title: A biliary HCO3- umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes.
abstract: Human cholangiocytes are continuously exposed to millimolar levels of hydrophobic bile salt monomers. We recently hypothesized that an apical biliary HCO3- umbrella might prevent the protonation of biliary glycine-conjugated bile salts and uncontrolled cell entry of the corresponding bile acids, and that defects in this biliary HCO3- umbrella might predispose to chronic cholangiopathies. Here, we tested in vitro whether human cholangiocyte integrity in the presence of millimolar bile salt monomers is dependent on (1) pH, (2) adequate expression of the key HCO3- exporter, anion exchanger 2 (AE2), and (3) an intact cholangiocyte glycocalyx. To address these questions, human immortalized cholangiocytes and cholangiocarcinoma cells were exposed to chenodeoxycholate and its glycine/taurine conjugates at different pH levels. Bile acid uptake was determined radiochemically. Cell viability and apoptosis were measured enzymatically. AE2 was knocked down by lentiviral short hairpin RNA. A cholangiocyte glycocalyx was identified by electron microscopy, was enzymatically desialylated, and sialylation was quantified by flow cytometry. We found that bile acid uptake and toxicity in human immortalized cholangiocytes and cholangiocarcinoma cell lines in vitro were pH and AE2 dependent, with the highest rates at low pH and when AE2 expression was defective. An apical glycocalyx was identified on cholangiocytes in vitro by electron microscopic techniques. Desialylation of this protective layer increased cholangiocellular vulnerability in a pH-dependent manner.
  
  
  CONCLUSIONS
  A biliary HCO3- umbrella protects human cholangiocytes against damage by bile acid monomers. An intact glycocalyx and adequate AE2 expression are crucial in this process. Defects of the biliary HCO3- umbrella may lead to the development of chronic cholangiopathies.
authors.affiliation: Tytgat Institute for Liver and Intestinal Research, Department of Gastroenterology and Hepatology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
authors.forename: Simon
authors.initials: S
authors.lastname: Hohenester
authors.forename: Lucas Maillette de Buy
authors.initials: LM
authors.lastname: Wenniger
authors.forename: Coen C
authors.initials: CC
authors.lastname: Paulusma
authors.forename: Sandra J
authors.initials: SJ
authors.lastname: van Vliet
authors.forename: Douglas M
authors.initials: DM
authors.lastname: Jefferson
authors.forename: Ronald P Oude
authors.initials: RP
authors.lastname: Elferink
authors.forename: Ulrich
authors.initials: U
authors.lastname: Beuers
mesh_terms: D027321:Anion Transport Proteins / Q000235:genetics / Q000378:metabolism; D017920:Antiporters / Q000235:genetics / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology; D001647:Bile Acids and Salts / Q000493:pharmacokinetics* / Q000633:toxicity*; D001650:Bile Duct Neoplasms; D001653:Bile Ducts, Intrahepatic / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D045744:Cell Line, Tumor; D018281:Cholangiocarcinoma; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005110:Extracellular Space / Q000378:metabolism; D055785:Gene Knockdown Techniques; D019276:Glycocalyx / Q000378:metabolism; D006801:Humans; D006863:Hydrogen-Ion Concentration / Q000187:drug effects; D008854:Microscopy, Electron; D009439:Neuraminidase / Q000494:pharmacology; D064606:SLC4A Proteins; D017693:Sodium Bicarbonate / Q000494:pharmacology*
chemical_list: D027321:Anion Transport Proteins; D017920:Antiporters; D001647:Bile Acids and Salts; D064606:SLC4A Proteins; D017693:Sodium Bicarbonate; D009439:Neuraminidase