BMP-7 Inhibits The Progression Of Diabetic Nephropathy By Up-regulating SnoN Expression Through Smad1/5

Existing studies and preliminary experiments of our research have confirmed that the decrease of SnoN?Ski-related novel protein N?protein level in the process of renal tubulointerstitial fibrosis is affected by gene transcription and ubiquitination degradation.We can imagine from two aspects of gene transcription and protein stability,increase or recovery of diabetic nephropathy disease in the process of renal tubular epithelial cells endogenous SnoN protein expression level,in order to delay or treatment of DN.Based on previous studies,it is found that the effect of Bone morphogenetic protein 7?BMP-7?in the treatment or delay of fibrosis disease is closely related to the mechanism of SnoN and the factors and pathways regulating SnoN expression.Moreover,we discovered that in the process of DN fibrosis,BMP-7was able to antagonize the fibrogenic effect of Transforming growth factor beta1?TGF?-1?by up-regulating the expression level of SnoN protein.However,this relationship,as well as the acting links and molecular mechanism,need to be confirmed by further studies.Objective:Discussed from gene transcription and protein stability under the condition of high sugar BMP 7,SnoN expression regulation of renal tubular epithelial cells and molecular mechanism,the role of the link clear BMP-7 on SnoN expression regulation of intracellular signaling pathways,confirm whether BMP 7 by reducing SnoN ubiquitin protein levels and renal tissues SnoN protein degradation of strength and restore SnoN protein levels.To further elucidate the pathogenesis of renal tubulointerstitial fibrosis in DN and to search for new effective therapeutic targets.Methods:?1?Cell experiment:the phenotype of RTECs cells was identified by Western blot,and then treated as follows:?1?through the normal sugar[DMEM?sugary tendency for 5.5 mmol/L?+2%FBS]and high sugar[DMEM?sugary tendency for 5.5 mmol/L+19.5mmol/L?+2%FBS]culture NRK-52 E cell expression of BMP-7 plasmid transfection,Western blot test each cell BMP-7,epithelial markers E-Cadherin,protein markers waveform?Vimentin?and extracellular matrix protein collagen protein III.?2?rhBMP-7 was stimulated at the concentrations of 12.5ng/ml,25ng/ml,50ng/ml,100ng/ml and 200ng/ml of RTECs cells cultured with high glucose,and the expression of E-Cadherin and Collagen III proteins in each group was detected by Western blot.The expression of E-Cadherin and Collagen III proteins in each group at 6h,12h,24h and 48h under the stimulation of rhBMP-7 at the optimal concentration was detected by Western blot.Changes in SnoN,Smad1,Smad5 and p-Smad1/5^?ser463/465?protein levels were detected by Wetern blot,SnoN mRNA levels were detected by real-time PCR,and changes in E-Cadherin,?-SMA and SnoN protein expressions were detected by immunofluorescence.?3?rhBMP-7 stimulation was given to RTECs cells cultured with high glucose,and rhBMP-7 was co-transfected to knock down or overexpress Smad1/5 plasmid.The Smad1,Smad5 and SnoN mRNA levels were detected by Real-time PCR,and the expression levels of SnoN,Smad1,Smad5,p-Smad1/5^{?ser463/465?,}E-Cadherin,Vimentin and Collagen III were detected by Western blot.?2?Animal experiments:Copy of 18 C57BL mice model of type 1 diabetes?DM group?,matching the same number is the same age of rat normal mice as Control group?NC?,4 weeks later,then two groups of mice were equal randomly again in NC+BMP-7groups,NC+Vector group,NC+Control group,the DM+BMP-7 groups,DM+Vector group and DM+Control group,including BMP-7 in the NC and DM mice group and the Vector group,respectively,in mice through the tail vein injection quality for 15 ug BMP-7 expression plasmid or corresponding empty plasmid,The injection was given once a week and executed six weeks later.The expression levels of BMP-7,?