1. ¹Scott and White Digestive Diseases Research Center, Temple, TX, USA
2. ²Department of Medicine, Scott and White Hospital and Texas A&M Health Science Center, College of Medicine, Temple, TX, USA
3. ³Department of Anatomy, University of Rome, Rome, Italy
4. ⁴Section of Technical Pathology, Scott and White Hospital and Texas A&M Health Science Center, College of Medicine, Temple, TX, USA
5. ⁵Section of Molecular Genetics, Scott and White Hospital and Texas A&M Health Science Center, College of Medicine, Temple, TX, USA
6. ⁶Department of Pathology, Scott and White Hospital and Texas A&M Health Science Center, College of Medicine, Temple, TX, USA
7. ⁷Department of Obstetrics and Gynecology, Scott and White Hospital and Texas A&M Health Science Center, College of Medicine, Temple, TX, USA
8. ⁸Department of Experimental Medicine, University of L'Aquila, L'Aquila, Italy
9. ⁹Division of Research and Education, Scott and White Hospital, Temple, TX, USA
10. ¹⁰Division of Research, Central Texas Veterans Health Care System, Temple, TX, USA
11. ¹¹Department of Molecular Cardiology, Scott and White Hospital, Temple, TX, USA
12. ¹²Department of Health Science, IUSM, University of Motor Sciences, Rome, Italy
13. ¹³Department of Gastroenterology, University of Rome, Rome, Italy
14. ¹⁴Department of Systems Biology and Translational Medicine, Scott and White Hospital and Texas A&M Health Science Center, College of Medicine, Temple, TX, USA

Correspondence: Dr S Glaser, PhD, Department of Medicine, Scott and White Hospital and Texas A&M Health Science Center College of Medicine, 702 SW HK Dodgen Loop, Temple, TX, 76504, USA. E-mail: sglaser@tamu.edu; Dr G Alpini, PhD, Division of Research, Central Texas Veterans Health Care System, Scott and White Hospital and Texas A&M Health Science Center College of Medicine, Medical Research Building, 702 SW HK Dodgen Loop, Temple, TX, 76504, USA. E-mail: galpini@tamu.edu or galpini@medicine.tamhsc.edu

¹⁵These authors contributed equally to this work.

Received 10 October 2008; Revised 4 December 2008; Accepted 5 December 2008; Published online 9 February 2009.

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 m², 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.