Inhibition Of SmgGDS Induces Injury Through The Rac1-Nox4-ROS Pathway In Renal Tubular Epithelial Cells

The research backgroundRenal fibrosis is a common pathological feature of progressive chronic kidney disease(CKD)of almost all different etiologies.The EMT process of renal tubular epithelial cells is considered to be a link in the process leading to renal fibrosis.ROS is involved in the mechanism of fibrosis in the progression of CKD and can induce EMT and the production of proinflammatory cytokine IL-1β in renal tubular epithelial cells.Therefore,excessive ROS is increasingly considered as an important factor that causes oxidative stress injury in the kidney and promotes the development of EMT and inflammation in renal tubular epithelial cells.The primary source of renal ROS is NADPH oxidase(Nox)production.Therefore,inhibiting the production of nox-derived ROS in renal tubular epithelial cells may be a breakthrough in the prevention and treatment of renal fibrosis.NADPH oxidase 4(Nox4)was initially identified in the kidney and identified as a subtype of Nox,which is highly expressed in the kidney.Nox is composed of five subunits:catalytic subunit gp9lphox,transmembrane subunit p22phox,cytoplasmic subunit p47phox,p67phox and small GTP-binding protein Rac1.which are assembled into a multi-subunit oxidase complex.Rac could be a catalyst for Noxl,Nox2,Nox4,Nox5.However,there is controversy about the role of Racl in Nox4 activation.Small GTP binding protein dissociation stimulus factor(SmgGDS)is a guanine nucleotide exchange factor that converts small GTP binding protein from GDP binding form to GTP-binding form.SmgGDS inhibit the action of Racl by binding to the Racl nuclear localization signal sequence,promoting nuclear translocation and proteasomal system degradation of Racl.Based on the role of Racl in the activation of Nox family,under the regulation of SmgGDS,Racl,as a link in the production of ROS by NADPH oxidase,may become a new therapeutic target for prevention and treatment of ROS induced renal injuryThe research methods1.Construction of HK-2 cell vector with low SmgGDS expression:after transfection of three kinds of siRNA-SmgGDS,total RNA and total protein of HK-2 cell were extracted.Western blot and qRCR were used to test the protein and mRNA expression levels of SmgGDS.2.Determination of ROS:HK-2 cells were inoculated on a 6-well plate culture plate,transfected with siRNA-SmgGDS,and treated with 50 KM,100 lpM NADPH oxidase inhibitor Apocynin and Racl inhibitor NSC23766 for 30min or 4h.Fluorescence staining was followed by collecting fluorescence photos whin fluorescence inverted microscope.3.Determination of protein and mRNA expression levels of Rac1 and Nox4:after transfection with siRNA-SmgGDS for 36H,50μM of NSC23766 was added to pretreat HK-2 cells for 36H to extract the total protein.The protein expression levels of Racl and Nox4 were determined by Western blot.After transfection with siRNA-SmgGDS for 24H,50 μM of NSC23766 was added to pretreat HK-2 cells for 24H to extract total RNA.The mRNA expression levels of Nox4 were tested by qRCR4.Determination of mRNA levels of downstream a-SMA,E-cadherin and IL-1p:after transfection of siRNA-SmgGDS for 24H,50μM and 100 μM of NSC23766 was added to pretreat HK-2 cells.The mRNA levels of E-cadherin,α-sma and IL-1βwere determined by qRCR.5.Statistical methods:the above experiments were repeated 3 times under the same conditions.SPSS 20.0 was used for all experimental data for statistical analysis.Univariate analysis of variance(ANOVA)was used for the comparison between multiple groups of mean factors.When P<0.05,the difference was considered statistically significant.The results1.Successful construction of HK-2 cell vector with low SmgGDS expression:the mRNA and protein expression levels of SmgGDS in three siRNA-SmgGDS were significantly decreased compared with the control group.It indicated that the construction of HK-2 cell vector with low expression of SmgGDS was successful.2.The production of ROS induced by the low expression of SmgGDS of HK-2 cells was inhibited by Apocynin and NSC23766:HK-2 cells transfected with siRNA-SmgGDS produced much more ROS than the negative control group.Both Apocynin and NSC23766 decreased ROS production.And ROS production decreased in a concentration-dependent manner.These results indicate that Racl and NADPH oxidases play an important role in the ROS production induced by SmgGDS.3.SmgGDS regulates the expression of Nox4 through Racl in HK-2 cells:compared with the control group,the protein expression levels of Racl,the mRNA and protein levels of Nox4 were significantly increased after the transfection of siRNA-SngGDS.However,50μM of NSC23766 reduced the protein levels of Racl and Nox4 and mRNA levels of Nox4 caused by transfection with siRNA-SmgGDS.These data suggest that SmgGDS may regulate the expression of Nox4 by Racl in HK-2 cells.4.SmgGDS-Rac1 pathway induced EMT and expression of inflammatory cytokines in HK-2 cells:after transfection with siRNA-SmgGDS,the expression levels of a-SMA and IL-1β mRNA increased,while the expression of E-cadherin mRNA decreased.In addition,50μM and 1OOμM of NSC23766 was used to inhibit mRNA levels of IL-1β elevated by transfection of siRNA-SmgGDS in a concentration-dependent manner.Using 50μM of NSC23766 reverses the a-SMA increase and E-cadherin decrease induced by interference with SmgGDS.It was proved that SmgGDS-Racl pathway plays an important role in EMT and inflammatory response of HK-2 cells.ConclusionIn HK-2 cells,SmgGDS regulate the expression of Nox4 through Racl,thereby affecting ROS production and downstream EMT markers and inflammatory factors.