| Background: Chronic kidney disease(CKD)patients’ progressive loss of renal function renal function is associated not only with development of glomerulosclerosis,but also with that of interstitial fibrosis.The essence of fibrosis is the scar repair.Although it has been reported a lot of the molecular mechanisms of renal tubulointerstitial fibrosis,when those were translated into clinical application,successful targets are still lacking.Therefore,molecular mechanisms of effective targeted therapy on the renal fibrosis still need to study.Leucine-rich-alpha-2-glycoprotein 1(LRG1)is a kind of secreted and leucineriched glycoprotein,is closely related to the occurrence and development of a wide variety of tumor.Recent researches have demonstrated that LRG1 expressed in the retina endothelial cell and promoted angiogenesis to increase the occurrence and development of diabetic retinopathy.Also,our previous study found LRG1 in kidney is mainly expressed in glomerular endothelial cells,can promote angiogenesis and increase the progress of diabetes via transforming growth factor-β signaling pathway.It was unexpectedly found that LRG1 was not only expressed in endothelial cells,but also in renal tubular epithelial cells.No studies have explored whether LRG1 is related with renal tubulointerstitial fibrosis and involved in the occurrence and progression of CKD.Objective: To clarify the relationship between LRG1 and progression of renal tubulointerstitial fibrosis and to elucidate its molecular mechanism.Methods: Correlation between LRG1 and occurrence of CKD was established though exploring the public database,detecting CKD patients’ samples.Unilateral ureteral obstruction(UUO)mice model and aristolochic acid nephropathy(AAN)in mice with Lrg1 gene knockout or inducible tubule-specific overexpression of Lrg1 were used to establish renal fibrosis models.The degree of fibrosis was eveluated,and the regulatory role of LRG1 in the process of renal fibrosis and the possible signaling mechanisms were identified.In vitro,renal tubular epithelial cells(HK-2)were overexpressed with LRG1 via lentivirus in a coculture system with primary renal interstitial fibroblasts(HKF),and 5ng/m L TGF-β1 was given to stimulate the cells.Smad3 activation of HKF in the coculture system was observed,and the expression of genes related fibrosis was analyzed.The online database(http://amp.pharm.mssm.edu/Enrichr)were used to predict the transcription factors in the promoter region of mouse Lrg1,followed by Ch IP-PCR validation to clarify the mechanisms that regulate the elevated expression of LRG1 during fibrosis.Results: Public data analysis showed that LRG1 m RNA expression was upregulated in renal interstitium of CKD patients including diabetic nephropathy,lupus nephritis,etc.,and was negatively correlated with renal function.The serum LRG1 concentration in CKD patients were higher than that in healthy control,and showed a negative linear relationship with renal function.Single cell RNA-sequencing data and in situ hybridization analysis showed that LRG1 expression localized in renal tubular epithelial cells,and the inflammatory cytokine TNF-α could induce its expression.The specific overexpression of LRG1 by renal tubular epithelial cells aggravated the degree of fibrosis in UUO mice,and the knockout of LRG1 alleviated renal fibrosis.Ch IP-PCR confirmed that NF-κB activated LRG1 transcriptional expression in renal tubular epithelial cells under TNF-α stimulated.Co-culture system showed that TGF-β/Smad3 signaling pathway were activated both in fibroblasts and renal tubular epithelial cells overexpressed LRG1 through the autocrine/paracrine manner.Conclusions: The inflammatory response caused by kidney injury promoted the up-regulation of LRG1 expression in renal tubular epithelial cells.LRG1 mediated the crosstalk of renal tubular epithelial cell and mesenchymal fibroblast through the TGF-β1/Smad3 signaling pathway,and promoted the occurrence of fibrosis.LRG1 is a promising target for anti-renal fibrosis therapy. |
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