| Background Diabetic kidney disease(DKD)currently has been the leading cause of end-stage kidney disease,while its actual mechanism remains unclear.Several clinical studies confirmed that hyperuricemia is closely related to the onset of DKD.In series of previous studies on DKD anima l models,we also confirmed that hyperuricemia is one of the independent risk factors for tubulointerstitial injury.In in vitro cultivation of human renal tubular epithelia against high glucose and high uric acid,expression of NLRP3 inflammasome is upregulated,and cells tend to be epithelial-mesenchymal trans-differentiation(EMT),an early process for fibrosis.Nevertheless,whether excess activation of the NLRP3 inflammasome underlying EMT and further fibrosis of tubular epithelia is still unclear.For the present study,we hence explored the possible direct link between them by using Mcc950,a specific inhibitor for NLRP3 inflammasome,with the aim to looking for new targets for DKD treatment.Objective To explore effects of Mcc950,a specific inhibitor of NLRP3 inflammasome,on the morphology and epithelial-mesenchymal transition of HK-2 cel s cultured in different concentrations of glucose and uric acid.Methods 1.HK-2 cells were cultured and subcultured to provide samples for the experiment;2.Using the three-factor analysis design method of 2 x 3 x 2,according to different glucose concentration(5.5mmol/L,25mmol/L),uric acid concentration(0mmol/L,0.2mmol/ L,0.4mmol/L)and the presence and absence of Mcc950,the experiment was divided into 12 groups;3.According to the experimental group,in 6-well plate,respectively,the treatment of HK-2 cells for 72 hours;4.The epithelial marker E-cadherin and muscle fibroblast markers FN?Vimentin??-SMA were detected with immunofluorescence and the expression of the above markers were analyzed by in each group.Results 1.As the concentration of glucose and uric acid increase,the HK-2 cells lose their original circular or circular form,in a shuttle-shaped,irregularly long strip or multi-angled.Under the fluoresce nce microscope,the green fluorescence shown in the four markers,E-cadherin,FN,Vimentin,?-SMA.,appears in the cytoplasm,in line with the positioning of the molecules in the cel.1)Simple effect(main effect): There was no significant difference in E-cadherin expression in normal-glucose concentration and high-glucose concentration environment(P>0.05);at both glucose concentrations,E-cadherin expression decreased with increased UA concentration(P <0.05);and Mcc950 increased the expression of E-cadherin in both glucose concentratio ns environment(P <0.05)2)Interaction effects: different glucose and uric acid concentrations have no synergy for E-cadherin expression(P >0.05);Mcc950 improves E-cadherin expression more significantly in a highglucose environment(P <0.05);and Mcc950 intervention does not affect E-cadherin expression at different uric acid concentrations(P>0.05).3.HK-2 cell muscle fibroblast markers FN,Vimentin,?-SMA: 1)Simple effect(main effect): the three muscle fibroblast markers FN,Vimentin,?-SMA expression increased in high-glucose concentration environment(P<0.05);With the increase of uric acid concentration,the expression of the three muscle fibroblast markers increased(P<0.05),by the intervention of Mcc950,the expression of the three muscle fibroblast markers decreased significantly(P <0.05). 2)Interaction effect: different glucose and uric acid concentrations had no synergy in the expression of the three muscle fibroblast markers(P>0.05);Mcc950 inhibited the expression of the three muscle fibroblast markers in a high-glucose,high uric acid environment(P <0.05).Conclusions 1.High concentrations of glucose and uric acid can improve the expression of fibroblast markers in HK-2 cells,High concentrations of uric acid can inhibit the expression of epithelial markers,and intervention of Mcc950 can inhibit these changes.2.High concentrations of glucose and uric acid can promote HK-2 cell EMT,but there is no synergy between them.Mcc950 inhibits the HK-2 cell EMT process mediated by high concentration of glucose and uric acid.3.NLRP3 inflammatory pathway plays an important role in the process of HK-2 cell EMT mediated by high sugar and high uric acid. |
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