Side Chain Structure Determines Unique Physiologic And

AUTOIMMUNE, CHOLESTATIC AND BILIARY DISEASE Side Chain Structure Determines Unique Physiologic and Therapeutic Properties of norUrsodeoxycholic Acid in Mdr2؊/؊ Mice Emina Halilbasic,1 Romina Fiorotto,2 Peter Fickert,1 Hanns-Ulrich Marschall,3 Tarek Moustafa,1 Carlo Spirli,2 Andrea Fuchsbichler,4 Judith Gumhold,1 Dagmar Silbert,1 Kurt Zatloukal,4 Cord Langner,4 Uday Maitra,5 Helmut Denk,4 Alan F. Hofmann,6 Mario Strazzabosco,2,7,8 and Michael Trauner1

24-norursodeoxycholic acid (norUDCA), a side chain–modified ursodeoxycholic acid derivative, has dramatic therapeutic effects in experimental cholestasis and may be a promising agent for the treatment of cholestatic liver diseases. We aimed to better understand the physiologic and therapeutic properties of norUDCA and to test if they are related to its side chain length and/or -relative resistance to amidation. For this purpose, Mdr2؊/؊ mice, a model for sclerosing cholan gitis, received either a standard diet or a norUDCA-, tauronorursodeoxycholic acid (tauro- norUDCA)-, or dinorursodeoxycholic acid (dinorUDCA)-enriched diet. Bile composition, serum biochemistry, liver histology, fibrosis, and expression of key detoxification and transport systems were investigated. Direct choleretic effects were addressed in isolated bile duct units. The role of Cftr for norUDCA-induced choleresis was explored in Cftr؊/؊ mice. norUDCA had pharmacologic features that were not shared by its derivatives, including the increase in hepatic ؊ and serum bile acid levels and a strong stimulation of biliary HCO3 -output. norUDCA directly ؊ stimulated fluid secretion in isolated bile duct units in a HCO3 -dependent fashion to a higher ؊ extent than the other bile acids. Notably, the norUDCA significantly stimulated HCO3 -output also in Cftr؊/؊ mice. In Mdr2؊/؊ mice, cholangitis and fibrosis strongly improved with norUDCA, remained unchanged with tauro-norUDCA, and worsened with dinorUDCA. Ex- pression of Mrp4, Cyp2b10, and Sult2a1 was increased by norUDCA and dinorUDCA, but was unaffected by tauro-norUDCA. Conclusion: The relative resistance of norUDCA to amidation may explain its unique physiologic and pharmacologic properties. These include the ability to undergo cholehepatic shunting and to directly stimulate cholangiocyte secretion, both resulting ؊ in a HCO3 -rich hypercholeresis that protects the liver from cholestatic injury. (HEPATOLOGY 2009;49:1972-1981.)

biliary fibrosis.3,4 These unique characteristics of See Editorial on Page 1795 Mdr2Ϫ/Ϫ mice are of particular interest because increasing ue to the limited efficacy of current medical evidence suggests a key role of MDR3 (ABCB4, the hu- treatment of chronic diseases of the biliary tree, man orthologue of rodent Mdr2) defects in human cho- such as sclerosing cholangitis,1 there is an urgent lestatic liver diseases ranging from neonatal cholestasis to D biliary cirrhosis in adults.5 More recently, MDR3 variants need to develop novel treatment strategies and to test them in animal models for human cholangiopathies.2 have been observed in patients with primary sclerosing Mice lacking the canalicular phosphatidylcholine flop- cholangitis6 and idiopathic biliary fibrosis,7 highlighting pase Mdr2(Abcb4)(Mdr2Ϫ/Ϫ mice) develop chronic cho- the importance of MDR3 as a modifier gene in disease lestatic liver disease including sclerosing cholangitis and course and severity of cholangiopathies.

Abbreviations: dinorUDCA, dinorursodeoxycholic acid; IBDU, isolated bile duct unit; K19, keratin 19; norUDCA, 24-norursodeoxycholic acid; ␣-SMA, ␣-smooth muscle actin; tauro-norUDCA, tauronorursodeoxycholic acid; UDCA ursodeoxycholic acid. From the 1Laboratory of Experimental and Molecular Hepatology, Department of Gastroenterology and Hepatology, University Clinic of Internal Medicine, Medical University of Graz, Graz, Austria; the 2Section of Digestive Diseases, Department of Internal Medicine, School of Medicine, Yale University, New Haven, CT; 3Karolinska University Hospital Huddinge, Stockholm, Sweden; 4Institute of Pathology, Medical University of Graz, Graz, Austria; the 5Department of Organic Chemistry, Indian Institute of Science, Bangalore, India; the 6Department of Medicine, University of California, San Diego, CA; 7CeLiveR, Ospedali Riuniti di Bergamo, Bergamo, Italy; and the 8Department of Clinical Medicine and Prevention, University of Milano-Bicocca, Milan, Italy. Received September 18, 2008; accepted January 26, 2009.

