Molecular Mechanism Involved In Regulation Of SnoN Expression In Renal Tubular Epithelial Cells Of Diabetic Rats

Along with the progress of diabetic nephropathy(DN),Transforming growth factor ?1(TGF-?1/Smads pathway plays an important role in development of tubulointerstitial fibrosis.Transcriptional co-repressor Ski-related novel protein N(SnoN),as a negative regulator of TGF-?1/Smads pathway,takes rigorously charge of intracellular transduction effects on this pathway.Our previous studies showed the expression of SnoN protein in renal tubular epithelial cells of diabetic rats reduced with the development of DN,accompanied by the deposition of ECM in renal tubular interstitium and phenotype transformation of the renal tubular epithelial cells.In vitro studies showed that though the reducion of SnoN protein induced by high-glucose cultivation,the expression of fibronectin(FN)and a-smooth muscle actin(a-SMA)was upregulated in renal tubular epithelial cells.Furthermore,the expression of FN and a-SMA enhanced and the renal tubular epithelial fibrosis aggravated due to SnoN knockdowned by siRNA.To sum up,downregulation of SnoN protein contributes to pathogenesis of DN.However,the mechanism of action emphasis on the expression of SnoN protein in renal tubular epithelial cells in is not clear,and remains amount of works.Objective This study aims to investigate the changes of SnoN mRNA and protein,and that of TGF-?1 expression in renal tissues of diabetes mellitus(DM)rats before-and-after treated with insulin,In order to further understand the relationship between expression of SnoN and TGF-?1/Smads pathway in the pathogenesis of DN.This study detected the changes of SnoN mRNA and protein expression in renal tabular epithelial cells(NRK-52E cells),in which the expression of Smad2 gene and Smad3 gene were reduced by the siRNA,simultaneously treated with high glucose,so as to clarify the mechanism of TGF-?1/Smads signaling on the regulation of SnoN protein expression.From gene transcription and protein stability,further investigate the regulation mechanism of reduce of SnoN protein in NRK-52E cells under high-glucose condition,for purpose of providing the theoretical and experimental basis for the effective treatment of DN.Methods 1.Establishment of diabetic model and grouping:the diabetic rat model was produced by injection of 55mg/kg streptozotocin via tail vein once.13 weeks later,the diabetic rats were treated with insulin to control blood glucose to normal level.The age of rats from normal and diabetic control groups were comparison.Blood glucose and 24h urine protein were measured.The pathological changes of kidneys and pancreas were detected using HE,and PAS and Masson staining.In addition,immunohistochemistry staining and Western blotting were employed to detect the protein expression of SnoN,TGF-?1,Smurf2,Arkadia,E-cadherin,?-sooth muscle actin(a-SMA),fibronectin(FN),Collagen?(Col-?),Collagen I(Col-?),respectively in kidney cortex.And Real time fluorescent quantitative-PCR(qRT-PCR)was employed to determine the expression of SnoN mRNA expression in kidney cortex.2.siRNA interference technology was used to knock down Smad2 and Smad3 protein expression in renal tubular epithelial cells.Western blot and qRT-PCR technologies were employed to detect the efficiency of knockdown.Then the renal tubular epithelial cells treated with normal glucose[DMEM(glucose 5.5 mmol/L)+ 2%FBS]or high-glucose[19.5mmol/L D-glucose + DMEM + 2%FBS]for 48h.Western blot was used to detect the proteins expression of SnoN,Smad2,Smad3,phosphorylated Smad2[Phospho-Smad2(Ser465/467)(p-Smad2)],phosphorylated Smad3[Phospho-Smad3(Ser423/425)(p-Smad3)],Akradia,E-cadherin,a-SMA and Col-?;qRT-PCR was employed to detect SnoN mRNA.3.NRK-52E cells were pre-treated with different doses of MG132 cultured in high glucose condition.In addition,immunofluorescence staining and western blot were employed to detect the protein expression of SnoN,Smad7,Smurf2,Akradia,E-cadherin,?-SMA,FN,Col-l in the renal tissue and NRK-52E cells.4.The diabetic model as previously described in Partl was employed in this study.NRK-52E cells were incubated with different doses of human recombinant BMP-7 and high dose of glucose.Immunohistochemistry or immunofluorescence and western blot were used to detect the expression of SnoN,BMP-7,TGF-?1 Phospho-Smad3(Ser423/425),Smad3,Phospho-Smad2(Ser465/467),Smad2,Smurf2,Arkadia,E-cadherin,a-SMA and Collagen ?;qRT-PCR was employed to detect SnoN mRNA.5.NRK-52E cells were incubated with different doses of human recombinant HGF and cultured with high dose of glucose.Immunohistochemistry or immunofluorescence and western blot were used to detect the expression of SnoN,extracellular signal-regulated kinase-1 and-2(ERK1/2)pathway,E-cadherin,a-SMA and Collagen?;qRT-PCR was employed to detect SnoN mRNA.Results 1.Biochemical indicators and morphological