Mechanism Study Of Macrophage Act1a Ffecting Renal Fibrosis By Reducing The Mesenchymal Transition Of Renal Tubular Epithelial Cells

Background and objective:Renal interstitial fibrosis(RIF)is a common pathological change of all kinds of chronic kidney disease while progressing to the end stage.Particularly,the main features of renal interstitial fibroblasts and collagen aggregation were increased.However,there is no good clinical treatment for renal fibrosis and the specific mechanism of its occurrence and development remain to be clarified.Studies have shown that the infiltration of macrophages and the cytokines secreted by macrophages play an important role in the process of renal fibrosis.In the process of renal fibrosis,renal tubular epithelial cells can also be transformed into myofibroblasts through the mechanism of transdifferentiation and participate in the process of renal fibrosis.Act1 is an adaptor protein activated by the NF-κB signaling pathway that is involved in the regulation of macrophage function.Our previous study preliminarily confirmed that down-regulation of macrophage Act1 alleviates renal fibrosis.However,it has not been reported whether macrophage Act1 improves renal fibrosis by regulating the transdifferentiation of renal tubular epithelial cells.Therefore,this study aims to further explore the role of down-regulation of macrophage Act1 on mesenchymal transformation of renal tubular epithelial cells and its molecular mechanism.Methods:1.Eight-week-old male WT mice and mice with down-regulated macrophage Act1 expression(Act1 mice)were selected to establish a renal fibrosis model through unilateral ureteral ligation;2.Masson staining,immunohistochemical indexes such as α-SMA,F4/80 and CD3 were used to further determine the correlation between the degree of renal fibrosis and the infiltration of macrophages.3.Immunofluorescence was used to observe the transformation of macrophages into myofibroblasts and the mesenchymal transdifferentiation of renal tubular epithelial cells;4.In vitro TGFβ1 was used to induce renal tubular epithelial cells(TCMK1)to establish in vitro Epithelial Cell Mesenchymal Transformation Model;5.TGFβ1-induced macrophage migration was observed by Transwell chamber.6.Through the co-culture system of renal macrophages and tubular epithelial cell lines,combined with Western Blot detection,whether the down-regulation of macrophages Act1 can affect the mesenchymal transdifferentiation of renal tubular epithelial cells induced by TGFβ1 was determined;7.Further RNA-seq transcriptome sequencing was performed to analyze the m RNA differential expression of the two macrophages in the co-culture system,and q RT-PCR was performed for verification.8.According to bioinformatics analysis and Western Blot,whether the two kinds of macrophages regulate the key signaling pathway of TGFβ1-induced renal tubular epithelial cell transdifferentiation through secreting differential proteins was determined.Results:(1)After down-regulation of macrophages Act1,the infiltration of macrophages in the renal interstitial tissue of the UUO model decreased;(2)The number of macrophages trans-differentiated in the renal interstitial tissue of the UUO model decreased after the down-regulation of macrophages Act1;(3)Down-regulation of macrophage Act1 inhibited TGFβ1-induced transdifferentiation of macrophages into myofibroblasts;(4)Down-regulation of macrophage Act1 inhibits TGFβ1-induced macrophage migration;(5)Down-regulation of macrophage Act1 inhibits mesenchymal transdifferentiation of renal tubular epithelial cells induced by TGFβ1;(6)Down-regulation of macrophage Act1 inhibited the expression and phosphorylation of key signaling proteins of JAK-STAT3 in TGFβ1-induced renal tubular epithelial cell transdifferentiation.Conclusion:Down-regulation of macrophage Act1 may improve renal fibrosis injury by inhibiting macrophage migration and renal tubular epithelial cell transdifferentiation.