Knockout of the secretin receptor (SR) in experimental primary sclerosing cholangitis reduces biliary hyperplasia and liver fibrois through decreased expression of epithelial-mesenchymal transitions (EMT) traits and cellular senescence in cholangiocytes

Background: Multidrug resistance-2 gene knockout mice (Mdr2-/-) mimic the key features of human primary sclerosing cholangitis (PSC) such as enhanced biliary damage, intrahepatic biliary mass (IBDM) and liver fibrosis. Enhanced biliary epithelial-mesenchymal transition (EMT) and senescence may increase the activation of hepatic stellate cell (HSCs) that contribute to the overall enhanced fibrotic reaction seen in PSC. We have previously shown that SR (only expressed by cholangiocytes) regulates biliary damage and hepatic fibrosis, since overexpression of SR increases IBDM and liver fibrosis, whereas knockout of SR decreases IBDM and fibrosis in cholestatic rodents. No data exists regarding the role of SR in the modulation of EMT and cellular senescence in the course of PSC. Thus, the aim of this study was to determine the effects of knockout of SR in Mdr2-/- mice on biliary hyperplasia, EMT, cellular senescence and liver fibrosis. Methods: Mdr2-/-/SR-/- mice (DKO) were generated by pair-breeding until homozygote DKOs were obtained. Male mice (matching WT, Mdr2-/- and DKO) were sacrificed at 12 wk of age, and serum, total liver samples, isolated cholangiocytes and HSCs (isolated by laser capture microscopy) were collected. We measured serum chemistry by EIA, and liver necrosis, inflammation and lobular damage by H&E staining. We analyzed IBDM by immunochemistry for CK-19 and liver fibrosis by Sirius Red staining in liver sections. We measured the expression of fibrotic markers (fibronectin, collagen-1a and TGF-b1/TGF-b1R), mesenchymal markers (S100A4, Vimentin and N-Cadherin) and epithelial marker (E-cadherin) by qPCR in total liver, isolated cholangiocytes and/ or HSCs and by immunofluorescence in liver sections. Results: Necrosis, inflammation and lobular damage were increased in Mdr2-/- mice compared to normal WT mice but were reduced in DKO mice. In Mdr2-/- mice, serum liver enzymes (increased compared to normal WT mice) were ameliorated in DKO mice. In Mdr2-/- mice, there was increased IBDM and hepatic fibrosis, and enhanced expression of fibrosis markers in both cholangiocytes and HSCs compared to normal WT mice, which was reduced in DKO mice. There was increased expression of mesenchymal markers and decreased E-cadherin expression in cholangiocytes from the Mdr2-/- compared to normal WT mice, changes that were reversed in DKO mice compared to Mdr2-/- mice. Similarly, the increase in cellular senescence observed in cholangiocytes from Mdr2-/- mice was decreased in DKO mice compared to Mdr2-/- mice. Conclusion: Our findings show that knockout of SR in Mdr2-/- mice reduces biliary damage and liver fibrosis, decreases EMT and cellular senescence and increases the expression of the epithelial marker, E-cadherin. Thus, targeting SR could represent a therapeutic strategy for ameliorating biliary damage and liver fibrosis in PSC.