-SMA and Collagen III protein in each group were detected by immunohistochemistry and immunofluorescence.The levels of SnoN mRNA in each group were detected by Real-time PCR.The expression levels of SnoN,Smad1,Smad5,p-Smad1/5^?ser463/465?,E-Cadherin,Vimentin and Collagen III in renal tissues of each group were detected by Western blot.The levels of SnoN protein ubiquitination were detected by immunoprecipitation.Results:?1?In vitro experiments:Western blot assay showed that the epithelial cell marker E-Cadherin was highly expressed in NG group and the mesenchymal cell marker Vimentin was poorly expressed,indicating that the cells were of epithelial origin,while the HG group showed low expression of E-Cadherin and high expression ofVimentin.?1?After BMP-7 overexpression,BMP-7 and E-Cadherin expression were significantly increased,while Vimentin and Collagen III expression were decreased.?2?In the case of high glucose,with the increase of rhBMP-7 stimulation concentration,the expression level of E-Cadherin protein gradually increased from 50ng/ml,while the Collagen III protein level gradually decreased from 25ng/ml.Therefore,rhBMP-7 stimulation cells of 100ng/ml were used in our subsequent experiments.Compared with RTECs cells cultured in high glucose culture,with the extension of rhBMP-7 stimulation time,the expression of E-Cadherin protein in the cells increased significantly at 48h,while the level of Collagen III protein decreased significantly,the expression of SnoN mRNA and protein increased,and the activation of p-Smad1/5^?ser463/465?increased.?3?For RTECs cells stimulated by rhBMP-7 at high glucose,Real-time PCR detected that Smad1 and Smad5 mRNA were significantly reduced,and SnoN mRNA was correspondingly reduced.Western blot showed that Smad1,Smad5,p-Smad1/5^?ser463/465?and SnoN protein levels were significantly reduced,and fibrosis lesions were further increased.On the contrary,after the RTECs cells stimulated by rhBMP-7 were co-transfected with plasmids expressing Smad1/5 gene under the condition of high glucose,Real-time PCR detected that Smad1 and Smad5 mRNA were significantly increased,and SnoN mRNA was also increased correspondingly.Western blot showed that Smad1,Smad5,p-Smad1/5^?ser463/465?and SnoN protein levels were significantly increased,and fibrosis was improved.?2?In vivo:Through a microscope groups of mice kidney morphology and immunohistochemical fluorescence staining found that compared with NC+Control group,the DM+Control groups of mice kidney glomerular sclerosis,inflammatory cells infiltration and fibrosis pathological changes,along with the extracellular matrix Collagen III deposits increased,?-SMA expression increased,and diabetic mice given BMP 7 after treatment dramatically improve the pathological changes,and Collagen III deposition reduction,?-SMA expression is also reduced.Real-time PCR and Western blot analysis showed that,compared with the NC+Control group,BMP-7 protein expression were decreased in the DM+Control group,while SnoN mRNA expression was increased and protein expression was decreased.However,compared with the DM+BMP-7 group and the DM+Control group,the mRNA and protein expressions of BMP-7 were increased,while the SnoN mRNA and protein expressions were further increased.Western blot analysis showed that the expression of p-Smad1/5^?ser463/465?and E-Cadherin in renal tissues of DM+BMP-7 group was higher than that of DM+Control group,while the expressionofCollagenIIIandVimentinweredown-regulated.Through immunoprecipitation,it was found that compared with the NC+Control group,the level of SnoN protein ubiquitination in renal tissues of the DM+Control group increased,while that of the DM+BMP-7 group decreased.Conclusion:1.In vitro,BMP-7 can activate the classical Smad1/5 signaling pathway to up-regulate SnoN mRNA andproteinlevels,preventrenaltubular epithelialcellsfrom transdifferentiating into mesenchymal cells and inhibit extracellular matrix deposition.2.At the overall level,BMP-7 can activate Smad1/5 protein,up-regulate SnoN mRNA and protein levels,and inhibit the ubiquitination and degradation of SnoN protein,so as to prevent diabetic renal fibrosis.