Relationship Between Epithelial Mesenchymal Transition And TGF-β1/p38MAPK Pathway In Renal Tubular Epithelial Cells Induced By High Glucose

Background:Diabetic kidney disease(DKD)is one of the most common microvascular complications,and is also the main cause of end-stage renal failure.Most of the previous studies on DKD have focused on glomerular lesions.It has been found that in DKD tubulointerstitial damage plays a very important role in its pathogenesis.Some literatures have reported that injury in renal tubulointerstitium is even earlier than that in glomerular in diabetic patients and the effect of renal tubular interstitial injury on renal function is closely related to prognosis.In recent years,the study has been found that the transformation of renal tubular epithelial cells into mesenchymal cells is the starting factor and important reason of renal interstitial fibrosis in DKD.The study found that transforming growth factor-β1(TGF-β1)is a very strong fibrotic factor and is most abundant in the kidney.TGF-β1 promotes cell hypertrophy and extracellular matrix accumulation in the mesangium,which decreases glomerular filtration rate and leads to chronic renal failure.Moreover,studies have found that high glucose environments induce TGF-β1 expression by enhancing oxidative stress.p38mitogen-activated protein kinase(p38 MAPK)is a subfamily of the MAPK family,the study found that the two are closely related to the fibrosis of DKD.Many studies have found that TGF-β1 has a significant interaction with p38 MAPK.On the one hand,TGF-β1 exerts its biological effects by enhancing the activity of p38 MAPK.On the other hand,p38 MAPK affects the DKD process by promoting the production of TGF-β1.The literature reports p38 MAPK may be a factor in the downstream of TGF-β1.So it is very important to explore the role of p38 MAPK and TGF-β1 in DKD.Therefore,this study induced EMT in rat renal tubular epithelial cells(NRK-52E)by high glucose,and explored the relationship between TGF-β1/p38 MAPK when the occurrence of EMT,providing theoretical and experimental basis for the prevention and treatment of DKD.Objective:1.Whether renal tubular epithelial cells are transformed into mesenchyme under high glucose induction.2.Relationship between TGF-β1/p38 MAPK pathway in mesenchymal transition of renal tubular epithelial cells.Methods:1.The morphological changes of the cells were observed and recorded by inverted phase contrast microscope in normal control group(5.5mmol / L glucose),mannitol control group(5.5mmol / L glucose + 24.5 mmol / L mannitol),30 mmol / L high glucose group(30mmol / L glucose)and 50mmol/L high glucose group(50mmol/L glucose).After treatment with p38 MAPK inhibitor and TGF-β1 neutralizing antibody in 50mmol/L high glucose medium,the tubular morphological changes of renal tubular epithelial cells were observed by inverted phase contrast microscope in H group(50mmol/L glucose +equivalent sterile PBS phosphate buffer + DMSO),HP group(50mmol/L glucose + p38 MAPK inhibitor + equal amount of sterile PBS phosphate buffer),HT group(50mmol / L glucose + TGF-β1 neutralizing antibody + equal volume of DMSO)and HPT group(50mmol/L glucose + p38 MAPK inhibitor + TGF-β1 neutralizing antibody).2.CCK-8(cell counting kit-8)was used to detect the cell proliferation rate of different concentrations of glucose and TGF-β1 neutralizing antibody.3.Western blot and immunofluorescence were used to detect E-cadherin,α-smooth muscle actin(α-SMA),transforming factor-β1(TGF-β1)and p-p38MAPK/p38 MAPK protein expression in normal control group,mannitol control group,30 mmol / L high glucose group,50mmol/L high glucose group,H group,HP group HT group and HPT group.Results:1.At 0h and 24 h,the cells were paved with stone-like arrangement;at 48 hours,the cells in the high glucose group were disordered,and the typical cobblestone-like arrangement disappeared,becoming a fusiform and irregular shape.The cells in H group,HP group,HT group and HPT group disappeared from the typical cobblestonelike arrangement and became fusiform and irregular.However,compared with H group,HP group and HT group,the HPT group had a disordered cell arrangement and the morphology was relieved.2.At 24 h and 48 h,the proliferation rate of cells in the high glucose group was significantly higher than that in the normal control group.After the TGF-β1neutralizing antibody was administered,high glucose-induced cell proliferation was inhibited,and concentration gradient-dependent effects existed within a certain range.3.Western blot and immunofluorescence results showed that compared with the control group,the E-cadherin protein was decreased in the high glucose group,and the expressions of α-SMA,TGF-β1 and p-p38 MAPK were increased,which had a statistically significant difference.Western blot and immunofluorescence results showed that the expression of E-cadherin protein and the expression of α-SMA protein and p-p38 MAPK protein were decreased in HP group,HT group and HPT group compared with H group.Compared with H group,there was no significant difference in TGF-β1 protein expression,the expression of TGF-β1 protein in HT group and HPT group was decreased,and the above differences were statistically significant.Conclusion:1.Rat renal tubular epithelial cells are transformed into mesenchyme under high glucose stimulation.TGF-β1 and p38 MAPK participate in the EMT process in renal tubular epithelialcells induced by high glucose.2.TGF-β1 neutralizing antibody and p38 MAPK inhibitor can improve high glucoseinduced EMT process in high glucose environment;TGF-β1 neutralizing antibody combined with p38 MAPK inhibitor can further delay the EMT process.3.p38 MAPK may be a downstream factor of TGF-β1.