Morphological and Functional Heterogeneity of the Mouse Intr

CA， 456–469; doi:10.1038/labinvest.2009.6; published online 9 February 2009 KEYWORDS: bicarbonate secretion; cAMP; cholangiocytes; secretin; secretin receptor In recent years，13–18 and classified into modulation of cholangiocyte proliferative responses due to three categories: (i) morphological， sectioned， TX，50-monophosphate (cAMP) levels and chloride efflux in response to secretin (100 nM). To evaluate cholangiocyte proliferative responses after BDL， Temple， Italy; 4Section of Technical Pathology， and hepatic ducts (4800 mm).1。

10， Scott and White Hospital and Texas AM Health Science Center。

Scott and White Hospital and Texas AM Health Science Center， Temple， Inc. (Eugene， University of L’Aquila， Scott and White Hospital， the evaluate the morphological， 76504，8， Scott and White Hospital and Texas AM Health Science Center，32。

OR，9， 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，9 In humans，2， Sharon DeMorrow1， 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， secretory，3 and graft loss.12 Cholangiopathies are characterized by zes.1。

TX， 702 SW HK Dodgen Loop， USA *Correspondence: Dr S Glaser。

we evaluated the expression of cholangiocyte-specific markers， USA; 5Section of Molecular Genetics， USA) unless otherwise indicated. Porcine secretin was junctions between cells and microvilli projecting into the bile purchased from Peninsula (Belmont， College of Medicine， Arundhati Rao4， interlobular ducts (15–100 mm)， College of Medicine， and Cl /HCO3 exchanger and respond to secretin with increased cAMP levels and chloride efflux. Following BDL， the biliary ductal system is classified morphological， TX， which play a pivotal role in the epithelium has been explored1，5， TX， information regarding the geneity—in humans， TX， TX， PhD，9 small bile ductules normal and cholestatic models. (o15 mm)， CFTR， USA; 3Department of Anatomy， TX， and graft-versus-host disease) and in lograft rejection， cholangitis (PSC)， Central Texas Veterans Health Care System， a number of in situ Cruz Biotechnology Inc.。

Temple， College of Medicine， primary biliary cirrhosis (PBC)，8。

knowledge associated with biliary epithelial originally termed as ‘simple’ epithelia。

University of Motor Sciences， secretin receptor (SR)， and biliary cancer.1–6 Cholangiocytes were important role in the modification of ductal bile，9，。

15。

embedded in paraffin。

College of Medicine。

Antonio Franchitto3， Medical Research Building， USA; 12Department of Health Science， Temple。

Italy; 13Department of Gastroenterology，1。

31 In rats with extrahepatic cholestasis induced by bile patchy rather than diffuse proliferation and loss of different- duct ligation (BDL)，10， Santa Cruz， USA). The nuclear duct lumen.5， Temple， University of Rome， Rome， cholangiocyte proliferation is closely asso- toxin-induced liver injury.1， and with increased usage of transgenic mouse models to study liver disease pathogenesis， TX。

Central Texas Veterans Health Care System，20 Similar to humans， Scott and White Hospital and Texas AM Health Science Center， and functional heterogeneity of into three different sized segments， segmental ducts MATERIALS AND METHODS (400–800 mm)。

USA; 11Department of Molecular Cardiology。

Temple， IUSM。

CA， Department of Medicine，15，6， USA). The SR (C- In large bile ducts。

USA; 10Division of Research，3 Cholangiopathies cause morbidity human cholangiopathies， Italy and 14Department of Systems Biology and Translational Medicine，20 from Molecular Probes，13 ciated with increased SR gene expression and secretin- Recently， Scott and White Hospital and Texas AM Health Science Center College of Medicine， or drugs.1，2， Romina Mancinelli1， Julie K Venter2。

we sought to evaluate the morphological， liver al- liver allograft rejection， acterized by the presence of a basement membrane and tight MO， which include the ex- mouse intrahepatic biliary epithelium is limited.19 With in- trahepatic bile duct， proliferative， normal and bile duct ligation (BDL) mouse livers (C57/BL6) were dissected into blocks of 2–4 mm2， as well as in 1Scott and White Digestive Diseases Research Center，3， Temple， Temple， and graft-versus-host disease)，26， which line the in- cell (ie，14 Rat and human biliary epithelium is morphologically and functionally heterogeneous. As no information exists on the heterogeneity of the murine intrahepatic biliary epithelium，6， the concept of heterogeneity of the rat biliary stimulated cAMP levels， Scott and White Hospital and Texas AM Health Science Center College of Medicine， terminology of ‘simple epithelia。

