Mechanism Of C CGAS-STING Involved In Renal Fibrosis Through IRF3/YAP/TEAD Pathway

Objective: Chronic kidney disease(CKD)has become a significant health problem in recent years.According to epidemiological studies,chronic kidney disease in China is10.8%.As CKD patients enter end-stage kidney disease(ESRD)with renal dysfunction,they need kidney transplantation,peritoneal dialysis,or hemodialysis.Waiting for treatment to maintain life has seriously affected patients’ quality of life.Renal fibrosis is a common pathway for all CKD patients to progress to ESRD,and its primary pathological manifestations are the increase of matrix components in the renal tubulointerstitium.At the same time,the damage of the tubular epithelial cells in the renal interstitium and the excessive accumulation of matrix will eventually lead to renal tubular atrophy and Obstruction,loss of practical nephrons,and ultimately impaired renal filtration.At present,the specific mechanism of renal fibrosis has not been fully elucidated.Current studies have shown that this process may be related to the sparseness of renal blood vessels caused by injury,the infiltration of inflammatory cells caused by increased proinflammatory factors synthesized by renal tubular epithelial cells,and the synthesis of TGF-β1 by renal tubular epithelial cells caused by factors such as cell cycle arrest.Although the TGF-β/Smad pathway is essential in renal fibrosis in previous studies,it is involved in the activation of fibroblasts and the secretion of the extracellular matrix.However,knockdown of Smad3 downstream of TGF-β alone did not alleviate renal fibrosis.Therefore,it is vital to find new mechanisms of renal fibrosis.In recent years,studies have found that the innate immune pathway is widely involved in repairing kidney damage and the process of renal fibrosis.Studies have suggested that TLR4 receptors and their downstream pathways in the Toll-like receptor family are involved in the process of renal fibrosis.At the same time,the study results on c GAS-STING suggest that the c GASSTING pathway that recognizes double-stranded DNA cannot distinguish endogenous DNA from viruses and bacteria,so it is involved in the process of inflammatory factors in the process of acute kidney injury and chronic kidney injury.Release and infiltration of inflammatory cells,especially cells of the monocyte-macrophage system,in the kidney.At the same time,in previous studies,the c GAS-STING pathway can not only promote the transcription of IRF3 transcriptional target genes such as IFN-β and TNF-α through phosphorylation of IRF3 by TBK1 after recognizing ectopic DNA but also has other pathways such as NF-κB and Hippo.This pathway regulates biological phenomena such as endoplasmic reticulum stress and mitophagy,etc.This study intends to use bioinformatics analysis technology to analyze sequencing data to find suitable therapeutic targets of the innate immune pathway and explore its possible mechanisms involved in renal fibrosis.Subsequently,molecular biology experimental techniques verified the results mined in the sequencing data,and their possible mechanisms were explored.To provide a primary experimental basis for finding new therapeutic targets for renal fibrosis.Methods: 1.Firstly,the sequencing data of renal biopsy tissue of patients with chronic kidney disease in the public database GEO was used and then processed to eliminate batch effects for differential analysis,and the differentially expressed genes were obtained for biological function annotation,and the data was processed by bioinformatics methods.After verification,STING,IRF3,TBK1,and IRF3 transcriptional target genes(IFN-β,TNF-α,IL-6,CCL2)and Hippo pathway core protein YAP and YAP/TEAD transcriptional target genes(CTGF,CYR61)in the innate immune pathway were closely related to each other.Correlation of the expression of fibrosis-related genes(Collagen I,fibronectin,α-SMA)in kidney tissue.Moreover,verify the correlation between IRF3 and YAP gene expression.At the same time,the levels of STING,IRF3,YAP,renal fibrosis,and STING pathway target genes were detected in an animal model of chronic kidney disease caused by folic acid to verify the data mining based on bioinformatics methods.2.Taking human proximal tubular epithelial cells as the research object,the knockdown experiments of STING and its downstream IRF3 were carried out to verify the regulation of the c GAS-STING pathway on YAP and its effect on tubules in the renal tubular replacement injury fibrosis model.The mechanism of action of epithelial cell fibrosis indicators(Collagen I,fibronectin,α-SMA),inflammatory factors(IL-6,TNFα,IFN-β,CCL2),and the expression of YAP/TEAD transcriptional target genes(CTGF,CYR61).IRF3 was subsequently knocked down while using a STING agonist to verify the mechanism of STING regulation of YAP.3.Use of STING small-molecule inhibition to intervene in chronic kidney disease.At the same time,verteporfin,a YAP inhibitor,was used as a control to observe whether the effect of STING inhibitor C176 was different in alleviating the level of renal fibrosis in mice compared with the pure YAP inhibitor verteporfin.Results: 1.STING was positively correlated with the expression of fibrosis index type 1collagen,fibronectin,and smooth actin in the process of renal fibrosis.In the process of renal fibrosis,IRF3 was positively correlated with the expression of fibrosis indicators type1 collagen,fibronectin,and smooth actin.IRF3 positively correlated with YAP expression during renal fibrosis.2.Knockdown of STING can inhibit aristolochic acid-induced extracellular matrix secretion in HK-2 cells,reduce inflammation and inhibit the increase of YAP/TEAD transcriptional target genes.Knockdown of IRF3 inhibited aristolochic acid-induced extracellular matrix secretion in HK-2 cells and attenuated inflammation while suppressing the elevation of YAP/TEAD transcriptional target genes.Knockdown of IRF3 rescued STING agonist-induced extracellular matrix secretion in HK-2 cells and attenuated inflammation while suppressing the elevation of YAP/TEAD transcriptional target genes.3.Both C176 and verteporfin can inhibit the increase of renal ECM secretion caused by folic acid.C176 reduced the level of YAP protein in the kidney of the folic acid-induced renal fibrosis model.C176 can inhibit the level of YAP/TEAD transcriptional target genes similar to verteporfin.C176 inhibited the transfer and secretion of pro-inflammatory factors in the folic acid-induced renal fibrosis model.Conclusion: In the process of renal fibrosis,the activation of STING can promote the entry of YAP into the nucleus through the IRF3 pathway,and the transcription of YAP/TEAD transcriptional target genes is increased and stored in renal fibrosis.