Effect Of NOD2 On Endothelial-to-mesenchymal Transition Induced By High Glucose In Glomerular Endothelial Cells

Background Diabetic nephropathy(DN)is one of the most important microvascular diseases of diabetes mellitus.Glomerular hyperfiltration occurs early in the course of the disease,followed by microalbuminuria,macroalbuminuria,and progressive renal dysfunction.It eventually develops into end stage renal disease(ESRD).In recent years,studies about the cell and molecular level have suggested many pathological mechanisms of diabetic nephropathy,including hemodynamic abnormalities,production of advanced glycation end products,inflammatory responses,oxidative stress,protein kinase C activation,and.reduced vitamin D receptors expression,podocyte injury,and endothelial cell damage,et al.Glomerular endothelial cells(GEn Cs)form the inner layer of the glomerular filtration membrane and maintain the integrity of the glomerular filtration membrane.It plays an important role in preventing urinary protein leakage and maintaining normal renal function.Hyperglycemia can cause endothelial cell damage,manifested as increased endothelial permeability,dystrophy and upregulation of adhesion factors,leading to proteinuria and impaired renal function.According to reports,high glucose-induced GEn Cs can undergo phenotypic changes,manifested as deletion of endothelial cell phenotype and appearance of mesenchymal cell phenotype.This transition is known as endothelial-to-mesenchymal transition(End MT).In recent years,some researchs believe that diabetic nephropathy may be caused by the innate immune response activated by hyperglycemia.Under the induction of high glucose,the nucleotide binding oligomerization domain(NOD2)in the pattern recognition receptors(PRRs)family on the surface of innate immune cells is activated,causing innate immune response,leading to the prgress of diabetic nephropathy.At present,the role of NOD2 in endothelial cell injury has not yet been reported.It is important to investigate whether NOD2 is involved in the high glucose-induced GEn Cs End MT to understand the pathogenesis of DN and its clinical treatment.Objective In this study,we cultured conditional immortalized cell lines of mouse GEn Cs,stimulated with high glucose to simulate the hyperglycemic environment in human,observed the expression levels of NOD2 and End MT related indicators in GEn Cs induced by high glucose,and stimulated by MDP.We used sh RNA silencing NOD2 expression to observe the effect of NOD2 on high glucose-induced End MT;meanwhile U0126-inhibitor of MEK1/2 were used to observe changes in End MT-related molecules to verify whether MEK/ERK signaling mediates End MT of GEn Cs.Overall,we investigated whether NOD2 mediates high glucose-induced End MT of GEn Cs through the MEK/ERK signaling pathwayMethods 1.To investigate the expression level of NOD2 and End MT related molecules in GEn Cs stimulated under high glucose 1.1 To observe the changes of NOD2 expression in glomerular endothelial cells stimulated by different concentrations of high glucose.Mouse GEn Cs were cultured in vitro and divided into four groups: normal control group(glucose 5.6 mmol/L),20 mmol/L glucose group,40 mmol/L glucose group,osmotic pressure control group (5.6 mmol/L glucose +34.4mmol/L mannitol.After 24 h of difficult culture-medium,we used Western Blot to detect cellular NOD2 expression levels.Note: This experiment involved high sugars are 40mmol/L.1.2 To observe the changes of NOD2 and End MT-related molecules(endothelial cell marker CD31,mesenchymal cell marker ?-SMA)under high glucose stimulation.The mouse GEn Cs were cultured at high-glucose with 12 h and 24 h,respectively.Western Blot was used to detect the protein expression of NOD2,CD31 and ?-SMA under high glucose,q RT-PCR was used to detect the expression of NOD2 m RNA,and immunofluorescence(IF)was used to observe the localization and distribution of NOD2 protein in cells.2.To explore the role of NOD2 in high glucose-induced End MT of GEn Cs 2.1 To explore the expression levels of CD31 and ?-SMA stimulated by MDP(exogenous ligand of NOD2)to verify whether high glucose has the same effect as NOD2.The mouse GEn Cs were stimulated with MDP(2ug/m L)at different times for 12 h and 24 h,respectively.Western Blot was used to detect the protein expression of CD31 and ?