GEF-H1/RhoA Signal Pathway On The Pathogenesis Of Type Ⅰ Nephronophthisis

Background and objectivesNephronophthisis(NPHP)is children’s most common monogenic disease leading to end-stage renal disease(ESRD).NPHP1 gene mutation is the most common type Ⅰ nephronophthisis.The characteristic pathological changes are mainly concentrated in the renal tubulointerstitium at the corticomedul-medulla junction,including cystic dilatation and/or cyst formation of renal tubules,inflammation,fibrosis and irregular thickening or thinning of the basement membrane.The pathogenesis of NPHP is still unclear,and there is a lack of adequate clinical treatment.GEF-H1 is a RhoA activator that binds to microtubules.When it dissociates from microtubules,GEF-H1 activates RhoA to bind to GTP.It has been found that RhoA is activated in a mouse model of NPHP,but the role of the GEF-H1/RhoA signaling pathway in the pathogenesis of NPHP remains unclear.This study aims to explore the role of the GEF-H1/RhoA signaling pathway in the pathogenesis of NPHP using the NPHP1 knockout(NPHP1KO)mouse model and NPHP1 knockdown(NPHP1KD)cell model successfully constructed by our group,in order to find new targets for the treatment of NPHP.Methods1.In vivoWestern blot and immunofluorescence detected the expression and distribution of GEF-H1 and the level of GTP-RhoA in kidney tissues of NPHP1KO mice.We then injected adeno-associated virus type 9 carrying a recombinant GEF-H1-cDNA plasmid(AAV-shGEF-H1)and a control plasmid(AAV-eGFP)into the kidneys of 4-5 weeks mice.We set up six groups:Wild-type mice group(WT),wild-type mice virus control group(WT-AAV-eGFP),wild-type mice virus knockdown group(WT-AAV-shGEF-H1),NPHP1 knockout mice group(NPHP1KO),NPHP1 knockout mice viral control group(NPHP1KO-AAV-eGFP),NPHP1 knockout mouse viral knockdown group(NPHP1KO-AAV-shGEF-H1)waiting until 24 weeks of age for sampling.Western blot and immunofluorescence were used to detect the interference efficiency of the virus.Finally,immunofluorescence and pathological staining were used to detect the cyst,inflammation,fibrosis and the involvement of extrarenal organs.In addition,the expression of downstream molecules GTP-RhoA and p-MLC2 was detected by Western blot and RhoA activity reagent,respectively.2.In vitroIn the NPHP1 knockdown(NPHPIKD)HK2 cells constructed by lentivirus transfection,the expression of GEF-H1 and GTP-RhoA was detected by Western blot and RhoA activity assay,respectively.Then,small interfering RNA(siRNA)was used to knock down GEF-H1,and the expression changes of E-cadherin andα-SMA were detected.3.Statistical methodExperiments were repeated at least three times independently,and data are expressed as mean±SEM.Student’s t-test and one-way ANOVA were used to compare the differences.P<0.05 was considered statistically significant.GraphPad Prism9.3.0 software was used for statistical analysis.Results1.In vivoWe observed increased expression and redistribution of GEF-H1 in the kidneys of NPHP1KO mice and increased activity of the downstream GTP-RhoA,indicating that GEF-H1/RhoA signaling is activated in NPHP1KO mice.Western blot and immunofluorescence showed that AAV successfully knocked down the expression of GEF-H1.Subsequently,we also observed that GEF-H1 knockdown alleviated cyst formation,fibrosis,and inflammation.As evidenced by a reduction in the number of cysts and tubular dilatation,HE staining for inflammatory cells,and Masson staining for collagen fibers,as well as inflammatory and fibrotic markers represented by F4/80 and α-smooth muscle actin,accompanied by a decreased expression of the downstream GTP-RhoA and p-MLC2.However,there was no significant difference in blood urea nitrogen,creatinine and indexes of extrarenal organs.2.In vitroGEF-H1 and GTP-RhoA expression was also increased in NPHP1KD HK2 cells,suggesting activation of the GEF-H1/RhoA pathway.Knockdown of GEF-H1 alleviated epithelial-mesenchymal transition(EMT)by increasing epithelial marker E-cadherin and reducing fibrosis marker α-SMA.ConclusionsGEF-H1/RhoA signaling was significantly activated in NPHP1KO mouse kidney tissues and NPHP1KD HK2 cells.Knockdown of GEF-H1 expression can block the RhoA/MLC2 signaling pathway and improve the pathological changes of the kidney in NPHP1KO mice.These results suggest that GEF-H1/RhoA signaling pathway plays an essential role in the pathogenesis of NPHP and provides new clues for the search for therapeutic targets for NPHP.