The Effect And Mechanism Of SGK1 On Pyroptosis-related Renal Tissue Of Mice With Bladder Outlet Obstruction

Objective: Posterior urethral valve(PUV)is the main cause of congenital obstructive nephropathy,which can lead to different degrees of bladder outlet obstruction.The renal inflammatory reaction is the basis for mediating the development of obstructive nephropathy and plays an important role in chronic kidney disease.Therefore,inflammatory injury of the kidney is the main cause of end-stage renal disease.Inflammatory mediators play an important role in renal injury,and their abnormal secretion is the key to the continuous progression of the disease.At the same time,it has been reported that renal inflammatory injury is mainly the inflammatory reaction of renal tubules.Previous studies have shown that pyroptosis plays an important role in the occurrence and development of inflammatory diseases.However,the mechanism of renal inflammatory injury induced by bladder outlet obstruction has not been studied.Serum and Glucocorticoid-regulated Kinase 1(SGK1)is a member of the serine/threonine kinase gene family,and it has been confirmed that overexpression of SGK1 is observed in obstructive nephropathy.EMD638683 is a highly selective SGK1 inhibitor,and it has been proved to have anti-inflammatory effects.However,the effect and mechanism of SGK1 inhibitor EMD638683 on renal inflammatory injury have not been reported.Methods: This article intends to use a new bladder outlet obstruction modeling method,namely urethral ostium suture method,to build a mouse Bladder Outlet Obstruction animal model,thereby causing lower urinary tract obstruction-mediated renal inflammatory injury.HE staining was used to observe the infiltration of inflammatory cells;PAS staining was used to observe the renal tissue lesions;Masson staining was used to observe the collagen deposition of renal tubules;TUNEL staining was used to detect the DNA damage of renal tissue cells;Immunohistochemistry and Western blot were used to detect the protein expression of F4/80,NLRP3,Caspase-1 and Gasdermin D-N terminal(GSDMD-N),IL-1β and SGK1,so as to explore the mechanism of renal inflammatory injury induced by bladder outlet obstruction.In cell-level studies,mouse Renal Tubular Epithelial Cells(mRTEC)were stimulated with different concentrations of aldosterone(ALD),and Western blot was used to observe the expression of SGK1,NLRP3,and Caspase-1 proteins;Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of inflammatory factor IL-1β in the supernatant,and to explore the inflammatory damage effect of aldosterone at different concentrations on mouse renal tubular epithelial cells.The renal tubular epithelial cells were intervened by EMD638683,the protein expression of SGK1,NLRP3,Caspase-1 and GSDMD-N was detected by Western blot,and the protein expression of IL-1β in the supernatant was determined by ELISA,so as to clarify the effect and mechanism of SGK1 inhibitor EMD638683 on renal inflammatory injury.Results: The mouse model of renal inflammatory injury induced by bladder outlet obstruction was established.HE staining showed that the infiltration of mouse renal interstitial inflammatory cells gradually increased with the increase of mouse obstruction time(1,2,and 3 weeks);Compared with the normal group,the inflammatory cell infiltration in the renal interstitial of the mice with obstruction for 3 weeks(BOO(3w))was obviously increased,and the renal tubular epithelial cells were degenerated.PAS staining showed that compared with the normal group,the damaged renal tubules were observed in the BOO(3w)group.Masson staining was used to evaluate the renal fibrosis induced by bladder outlet obstruction.In the obstruction for 1 week(BOO(1w))and 2 week(BOO(2w))group,the renal interstitial tissue of the mice could occasionally have collagen deposition;Compared with the normal group,the BOO(3w)group had obviously increased renal interstitial collagen.TUNEL staining showed that compared with the normal group of mice,the BOO(3w)group could observe a obvious increase in TUNEL positive cells.Immunohistochemical method was used to detect the expression of related proteins in kidney slices.In the BOO(3w)group,F4/80,SGK1,NLRP3,IL-1β and Caspase-1 were mainly expressed in the cytoplasm of renal tubular cells,and the expression of proteins was significantly higher than the normal group.The expression of related proteins in renal cortex was detected by Western blot.The expression of SGK1,NLRP3,Caspase-1,GSDMD-N and IL-1β protein in renal tissue of BOO(3w)group were significantly higher than the normal group.In the study,it was found that aldosterone with concentrations of 1 μmol/L,5 μmol/L and 10 μmol/L can significantly increase the expression of IL-1β in renal tubular epithelial cells,and found that its upstream molecules NLRP3,Caspase-1 protein were also significantly increased,as did the expression of SGK1 protein.EMD638683 interfered with aldosterone-stimulated renal tubular epithelial cells.Compared with the ALD group,the expression of SGK1 and cellular inflammatory factor IL-1β in the ALD+EMD638683 treatment group was significantly reduced,and the protein expression of NLRP3,Caspase-1,GSDMD-N were also significantly reduced.There was no significant difference between the ALD+EMD638683 treatment group and the normal group.It was confirmed that the SGK1 inhibitor EMD638683 can significantly reduce the expression of IL-1β,NLRP3 and Caspase-1,and the reduction of Caspase-1 also significantly reduced its downstream level molecule GSDMD-N Protein expression.Conclusion: The mouse BOO animal model was successfully established to induce renal inflammatory injury mediated by lower urinary tract obstruction,and pyroptosis plays an important role in renal inflammatory injury mediated by bladder outlet obstruction.The SGK1 inhibitor EMD638683 may play a role in its anti-inflammatory effect by inhibiting the NLRP3-Caspase-1-IL-1β/Pyroptosis pathway to block pyroptosis and the release of inflammatory factor IL-1β.