CTRP13 Is Implicated In High Glucose-induced Filtration Dysfunction Via The SIK1/CRTC2 Pathway In HLSEC

Objective: To investigate the molecular mechanism of serum complement C1q/tumor necrosis factor-related protein 13(CTRP13)involvement in high glucose-induced filtration dysfunction in human hepatic sinusoidal endothelial cells(HLSEC).Methods:HLSEC were cultured in vitro,lentiviral CTRP13 overexpression vector(LV-CTRP13)was constructed and infected with HLSEC,and the cells were divided into normal control(5.5 mmol/L glucose,NC),high glucose(25 mmol/L,HG),NC+LV-CTRP13,HG+LV-CTRP13,NC+ empty control(LV-CON)group,HG+LV-CON group,HG+LVCTRP13+HG-9-91-01 [salt-inducible kinase 1(SIK1)inhibitor] group,HG+LV-CON+HG-9-91-01 group,HG+LV-CTRP13+GW4064 [cyclic adenosine monophosphate-regulated transcriptional co-activator 2(CRTC2)inhibitor] group,and HG+LV-CON+GW4064 group.The cell viability was measured by CCK8,and the mRNA expression levels of CTRP13,SIK1,CRTC2 and P130 Crk-related substrate(P130Cas)in each group of cells were detected by quantitative real-time fluorescence PCR(q RT-PCR),and the expression levels of CTRP13,SIK1,p-SIK1,CRTC2 and p-CRTC2 in each group of cells were detected by Western blot.The protein expression levels of CTRP13,SIK1,p-SIK1,CRTC2,p-CRTC2 and P130 Cas in each group of cells were detected by Western blot.One-way ANOVA was used for comparison between groups.Results :1.In the high glucose state,mRNA and protein expression of CTRP13,SIK1,and P130 Cas were decreased and mRNA and protein expression of CRTC2 were increased in hepatic sinusoidal endothelial cells(P<0.05);compared with the NC group,mRNA of CTRP13,SIK1,and P130 Cas were decreased by 68%,69%,and 79%,and protein was decreased by 57.3%,39.2%,58.7%;12.9-fold elevated mRNA and 1.4-fold elevated protein for CRTC2.2.Fluorescence microscopy showed that there was green fluorescence in the cells of HG+LV-CTRP13 group versus HG+LV-CON group,and the expression of CTRP13,SIK1,and P130 Cas was elevated and the expression of CRTC2 was decreased in HLSECs of HG+LV-CTRP13 group(P < 0.05);compared with HG+LV-CON group,the expression of CTRP13,SIK1,and P130 Cas was elevated in HG+LV-The mRNA of CTRP13,SIK1,and P130 Cas was elevated 2.5-fold,5.5-fold,and 2-fold,and protein was elevated 59.3%,51%,and 61.9% in HLSECs in the CTRP13 group,and the mRNA of CRTC2 was decreased 49.3%and protein was decreased 24.7%.(P<0.05).3.LV-CTRP13 transfection increased the expression of CTRP13,SIK1,and P130 Cas and reversed the high expression of CRTC2 induced by high glucose.Elevated mRNA expression of CTRP13,SIK1,and P130 Cas,elevated protein expression of CTRP13,SIK1,pSIK1,p-CRTC2,and P130 Cas,and decreased mRNA and protein expression of CRTC2 in HLSECs in the HG+LV-CTRP13 group compared with the HG+CON group(P<0.05).The mRNA of CTRP13,SIK1,and P130 Cas was elevated 2.5-fold,5.5-fold,and 2-fold,and the protein of CTRP13,SIK1,p-SIK1,p-CRTC2,and P130 Cas was elevated 59.3%,51%,34.2%,20.4%,and 61.9%;the mRNA of CRTC2 was decreased 49.3%,and the protein was decreased 24.7%.4.HG-9-91-01(SIK1 inhibitor)intervention decreased the mRNA and protein expression of SIK1,P130 Cas,and p-SIK1(T182)and p-CRTC2(S171)in HLSECs of HG+LV-CTRP13+HG-9-91-01 group(P<0.05).Compared with the HG+LV-CTRP13 group,the mRNA of SIK1 and P130 Cas were reduced by 45.8% and 54.2%,and the protein of SIK1,p-SIK1,p-CRTC2,and P130 Cas were reduced by 37.8%,35.5%,19.4%,and 23.5% in the HLSECs of the HG+LV-CTRP13 group.The expression of CTRP13 in HG+LV-CTRP13 and HG+LV-CTRP13+HG-9-91-01 groups was basically the same,and the difference was not statistically significant(P>0.05)5.GW4064(CRTC2 inhibitor)intervention,mRNA and protein expression of CRTC2 was decreased and mRNA and protein expression of P130 Cas was increased in HLSECs of HG+LV-CTRP13+GW4064 group(P<0.05);compared with HG+LV-CTRP13 group,mRNA of CRTC2 was decreased and protein of P130 Cas was increased in HG+LVCTRP13+GW4064 group,the mRNA of CRTC2 was 88.1% lower and protein was 19.4%lower,and the mRNA of P130 Cas was 1.1-fold higher and protein was 51.5% higher in HLSECs;the expression of CTRP13 and SIK1 in HG+LV-CTRP13 group and HG+LVCTRP13+HG-9-91-01 group were basically the same,and the differences were not The differences were not statistically significant(P>0.05).Conclusions :CTRP13,SIK1,CRTC2,and P130 Cas are involved in high glucoseinduced filtration dysfunction in HLSECs,and CTRP13 overexpression inhibits high glucoseinduced filtration dysfunction in HLSECs,and this mechanism is achieved through the SIK1/CRTC2 pathway.