Thrombospondin 4 Inhibits Lipid Damage And Attenuates Renal Fibrosis By Upregulating The Expression Of Prostaglandin Endoperoxidase 2

Background:Renal fibrosis,the accumulation of scarring within the renal parenchyma,represents a common final pathway in nearly all chronic and progressive kidney diseases.Indeed,corticointerstitial dilatation is the best histologic predictor of renal function decline in chronic kidney disease(CKD),glomerular disease,and type I diabetic nephropathy.Post-injury fibrous matrix deposits may initially contribute to the tissue repair process and,after mild injury,are subsequently resorbed during tissue repair.However,during the course of injury in chronic CKD,fibrous matrix deposition continues uncontrolled,disrupting organ architecture,reducing blood supply,and impairing organ function.Fibrosis impairs the tissue's ability to repair itself,leading to kidney failure.Regardless of etiology,renal fibrosis is a common consequence of many chronic kidney diseases(CKD).Despite many promising experimental data,currently available strategies can only attenuate or delay the progression of CKD,but not reverse fibrosis.Thrombospondin 4(THBS4)belongs to the thrombospondin(thrombospondin,THBS)family of all calcium-binding glycoproteins.It plays an important role in various biological processes such as tissue remodeling and tissue remodeling..In our recent study,it was found that THBS4 plays a protective role in the development of renal fibrosis,but its specific mechanism of action has not yet been ascertained.Objective:The purpose of this study is to study the effect of THBS4 on renal fibrosis and its specific mechanism.Methods:Cell experiment:(1)Cells are plated.After they adhere to the wall,they are starved for 12 hours with serum-free medium,and then transforming growth factor-β 1(transforming growth factor-β 1,TGF-β 1)is added to establish an in vitro model of renal fibrosis.(2)Cells are plated,After it adheres to the wall,change the serum-free medium,put it into an anaerobic tank,and put an anaerobic gas producing bag produced by Mitsubishi Gas Chemical Co.,Ltd.in the anaerobic tank to construct an anoxic environment.After 24 hours,Take out the six-well plate and put it into the cell culture incubator to reoxygenate for 24 hours to construct the fibrosis model in vitro.Animal experiments:(1)6-8 week old male C57/BL mice were used.After the mice were anesthetized,an incision was made at the left rib and waist,and the left ureter was found and ligated with 4-0 suture to construct the hydronephrosis.Renal fibrosis model.Mice were sacrificed on days 0,3,7,and 14,and the kidneys of the mice were collected.HE staining and Masson staining were used to show the degree of renal fibrosis,and α-SMA was used to show myofibroblasts.Simultaneous detection of THBS4 expression in renal fibrosis(2)Using 6-8 week old male C57/BL mice,anesthetized the mice,made an incision on both ribs and waist to expose the kidneys,and used arterial clips to clamp the renal arteries on both sides to establish renal fibrosis caused by ischemia-reperfusion Model.The mice were killed on the 0th day,the 14 th day and the 28 th day respectively,HE staining and Masson staining were used to detect the degree of renal fibrosis,α-SMA was used to detect the activation of myofibroblasts,and THBS4 was used to detect its expression in renal fibrosisResults:(1)THBS4 expression is elevated in an in vitro model of renal fibrosis.Western blot and cellular immunofluorescence detection showed that the expression of THBS4 was increased in TGF-β1-stimulated in vitro renal fibrosis model and hypoxia-induced in vitro renal fibrosis model.(2)THBS4 was increased in the renal fibrosis model.The expression of THBS4 was increased in the in vivo model of renal fibrosis,and the expression of THBS4 was found to be increased in the mouse uuo model and IR model by western blot and tissue immunofluorescence.(3)In the humanhydronephrosis model,the expression of THBS4 was also increased.(4)Using si RNA In the renal fibrosis model constructed after knocking down the expression of THBS4,the related indicators of renal fibrosis(α-SMA and FN)increased.(5)In the construction of the renal fibrosis model using the constructed THBS4 overexpression cell line,the related indicators of renal fibrosis(α-SMA and FN)increased.-SMA and FN)decreased.(6)Through RNAseq,the lipid metabolism pathway and downstream protein prostaglandin endoperoxide synthase 2(prostaglandin G/H synthase 2,PTGS2)were found.(7)In the in vitro model of renal fibrosis,the expression of PTGS2 increased High,but lipid metabolism-related indicators(PPARG and PGC1α)decreased.(8)In mouse uuo model and IR model,the expression of PTGS2 increased,while lipid metabolismrelated indicators(PPARG and PGC1α)decreased.(9)In the construction of THBS4 knockout mice In the mouse uuo model,the fibrosis indicators(α-SMA and FN)of the mice increased,and the lipid metabolism-related indicators(PPARG and PGC1α)decreased.Conclusion:THBS4 inhibits lipid damage and alleviates renal fibrosis by up-regulating the expression of PTGS2.