Glucagon-like Peptide-1 Receptor Pathway Inhibits Extracellular Matrix Production By Mesangial Cells Through Store-operated Ca²⁺ Channel

Background: Diabetes is a serious challenge to global public health today.Among them,diabetic kidney disease(DKD)is one of the serious complications that cause adverse outcomes of diabetes.Studies have shown that abnormal accumulation of extracellular matrix in mesangial cells leading to glomerulosclerosis and loss of function is an important part of the progression of DKD.Recent studies demonstrated that glucagon like peptide-1 receptor pathway had renoprotective effects.However,the underlying mechanism(s)remains unclear.Our previous study demonstrated that store-operated calcium channel signaling suppressed extracellular matrix protein production by mesangial cells.The present study was aimed to determine whether glucagon-like peptide-1 receptor agonists can inhibit the production of extracellular matrix proteins in mesangial cells under high glucose conditions,and whether this renoprotective effect is related to the activation of glucagon like peptide-1 receptor and / or store-operated calcium channel(SOC)in order to understand the potential role of glucagon like peptide-1 receptor agonists in the treatment of diabetic kidney disease.Objective:1.To observe whether the glucagon like peptide-1 receptor agonist can inhibit the production of extracellular matrix proteins in mesangial cells under high glucose state.2.To study the effect of liraglutide on high glucose-induced store-operated calcium channel(SOC)in mesangial cells and its relationship with inhibition of extracellular matrix protein production.3.To investigate the effects of liraglutide on high glucose-induced glucagon like peptide-1 receptors in mesangial cells,and its relationship with SOC and extracellular matrix protein production.Methods:1.Human mesangial cells were cultured with different concentrations of the GLP-1 receptor agonist liraglutide under high glucose state.Western blot was used to detect the expression of extracellular matrix proteins,including fibronectin(FN)and collagen IV(Col IV).Then the effect of Exendin-4,another GLP-1R agonist,on the expression of extracellular matrix protein in HMCs was observed.2.Human mesangial cells were cultured with different concentrations of liraglutide under high glucose state.The expression of Orai1,a store-operated calcium channel(SOC)protein,was detected by Western blot,and Store-operated calcium entry(SOCE)was detected by intracellular calcium imaging to assess SOC activity.Then SOC function was inhibited by SOC-specific inhibitor GSK-7975 A,and Western blot was used to detect the changes of liraglutide on hyperglycemia-induced extracellular matrix protein production.3.Human mesangial cells were cultured with different concentrations of liraglutide under high glucose state,and detected the expression of GLP-1 receptor protein by Western blot.Then,the GLP-1 receptor was inhibited by g GLP-1 receptor antagonist Exendin 9-39,and Western blot was used to detect the changes of 100 n M liraglutide on hyperglycemia-induced extracellular matrix protein production and SOC channel protein Orai1 expression in human mesangial cells.4.SPSS 20.0 software was used for statistical analysis.All data are expressed as mean ± standard deviation(Mean ± SEM).Student t-test,is used for comparison between the two groups,and one-way analysis of variance(ANOVA)is used for comparison between multiple groups.P <0.05 is considered statistically significant.Results:1.High glucose stimulated the production of extracellular matrix proteins in human mesangial cells.Liraglutide treatment reduced high glucose-stimulated production of FN and Col IV for 48 hours,and it had a certain concentration dependence.Exendin-4,another glucagon like peptide-1 receptor agonist,also reduced the production of high glucose-stimulated FN and Col IV.2.High-glucose intervention inhibited the expression of Orai1 in human mesangial cells for 8 hours and 24 hours.Liraglutide treatment significantly upregulated the high glucose-induced reduction of Orai1 protein.This phenomenon was more obvious at 24 hours and had a certain concentration dependence.3.High glucose intervention significantly inhibited SOCE in human mesangial cells for 24 hours.The inhibition of high glucose on SOCE was significantly attenuated by 100 n M liraglutide.4.Liraglutide effects on FN and Col IV protein abundance in human mesangial cells induced by high glucose for 48 hours were significantly attenuated by GSK-7975 A,a selective blocker of SOC.5.The expression of glucagon-like peptide-1 receptor protein in human glomerular mesangial cells was significantly reduced under high glucose state.Liraglutide treatment for 48 hours improved the expression of glucagon-like peptide-1 receptor protein with a certain concentration dependence.6.In the presence of Exendin 9-39,a specific glucagon-like peptide-1 receptor antagonist,liraglutide failed to suppress HG-induced Orai1 protein reduction.And the inhibitory effects of liraglutide on extracellular matrix proteins(FN and Col IV)were not observed in the presence of Exendin 9-39.Conclusion:1.Glucagon like peptide-1 receptor agonists inhibit high glucose-induced extracellular matrix proteins production in mesangial cells by restoring SOC functions.2.Glucagon like peptide-1 receptor agonists can improve the SOC function of glomerular mesangial cells and inhibit the production of extracellular matrix proteins by activating the expression of glucagon-like peptide-1 receptors in a high glucose state.