Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium

Abstract

Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium, and with increased usage of transgenic mouse models to study liver disease pathogenesis, we sought to evaluate the morphological, secretory, and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry, normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2–4 μm2, embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Sizes of bile ducts and cholangiocytes were evaluated by using SigmaScan to measure the diameters of bile ducts and cholangiocytes. In small and large normal and BDL cholangiocytes, we evaluated the expression of cholangiocyte-specific markers, keratin-19 (KRT19), secretin receptor (SR), cystic fibrosis transmembrane conductance regulator (CFTR), and chloride bicarbonate anion exchanger 2 (Cl/HCO3 AE2) by immunofluorescence and western blot; and intracellular cyclic adenosine 3′,5′-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL, small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by fluorescence-activated cell sorting, and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections. In situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, with smaller cholangiocytes lining smaller bile ducts and larger cholangiocytes lining larger ducts. Both small and large cholangiocytes express KRT19 and only large cholangiocytes from normal and BDL mice express SR, CFTR, and Cl/HCO3 exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate. We conclude that similar to rats, mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree.

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Acknowledgements

We thank Dr Andreea Trache and Anna Webb of the Texas A&M University Health Science Center Microscopy Imaging Center for assistance with confocal microscopy. This work was supported by the Dr Nicholas C Hightower Centennial Chair of Gastroenterology to Dr Alpini from Scott and White Hospital, a VA Research Scholar Award, a VA Merit Award, and the NIH grants DK054811 and DK062975 to Dr Alpini, by a grant from MIUR (PRIN) 2007 no. 2007HPT7BA_001), University and Faculty funds (Professor Eugenio Gaudio), by MIUR (PRIN) grants no. 2007, prot. HPT7BA_003 to Professor Alvaro, and an NIH K01 grant award (DK078532) to Dr DeMorrow.

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Correspondence to Shannon S Glaser or Gianfranco D Alpini.

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Glaser, S., Gaudio, E., Rao, A. et al. Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium. Lab Invest 89, 456–469 (2009) doi:10.1038/labinvest.2009.6

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Keywords

  • bicarbonate secretion
  • cAMP
  • cholangiocytes
  • secretin
  • secretin receptor

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