| BackgroundDiabetic nephropathy(DN)is the most common cause of end-stage renal disease.The primary clinical manifestation is the presence of persistent microalbuminuria,followed by persistent proteinuria and overt diabetic nephropathy.Subsequently,glomerular filtration rate(GFR)decreased gradually,and 50% of patients developed into end-stage renal disease within 5 years.Although diabetic nephropathy is considered to be a disease of glomerular disease,more and more evidences show that the degree of renal function are more correlated with the degree of tubulointerstitial lesions.Although the majority of patients with diabetic nephropathy are initially due to the changes in glomerular proteinuria,renal interstitial changes play an important role in the long-term pathological changes of diabetic nephropathy.As more and more researches focus on pathological interstitial changes,which mainly focused on the role of cells in the pathogenesis of fibrosis,such as proximal tubular epithelial cells(PTC)or interstitial cells.Therefore,it is of great significance to study the changes of PTC and the role of PTC in renal interstitial fibrosis in diabetic nephropathy.Forkhead transcription factor O1(FoxO1)is an important regulatory factor,which regulates glucose and lipid metabolism,oxidative stress and redox signaling,cell cycle and apoptosis.Previous study found that upregulation of FoxO1 in the renal cortex of DN rats can alleviate glomerular lesions,including mesangial cells,podocytes and renal tubular injury.Thioredoxin interacting protein(TXNIP),also known as Vitamin D3 up-regulated protein-1(VDUP-1)or Thioredoxin binding protein 2(TBP-2),is thioredoxin(Trx)endogenous inhibitor,which plays an important role in regulating glucose and lipid metabolism,inflammation,diabetes,etc.Trx-Txnip system is important to maintain the redox state of cells.Studies shown that the expression of VDUP-1、collagen type IV alpha 1 chain(COL4A1)mRNA in the mesangial cells under high glucose increased rapidly,resulting in collagen type IV accumulation.Gene chip analysis showed that in the high glucose environment,the expression of thioredoxin interacting protein(TXNIP)was significantly increased in human proximal tubular cell line(HK-2 cells).Therefore,these findings lead us to speculate that FoxO1 may attenuate the high glucose induced ROS in the renal tubule,and alleviate the renal tubulointerstitial fibrosis in diabetic nephropathy by inhibiting the over activation of TXNIP.ObjectiveTo study the roles and mechanisms of FoxO1/Txnip on renal tubulointerstitial fibrosis under diabetic conditions.MethodsIn vitro experiments,FoxO1 knockout(KO)and overexpression(KI)HK-2 cell lines were constructed by CRISPR/CAS 9.The FoxO1 knockout cell were transfected with Txnip siRNA.The research has six groups: normal glucose concentration group(NG),high glucose(25mmol/L)group(HG),high glucose +FoxO1 knockout group(KO),high glucose +FoxO1 overexpression group(KI),high glucose +FoxO1 knockout +Txnip siRNA group(KO+Txnip siRNA)and high glucose+FoxO1 knockout+ negative control group(NC).The mRNA levels and the The protein expressions of FoxO1,pFoxO1,Txnip,FN,fibronectin,collagenⅣ were assessed by Real time PCR and western blot;flow cytometry was used to detect the level of ROS and oxidative stress indicators;the distribution of FoxO1 and Txnip was detected by immunofluorescenceIn vivo,kidney specific overexpression of FoxO1 transgenic mice were constucted by zygotes microinjection construct.Normal mice group(NG),diabetic group(DM)and transgenic group(TG)and transgenic diabetic group(TG-DM).The mRNA levels and the the protein expressions of FoxO1,pFoxO1,Txnip,FN,fibronectin,collagen Ⅳwere assessed by Real time PCR and western blot;FN and Col IV protein expression were detected with immunohistochemical.Kidney pathology in HE,Masson,PAS sections was examined by light microscopy.Ultrastructure of renal cortex was observed with electron microscope.Results1 FoxO1 expression: FoxO1 mRNA and protein expression levels were not significantly different in HK-2 from the NG and HG groups(mRNA)(P>0.05),but was increased in the KI group(P<0.05).Compared with NG group,FoxO1 protein expression in HG groups was not statistically significant(P > 0.05),while the ratio of p-FoxO1/FoxO1 was increased(P < 0.05),showing that high glucose has an inhibitory effect on FoxO1 transcription activity.Compared with HG group,the relative expression level of p-FoxO1/FoxO1 was decreased(P<0.05).2 Effects of FoxO1 on Txnip-Trx and ROS: Txnip mRNA and protein expression levels and ROS levels were elevated(P<0.05)in the HG group compared with the NG group.Compared with the HG group,the levels were lower in KI group(P<0.05)and higher in KO group(P<0.05).Compared with KO group,the level in KO+Txnip siRNA group decreased(P<0.05).3 Effect of FoxO1 on renal interstitial fibrosis: compared with NG group,the mRNA and protein expression of FN and Col IV in HG group increased(P<0.05).Compared with HG group,their mRNA and protein decreased in KI group and increased in KO group(P<0.05).Compared with KO group,and FN and Col IV increased in KO+Txnip siRNA group(P<0.05).4.Expression of FoxO1 in the renal tubule of mice: In the renal tubule of mouse,the mRNA and protein expression of FoxO1 did not differ between the NG and DM groups(P>0.05),but increased in FoxO1-Tg and FoxO1-Tg DM groups(all P<0.05).The p-FOXO1/FOXO1 ratio increased in the DM group,but decreased in the FoxO1-Tg DM group(all P<0.05).5 FoxO1 inhibited the activation of TXNIP signaling pathway in renal tubules of mice: Compared with NG group,the mRNA and protein level of Txnip in DM group was increased(P<0.05),and the level of Txnip in the FoxO1-Tg DM group was lower(P<0.05)than that in DM group.Compared with the NG group,the levels of Trx in the DM group were significantly lower(P<0.05),while the Trx level increased(P<0.05)in the FoxO1-Tg DM group compared with those in the DM group.6 FoxO1 alleviate renal tubule interstitial fibrosis in diabetic nephropathy mouse: Compared with NG group,the FN,collagen mRNA and protein expression DM group was significantly increased(P<0.05),showing that renal interstitial fibrosis occurred in mice with diabetic nephropathy.Compared with the DM group,the expression of FN,collagen mRNA and protein in the FoxO1-Tg DM group was decreased(P<0.05).7 FoxO1 can effectively alleviate renal injury in diabetic mice: Compared with NG group,the blood glucose levels of DM group and FoxO1-Tg DM group significantly increased at the end of the 12 th week.The levels of serum creatinine,urea nitrogen and 24 h urinary protein in DM group were significantly higher than those in NG group(P<0.05),and the levels of those in group FoxO1-Tg DM were significantly lower than those in group DM(P<0.05).Staining showed a larger amount of the Collagen and thickened glomerular basement membrane in the kidneys of diabetic mice.Conversely,renal pathology was ameliorated in the FoxO1-Tg DM group of mice.Conclusions1.HG decrease FoxO1 activity,which oxidative injury and renal tubular interstitial fibrosis.2.Overexpression of FoxO1 can improve proteinuria and alleviate tubulointerstitial fibrosis,which may be associated with the excessive activation of TXNIP/Trx/ROS pathway. |
PDF Full Text Request