-SMA under MDP stimulation.2.2 To verify transfection efficiency of NOD2-specific sh RNA on GEn Cs.The GEn Cs were transfected with sh RNA and the mouse GEn Cs were cultured in vitro and divided into three groups: normal control group,scramble group,and sh RNA interference group.Western Blot was used to detect the NOD2 expression levels.2.3.To observe transfection of sh RNA to affect the expression of CD31,?-SMA protein of GEn Cs after NOD2 expression.The sh RNA transfection of GEn Cs was divided into four groups: normal control group,high glucose group,high glucose + scramble group,and high glucose + sh RNA interference group.Western Blot was used to detect the protein expression of CD31 and ?-SMA.3.To investigate whether NOD2 mediates End MT of GEn Cs through MEK /ERK signaling pathway.3.1 To observe the expression level of MEK/ERK signaling pathway related molecules under high glucose stimulation.Western Blot was used to detect the changes of MEK1/2 and phosphorylated MEK1/2(p-MEK1/2)induced by high glucose.Correspondingly,GEn Cs were stimulated with MEK1/2 inhibitor U0126 for 1 hour and then high glucose was added for 24 hours.The groups were: normal control group,high glucose group,high glucose + U0126 group.Western Blot was used to detect the expression of ERK1/2 and phosphorylated ERK1/2(p-ERK1/2).3.2 To observe the changes of the expression level of MEK/ERK signaling pathway-related molecules after stimulation with specific sh RNA of NOD2.The groups were: scramble group and sh RNA interference group.The expression of MEK1/2,p-MEK1/2,ERK1/2,and p-ERK1/2 was detected by Western Blot.3.3 To observe the expression levels of CD31 and ?-SMA were changed after adding of MEK1/2 inhibitor U0126.Divided into normal control group,high glucose group,high glucose + inhibitor group.Western Blot was used to detect the expression of CD31 and ?-SMA protein.IF was used to detect the cell localization and protein expression distribution of CD31 and ?-SMA.Result 1.The expression of NOD2 protein and m RNA increased after GEn Cs was stimulated with high glucose,suggesting that NOD2 expression was increased under high glucose.With the high glucose and NOD2-specific ligand MDP stimulation of GEn Cs,the decrease of endothelial cell marker CD31 expression and the increase of mesenchymal cell marker ?-SMA expression were detected,and the difference was statistically significant(P<0.05).It suggests that high glucose may activate NOD2 to induce transition of GEn Cs.2.The expression of NOD2 decreased after sh RNA transfecting to GEn Cs,suggesting that sh RNA can effectively silence the NOD2 expression,and sh RNA can reverse the high glucose-induced downregulation of CD31 and the upregulation of ?-SMA,and the difference is statistically significant(P<0.05),suggesting that NOD2 can induce high glucose-stimulated mesenchymal transition of GEn Cs 3.The expression of phosphorylated MEK1/2(p-MEK1)increased after GEn Cs stimulated with high glucose,suggesting the activation of MEK1/2;sequently ERK1/2 is also activated;inhibiting MEK1/2 with U0126 can reduce phosphorylation of ERK1/2;These results show that the MEK/ERK pathway is activated by high glucose induction.When sh RNA was transfected into GEn Cs,the phosphorylation levels of MEK1/2 and ERK1/2 were significantly reduced in the presence of silencing NOD2 expression,suggesting that NOD2 is involved in the activation of MEK/ERK pathway.After treated with MEK1/2 inhibitor U0126,the expression of CD31 increased and the expression of ?-SMA decreased,and the difference was statistically significant(P<0.05),suggesting that U0126 can reverse NOD2-induced End MT.These suggest that NOD2 promotes high glucose-induced mesenchymal transition of GEn Cs through the MEK/ERK signaling pathway.Conclusion 1.Under the induction of high glucose,the expression of NOD2 in mouse glomerular endothelial cells increased,the expression of CD31 decreased and the expression of ?-SMA increased.2.Silencing NOD2 reversed high glucose-induced downregulation of CD31 and upregulation of ?-SMA.3.NOD2 mediates high glucose-induced endothelial-to-mesenchymal transition of glomerular endothelial cells through the MEK/ERK signaling pathway.