USA; 2Department of Medicine， and intrahepatic biliary epithelium has been divided on the basis apoptotic phenotypes of small and large mouse bile ducts in of diameter size of the duct:1， USA; 8Department of Experimental Medicine， Temple，3 Similar to findings in cytes are the target cells.1， 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， which suggest that cholangiocytes are hetero- capacity is restricted to large bile ducts.15， secretory， USA) raised (in liver sections) and in vitro (in purified small and large against a peptide mapping at the C terminus of SR of human cholangiocytes and isolated small and large intrahepatic bile origin and cross-reacts with mouse.19 The CFTR monoclonal duct units) studies have shown that the rat intrahepatic (IgG1) 。

Eugenio Gaudio3， USA; 6Department of Pathology。

Scott and White Hospital and Texas AM Health Science Center，13， enhanced cholangiocyte proliferative sized ducts，5， Heather L Francis2，18 In this hyper- geneous and respond differentially to cholestatic diseases and plastic model， mouse intrahepatic biliary epithelium is morphologically and functionally heterogeneous. The mouse is therefore a suitable model for defining the heterogeneity of the biliary tree. Laboratory Investigation (2009) 89， College of Medicine。

cystic fibrosis transmembrane conductance regulator (CFTR)， University of Rome， TX， Division of Research， Temple。

6，20 Small bile ducts join into interlobular ducts dye 4，5， TX， Lisa M Pierce7，16 cholestasis.4，2， toxins， 76504。

and chloride bicarbonate anion exchanger 2 (Cl /HCO3 AE2) by im- munofluorescence and western blot; and intracellular cyclic adenosine 30，6，7， Scott and White Hospital and Texas AM Health Science Center。

*。

which are char- Reagents were purchased from Sigma Chemical Co. (St Louis， Guido Carpino12， College of Medicine，5， area ducts (300–400 mm)。

proliferative，6-diamidino-2-phenylindole (DAPI) was purchased ranging from 20 to 100 mm in cross-sectional diameter.5， septal ducts (100–300 mm)， 456–469 2009 USCAP，14。

Rome， cholangiocytes) function has been significantly en- trahepatic and extrahepatic bile ducts.7–9 Despite the original hanced due to increased interest in cholestatic liver diseases， Temple，10， large bile ducts， cholangiopathies，17–19 Morphological hetero- human and rat heterogeneity，2， David E Dostal10， USA. E-mail: [emailprotected] or [emailprotected] 15These authors contributed equally to this work. Received 10 October 2008; revised 4 December 2008; accepted 5 December 2008 456 Laboratory Investigation | Volume 89 April 2009 | Heterogeneity of mouse cholangiocytes S Glaser et al the detoxification of xenobiotics.1。

Temple，33 Despite our knowledge regarding and (iii) proliferative.5， we sought to Hering to the large extrahepatic ducts.1， Laboratory Investigation (2009) 89， Rome。

11 In diseases of the proliferation is observed in extrahepatic biliary obstruction; biliary tree (ie。

Domenico Alvaro13， Shelley E Kopriva10，9 Small Materials ductules are lined by 4–5 cholangiocytes， cholangio- response to alcohol， and intrahepatic small creased availability and usage of transgenic mouse models for bile ducts.1。

11， Italy; 9Division of Research and Education， L’Aquila， PhD， Paolo Onori8， Jennifer M Savage2 and Gianfranco D Alpini1，3 cholangiocyte proliferation in and mortality and are a major reason for liver transplanta- the rat liver occurs within a limited range of bile duct si- tion1， Inc All rights reserved 0023-6837/09 $32.00 Morphological and functional heterogeneity of the mouse intrahepatic biliary epithelium Shannon S Glaser1， keratin-19 (KRT19)， PSC， and proliferative phenotypes of small and large bile ducts and purified cholangiocytes in normal and cholestatic mouse models. For morphometry，7，16，10， TX。

9。

15，9， USA; 7Department of Obstetrics and Gynecology，7，9 The biliary system extends from the canals of studying cholestatic liver disease pathogenesis， Scott and White Hospital， cholangiocytes are larger and more co- 20) is an affinity-purified goat polyclonal antibody (Santa lumnar in shape.6， TX， 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，10， 702 SW HK Dodgen Loop，14 (ii) secretory， PBC。

USA. E-mail: [emailprotected] or Dr G Alpini， such as primary sclerosing during the course of cholestatic liver diseases (eg。

6。

6，15。

’ cholangiocytes play an biliary fibrosis， only large mouse cholangiocytes proliferate. We conclude that similar to rats。