changes in rat model showed that DM model was successful and stable.Compared with normal control group,the expression of SnoN protein was reduced but mRNA was increased,with increase of TGF-?1,Smurf2 and Arkadia,a-SMA,FN,Col-? Col-I,and decrease of E-cadherin in DM group.Up-regulation of SnoN protein expression and down-regulation of mRNA expression in DM rats after the blood glucose was controlled,while TGF-?1 Smurf2,and Arkadia protein expression was down-regulated,accompanied by improvement in renal fibrosis and decrease of urinary protein,and the expression of E-cadherin was increased but that of a-SMA,FN,Col-?,and Col-? was reduced.2.High glucose enhanced the expression of SnoN mRNA but reduced the expression of the protein in renal tubular epithelial cells.The activated TGF-?1/Smads signal and the expression of Arkadia promoted EMT and the synthesis of ECM.NRK-52E cells with transfection of Smad2 siRNA and Smad3 siRNA under high glucose condition resulted in a decrease of Smad2,Smad3 mRNA and total proteins expression,as well as a reduced the SnoN mRNA expression;Compared with single glucose treatment group,SnoN protein decreased after transfection of Smad2 siRNA,phosphorylation of Smad3 protein and Arkadia expression increased,but promoted EMT and the synthesis of ECM;Instead,transfection of Smad3 siRNA increased SnoN protein and Smad2 protein activation,reduced Arkadia protein,and associated with inhibition of EMT and the synthesis of ECM.3.MG132 inhibited the protein expressions of a-SMA and Col-I in a dose-dependent manner induced by high glucose in NRK-52E cells,but it had no effect on the protein expression of Smurf2 and Arkadia.Contrary,MG132 increased the protein expression of SnoN,Smad7 and E-cadherin.4.In vivo result showed that,with DN progression,BMP-7 in rat renal tissues was down-regulated,TGF-?1/Smads pathway was activated,and expression of Smurf2 and Arkadia was increased.SnoN mRNA expression increased but protein expression decreased,which was accompanied by renal tubular epithelial mesenchymal transition(EMT),extracellular matrix(EMC)accumulation in renal interstitium,and severly impaired renal function.In vitro results showed that,exogenous BMP-7 increased the transcription and protein expression of SnoN and inhibited high dose glucose-induced NRK-52E cells phenotype conversion and ECM production.However,TGF-?1/Smads pathway and proteins expression of Smurf2 and Arkadia was not influenced.5.Exogenous HGF could increase the expression of SnoN at the levels of transcription and translation,and inhibit the NRK-52E cellular phenotype conversion and ECM generation induced by high glucose,possibly through ERK1/2-mediated increased expression of SnoN pathway in renal tubular epithelial cells.Conclusion1.Under the condition of DM,SnoN mRNA expression was increased by high expression of TGF-?1.But simultaneously increased Smurf2 and Arkadia,the E3 ubiquitin ligase,involved in SnoN protein degradation mediated by the activation of TGF-?1/Smads pathways.The effects on the regulation of SnoN expression showed that,protein degradation was stronger than the transcription activation induced by TGF-?1,which contributed a significant reduction in SnoN protein expression,which eventually resulted in transformation of renal tubular epithelial cell phenotype and an increase in extracellular matrix the in kidney tissues of DM rats.This experiment further concluded that high glucose indeed down-regulated the SnoN protein expression in tubular epithelial cells,and proved a dual regulatory mechanism of TGF-?1 on SnoN expression.Glucose control by insulin could restore the protein expression of SnoN in kidney of diabetic rats,which could slow down DN progress.2.Activated Smad2 or Smad3 is involved in the increased expression of SnoN in transcription,but the activation of Smad3 prompts SnoN protein degradation which accelerates the process of DN,and Smad2 inhibits SnoN protein degradation which slows down the development of DN.3.MG132 inhibits the Smad7 protein degradation-mediated by high glucose,which could reduce the development of the renal tubule-interstitium.This may be one of the mechanisms associated with MG132 treatment of DN.Its mechanism may not relate to reducing the E3 ubiquitin ligase Smurf2,Arkadia expression,but by inhibiting the activity of proteasome needed in the degradation of target protein.4.BMP-7 might improve DN and renal fibrosis by enhancing SnoN mRNA and protein expression in renal tubular epithelial cells,rather than directly inhibiting TGF-?1/Smads signaling pathway or by E3 ubiquitin ligase protein-mediated SnoN degradation.5.Effects of HGF on anti-fibrosis may through promoting SnoN mRNA expression by activating ERK1/2 pathways in the process of DN,and through protecting against renal tubular epithelial and stromal cells from EMT and ECM deposition under high glucose condition.