Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium

Abstract

Rat and human biliary epithelium is morphologically and functionally heterogeneous. Since 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.MethodsFor morphometry, normal and BDL mouse livers (C57/BL6) were dissected into blocks of 2-4 μm2, embedded in paraffin, sectioned, and stained with H&E. 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-/HCO-3 AE2) by immunofluorescence and western blot; and intracellular cAMP levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after bile duct ligation (BDL), small and large cholangiocytes were isolated from BDL mice. The proliferation status was determined by analysis of the cell cycle by FACS and bile duct mass was determined by the number of KRT19-positive bile ducts in liver sections.ResultsIn situ morphometry established that the biliary epithelium of mice is morphologically heterogeneous, which smaller cholangiocyte 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-/HCO-3 exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL, only large mouse cholangiocytes proliferate.ConclusionSimilar to rats, mouse intrahepatic biliary epithelium is morphologically, and functionally heterogeneous. The mouse is a suitable model for defining the heterogeneity of the biliary tree.

Differential transcriptional characteristics of small and large biliary epithelial cells derived from small and large bile ducts

“…As previously described (15), cells were incubated at 37°C in 5% CO 2 atmosphere in D-MEM (Gibco) supplemented with 10% FBS, 1% pen/strep, and 1% L-glutamine. For experiments (n ϭ 4), cells were plated at 5 ϫ 10 6 cells in 10-cm culture dishes for mock infection or infection with a multiplicity of infection of 35 infectious units of AdHNF6 or AdLacZ in 1 ml of media for 60 min, following which media was added to a final 10-ml volume.…”

“…See text for definitions of gene acronyms. previously characterized (38) and shown to display morphological, phenotypic, and functional characteristics of freshly isolated large and small cholangiocytes (15). The baseline profile in the expression of hepatocyte transcription factors HNF6 (OC1) and its paralog OC2 (18), HNF1b, HNF1a, HNF4a, Foxa2, and C/EBPb was assessed by real-time PCR in SV40LG and SV40SM cell lines.…”

“…The intrahepatic bile duct size ranges from large ducts emanating from the confluence of the extrahepatic bile ducts at the liver hilum to progressively smaller intrahepatic ducts (19). In experimental models, small BEC refer to BEC lining the small bile ducts, whereas large BEC line larger biliary ducts (3,15). Small and large rodent BEC have distinct morphometry (15), gene expression profile (38), as well as proliferative, apoptotic, and secretory responses to experimental stimuli (1,14,15).…”

“…In experimental models, small BEC refer to BEC lining the small bile ducts, whereas large BEC line larger biliary ducts (3,15). Small and large rodent BEC have distinct morphometry (15), gene expression profile (38), as well as proliferative, apoptotic, and secretory responses to experimental stimuli (1,14,15). This BEC heterogeneity is clinically relevant in that the large and small ducts are differentially targeted in human cholangiopathies (37), stressing the importance of understanding the molecular mechanism regulating their functional diversities.…”

CCN1 induces hepatic ductular reaction through integrin αvβ5–mediated activation of NF-κB

“…Freshly isolated primary cholangiocytes were purified by immunoaffinity separation with an mAb, rat IgG2a (provided by R. Faris, Brown University, Providence, Rhode Island, USA), against an antigen expressed by mouse cholangiocytes as described previously (70). Cells in the preparation were 98.5% ± 0.4% positive for the IgG2a antigen as assessed by immunocytochemistry.…”