Abstract
Background: Chronic kidney disease (CKD) is thus deemed to a global health problem. Renal fibrosis, characterized by accumulation of extracellular matrix (ECM) components in the kidney, is considered a common pathway leading to CKD. Regulator of calcineurin1 (RCAN1), identified as a competitive endogenous inhibitor of the phosphatase calcineurin, participates in ECM deposition in various organs. However, the role of RCAN1 in renal fibrosis remains unclear. Methods: Unilateral ureteral obstruction (UUO), a well-known model to induce renal fibrosis, was performed on mice for a week. To overexpress RCAN1.4 in vivo, Recombinant adeno-associated virus (rAAV) 9-packed RCAN1.4 over-expression plasm was employed in mice kidney. Lentivirus-packed RCAN1.4 over-expression plasm was employed to transfer into HK-2 and NRK-49F cells in vitro. Hematoxylin and eosin (H & E) were performed to assess histopathological kidney injury, Sirus-red and Masson’s trichrome staining were carried out for observing collagen deposition. Western blotting was also used to determine the expression levels. Results: RCAN1 isoform 4 (RCAN1.4) expression was impaired after suffering UUO-induced renal fibrosis in vivo and TGF-β1-induced renal fibrosis in vitro. Furthermore, knocking in of RCAN1.4 in mice kidney, transfected with rAAV9-packed RCAN1.4 over-expression plasm, reduced the expression levels of hallmarks of EMT. In vitro, the apoptosis-related proteins, including the ratio of Bax/Bcl-2 and cleaved-caspase3, were elevated in cells transfected with RCAN1.4 overexpression plasmid. Moreover, over-expression of RCAN1.4 recovered the renal function and attenuated ECM deposition. In addition, RCAN1.4 regulated NFAT2 nuclear distribution by inhibiting calcineurin pathway. Conclusions: The expression of RCAN1.4 was attenuated both in UUO-induced fibrotic kidney of mouse and in TGF-β1-induced fibrotic model in vitro. Moreover, overexpression of RCAN1.4 could reverse renal fibrosis, attenuate ECM related protein accumulation, promote apoptosis of myofibroblast via inhibiting CaN/NFAT2 signaling pathway. Taken together, our study demonstrated that targeting RCAN1.4 may be therapeutic efficacy in renal fibrosis Funding: This study was supported by the National Natural Science Foundation of China (No.82000639) and Innovation Project of Medical School of Wuhan University (TFZZ2018017). Declaration of Interest: The authors declare that they have no conflicts of interest. Ethical Approval: This experiment was authorized by the Ethics Committee of Renmin Hospital of Wuhan University, and the procedures were carried out adhere to the principles of Animal Care of Wuhan University (Wuhan, China).
Highlights
Chronic kidney disease (CKD) is the 16th cause of death worldwide [1]
In this study, we demonstrated that Regulator of calcineurin1 (RCAN1).4 expression was down-regulated in renal fibrosis in mouse and in activated renal interstitial fibroblasts
The results showed that TGFβ1 induced renal fibrosis in vivo and in vitro through inhibiting the RCAN1.4 expression
Summary
Chronic kidney disease (CKD) is the 16th cause of death worldwide [1]. The incidence rate of CKD is estimated at 9.1% and deemed to a global health problem [2]. Activated renal interstitial fibroblasts, which are called myofibroblasts and are characterized by the over-expression of αsmooth muscle actin (α-SMA) and type I collagen (COL1a1), can be induced by TGF-β1 [6, 10, 11]. RCAN1.4 has been identified as a competitive endogenous negative regulator of the phosphatase calcineurin which is associated with the activation of nuclear factor of activated T cells (NFAT) [15, 16]. Our studies try to investigate the critical role of RCAN1.4 in renal interstitial fibroblasts activation and kidney fibrosis. We supposed that, as an endogenous negative regulator of Calcineurin, RCAN1.4 had a critical potential role in regulating ECM accumulation through Calcineurin /NFAT signaling pathway. We investigated the mechanism of RCAN1.4 in regulating renal interstitial fibroblast activation and renal fibrosis. Showed that the signal of RCAN1 was decreased in the fibrotic kidney tissue of UUO mice compared with the sham group
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