Double knockout of secretin and secretin receptor exacerbates biliary damage and decreases biliary proliferation and ductal secretion during extrahepatic cholestasis: protective role of bicarbonate secretion during biliary disorders
Shannon S. Glaser1, 2, Fanyin Meng1, 2, Heather L. Francis1, 2, Julie Venter5, Laura Hargrove2, Holly A. Standeford3, Syeda H. Afroze5, Paolo Onori4, Kelly McDaniel3, Micheleine Guerrier5, Eugenio Gaudio4, Gianfranco Alpini1, 2
1Research and Medicine, Central Texas Veterans Health Core System and Texas A&M HSC College of Medicine, Temple, TX; 2Scott & White Digestive Diseases Research Center, Scott & White, Temple, TX; 3Research, Central Texas Veterans Health Care System, Temple, TX; 4Deportment of Anatomical, Histological, Forensic Medicine and Orthopedics Sciences, Universitá, Sapienza, Rome, Italy; 5Medicine, Texas A&M HSC COM, Temple, TX
Secretin (SEC) stimulates ductal secretion by interaction with its receptor (SR) by activation of the SR→CFTR→Cl-/HC〇3- AE2 exchanger pathway. Cholangiocyte proliferation/loss and changes in ductal secretion are key aspects of cholangiopathies. We have shown that activation of the SEC/SR axis sustains biliary proliferation in bile duct ligated (BDL) rats. The knockdown of either SEC or SR results in only partial inhibition of biliary proliferation and slight liver damage, which may be due to confounding effects of individual knockdown of SEC and SR. To pinpoint the role of the SEC/SR axis in regulating biliary proliferation and function, we established a double knockout mouse model for both SEC and SR (SEC/SR KO). Our hypothesis is that the lack of a protective bicarbonate umbrella (provided by the SEC/SR axis) in bile ducts leads to increased hepatobiliary damage. The aim of our study was to evaluate cholangiocyte proliferation and function in SEC/SR K〇 with extrahepatic cholestasis. Methods: Studies were performed in normal and BDL (7 day) SEC KO, SR KO and double SEC/SR KO mouse models. Biliary proliferation was assessed by measurement of: (i) intrahepatic bile duct mass (IBDM) in liver sections; and (ii) PCNA expression in isolated cholangiocytes by real-time PCR/immunoblots. Necrosis and inflammation were evaluated in H&E-stained liver sections. Biliary function was evaluated by measurement of the expression of CFTR and Cl-/HC〇3 - AE2.We also evaluated the expression of Bax in all experimental groups. Results: There was an increased reduction in IBDM and PCNA expression in BDL SEC/SR KO mice compared to BDL SEC or SR KO mice. There was more periportal necrosis and increased portal inflammation in BDL SEC/SR KO mice compared to BDL SEC or SR KO mice. We observed spontaneous damage in the liver sections from normal SEC/SR KO, which was associated with a significant increase in the expression of the pro-apoptotic protein Bax. In BDL SEC/SR KO mice, there was a greater reduction in both CFTR and Cl-/HCO3 - AE2 expression (compared to BDL SEC or SR KO mice) indicating a reduction of biliary growth and enhanced biliary damage due to lack of bicarbonate secretion. Summary/Conclusion: Knockdown of the SEC/SR axis down-regulates both biliary proliferation and the expression of CFTR and Cl-/HCO3 - AE2, which is associated with increased periportal necrosis, portal inflammation and biliary damage. These findings suggest that the complete inhibition of the SEC/SR axis triggers increased hepatic damage due to a lack of a: (i) functional SEC/SR/CFTR/Cl-/HC〇3 - AE2 system; and (ii) protective bicarbonate umbrella during extrahepatic cholestasis.
The following people have nothing to disclose: Shannon S. Glaser, Fanyin Meng, Heather L. Francis, Julie Venter, Laura Hargrove, Holly A. Standeford, Syeda H. Afroze, Paolo Onori, Kelly McDaniel, Micheleine Guerrier, Eugenio Gaudio, Gianfranco Alpini