atg5-si RNA,the recovery effect of TP on fibrosis was significantly impaired.4.We performed mi RNA chip analysis and found that mi R-141-3p was significantly increased in HG group,while reduced in HG+TP group.Mi R-141-3p directly acts on the 3’UTR region of PTEN.After transfection of PTEN-si RNA,the recovery effect of TP on autophagy and fibrosis was obviously inhibited.5.Transfection of mi R-141-3p inhibitor increased the expression of PTEN,inhibited downstream Akt/m TOR pathway,increased degree of autophagy and alleviated fibrosis.And we got opposite results after transfecting mi R-141-3p mimics.ConclusionHFD/STZ modeled DKD rats showed increased blood glucose and fibrosis levels,along with inhibited autophagy.TP treatment reduced urinary protein levels in DKD rats,effectively restored autophagy and relieved fibrosis.No side effects of TP on liver function and blood routine were found.High glucose led to a rise of mi R-141-3p,which acting on the 3’UTR region of PTEN.Mi R-141-3p inhibited the expressions of PTEN,activated downstream Akt/m TOR pathway,and then inhibited cell autophagy and promoted fibrosis.TP inhibited mi R-141-3p expression through PTEN/Akt/m TOR pathway to restore autophagy and alleviate fibrosis in DKD.