Anp32e Promotes Renal Interstitial Fibrosis By Regulating TGF-β1/Smad3

BackgroundRenal interstitial fibrosis is a common pathological feature of chronic kidney disease(CKD)caused by almost all kinds of causes,and is also a key factor affecting the prognosis of CKD.In recent years,a large number of studies have confirmed that a variety of cells and molecules play an important role in the occurrence and development of renal interstitial fibrosis,but the specific molecular mechanism is still unclear,and there is a lack of effective treatment.Through high-throughput sequencing analysis of m RNAs in renal tissues of unilateral ureteral obstruction(UUO)mice,we found for the first time that there was a significant difference in m RNA expression of Anp32 e in the UUO group and the sham group.Subsequently,we found through experimental verification that: Anp32 e upregulated and promoted renal interstitial fibrosis in UUO mice kidneys and TGF-β1-stimulated mouse renal tubular epithelial cells.The mechanism may be related to the involvement of Anp32 e in the TGF-β1/Smad3 signaling pathway(The results have not yet been published and are the content of the thesis of the 2020 Master’s degree student of our group).However,as a profibrotic factor,how does the high expression of Anp32 e affect renal interstitial fibrosis in the absence of TGF-β1stimulation or induction? What is the relationship between Anp32 e and TGF-β1/Smad3 signaling pathway? And whether Anp32 e protein is also differentially expressed in the renal tissues of patients with renal interstitial fibrosis? All these questions deserve further exploration.ObjectiveIn this study,we will construct animal and cell models with exogenous overexpression of Anp32 e to further explore the influence of Anp32 e protein high expression on renal interstitial fibrosis and its molecular mechanism.We also collected kidney tissues from clinical chronic nephritis patients to analyze the correlation between Anp32 e expression and RIF,which will provide a new vision for the treatment of renal fibrosis.Methods(1)Anp32e overexpressing lentivirus mice(LV-Anp32e)were constructed.HE and Masson staining were performed to detect pathological changes in the kidney,immunofluorescence was performed to detect the expression of GFP protein,immunohistochemistry and Western blot were used to detect the expression of Anp32 e,Fn and Col-I proteins.(2)Anp32e sh RNA lentivirus mice were constructed and subjected to UUO surgery.HE and Masson staining were performed to detect pathological changes in the kidney,immunohistochemistry and Western blot were performed to detect the expression of Anp32 e,Fn and Col-I proteins.(3)Anp32e overexpression plasmid was transfected in BUMPT cells cultured in vitro.Western blot was performed to detect the expression of Anp32 e,Fn and Col-I proteins,q RT-PCR was performed to detect Fn and Col-I m RNA levels.(4)Anp32e overexpression plasmid was transfected in BUMPT cells and treated with Smad3 phosphorylation inhibitor(SIS3),and the protein expressions of Smad3,Fn and Col-I were detected by Western blot.(5)Anp32e overexpression plasmid with Flag tag was transfected in BUMPT cells,and the interaction between Anp32 e and Smad3 was detected by immunoprecipitation.(6)BUMPT cells were transfected with Anp32 e overexpression plasmid and treated with TGF-β1 inhibitor(SB431542),and the expression of Smad3,Fn and Col-I was detected by Western blot.(7)The renal biopsies and clinical data of 9 patients with chronic glomerulonephritis(CGN)who were hospitalized in the Department of Nephrology of Xiangya Second Hospital were collected,the normal paraneoplastic tissues of 7 patients who underwent nephrectomy in the Department of Urology of Xiangya Second Hospital were used as control.HE and Masson staining were used to detect renal pathology,and immunohistochemistry was used to detect Anp32 e protein expression in renal tissues.The correlation between Anp32 e expression level and fibrosis area was analyzed by linear regression.Results(1)Overexpression of Anp32 e protein in mouse kidney and BUMPT cells upregulated the expression of Fn and Col-I;In UUO mice,knockdown of Anp32 e reduced tubular injury and inhibited the deposition of Fn and Col-I.(2)Overexpression of Anp32 e protein promoted the phosphorylation of Smad3 in mouse kidney and BUMPT cells;Smad3 phosphorylation was inhibited by knocking down Anp32 e in UUO mice and BUMPT cells stimulated by TGF-β1.Inhibition of Smad3 phosphorylation reduced Anp32e-induced fibrosis related proteins expression in BUMPT cells.Immunoprecipitation results showed that there was no interaction between Anp32 e and Smad3.(3)Overexpression of Anp32 e protein in mouse kidney and BUMPT cells promoted the expression of TGF-β1 protein;Knockdown of Anp32 e protein decreased TGF-β1 protein expression in UUO mice.Blocking TGF-β1 signaling in BUMPT cells inhibited Anp32e-induced Smad3 phosphorylation and Fn and Col-I expression.(4)In the CGN group,the expression of Anp32 e protein was significantly upregulated,accompanied by obvious renal tubular epithelial cell atrophy,brothlike margin disappearance,tubule dilatation and interstitial collagen fiber deposition,and the expression level of Anp32 e protein was positively correlated with the area of renal interstitial fibrosis.Conclusion(1)Anp32e is a pro-fibrotic factor,and its high expression can promote renal interstitial fibrosis.(2)Anp32e positively regulates Smad3 signaling by promoting TGF-β1 expression,thereby promotes renal interstitial fibrosis.(3)Anp32e expression upregulated in the renal interstitial fibrosis area of patients with chronic nephritis,and the expression level was positively correlated with the area of renal interstitial fibrosis.