1972 HEPATOLOGY, Vol. 49, No. 6, 2009 HALILBASIC ET AL. 1973

We have recently demonstrated that cholangitis and erties of norUDCA are related to its relative resistance to biliary fibrosis in Mdr2Ϫ/Ϫ mice are reversed by 24- N-acyl amidation or its side chain length, we compared norursodeoxycholic acid (norUDCA), a C23 homo- norUDCA with tauro-norUDCA (a synthetic taurine logue of ursodeoxycholic acid (UDCA) with one less conjugate of norUDCA) and dinorUDCA (a C22 homo- methyl group in its side chain,8 suggesting that side logue of UDCA and norUDCA with only three carbon chain–modified bile acids may represent a novel and atoms in its side chain) in the Mdr2Ϫ/Ϫ mouse model of promising treatment strategy for cholestatic liver dis- sclerosing cholangitis and biliary fibrosis. Moreover, this eases. The potential therapeutic mechanisms, hypoth- study was designed to test the hypothesis that the phar- esized so far to explain the pharmacodynamic effects of macologic effects of norUDCA depend on its ability to recir- Ϫ Ϫ norUDCA in Mdr2 / mice, include increased hydro- culate between bile ducts and hepatocytes as a consequence philicity (and decreased cytotoxicity) of the circulating of cholehepatic shunting and/or its direct stimulatory effects bile acid pool, induction of detoxification pathways and on cholangiocyte secretion. This information should be crit- elimination routes for bile acids, as well as potential direct ical for the design of novel drugs for the treatment of chole- 8 anti-inflammatory and anti-fibrotic effects. The therapeutic static liver diseases. effects of norUDCA may be related to its side chain structure 8,9 that strongly influences norUDCA metabolism. The met- Materials and Methods abolic fate of norUDCA differs substantially from that of other natural bile acids, including UDCA, because of its Ϫ/Ϫ minimal N-acyl amidation (conjugation) with taurine or gly- Animal Experiments. Mdr2 mice (FVB/N back- 8,9 ground) were obtained from Jackson Laboratory (Bar cine together with its considerable hydroxylation and glu- Ϫ/Ϫ curonidation. In contrast to conjugated bile acids, Harbor, ME). Eight-week-old male Mdr2 mice re- unconjugated norUDCA is a weak organic acid with only ceived either standard chow (Ssniff, Soest, Germany) two hydroxyl groups that can be reabsorbed from bile by or a diet containing norUDCA, tauro-norUDCA, or cholangiocytes and subsequently be resecreted by hepato- dinorUDCA (0.5% wt/wt, the dosage that corresponds cytes thereby undergoing a cholehepatic shunt, a process that approximately to 15 mg/mouse/day) over 4 weeks. Ϫ 10 Congenic B6.129P2-Cftrtm1Unc mice, which possess is associated with a HCO3 -rich hypercholeresis. Flushing Ϫ the S489X mutation that blocks transcription of of bile ducts by HCO3 -rich bile may dilute and inactivate 13 toxic biliary contents, thereby protecting bile ducts from fur- Cftr, were fed with Peptamen (Nestle Clinical Nutri- Ϫ/Ϫ 8 Ϫ ther injury in Mdr2 mice. HCO3 -rich hypercholeresis tion, Deerfield, IL) or with Peptamen containing has also been described for UDCA (the parent compound of norUDCA (in a dosage equivalent to that used in Ϫ Ϫ norUDCA), although this process only occurs in vitro once Mdr2 / mice) for 1 week. All animals were housed taurine stores have become depleted and the concentration under a 12:12-hour light/dark cycle and permitted ad of unconjugated UDCA prevails over that of its taurine con- libitum consumption of water and diet. The experi- jugate.11 Recent studies have shown that UDCA is also able mental protocols were approved by the local Animal to directly stimulate fluid secretion by cholangiocytes Care and Use Committees according to criteria out- through Cftr-mediated ATP secretion and apical purinergic lined in the Guide for the Care and Use of Laboratory signaling,12 but the potential effects of norUDCA remain to Animals prepared by the U.S. National Academy of be elucidated. Sciences (National Institutes of Health publication 86- To test whether the physiologic and therapeutic prop- 23, revised 1985).

Supported by grant P19118-B05 from the Austrian Science Foundation and a GEN-AU project grant (GZ 200.139/1-VI/1/2006) from the Austrian Ministry for Science (both to M. T.) and by the Swedish Research Council and The Swedish Medical Association (to H. U. M). E. H. was also supported by the Ph.D. program of the Medical University of Graz. M. S., C. S., and R. F. were supported by Fondazione S. Martino, Bergamo; National Institutes of Health Grant DK079005; and Yale University Liver Center (National Institutes of Health Grant DK34989). R. F. was also supported by Cystic Fibrosis Foundation (CFF-FIOROT0810). C. S. is a recipient of an ALF/AASLD Liver Scholar Award. The synthetic work was supported by a grant from JNCASR, Bangalore (to U. M.). A. F. H. was supported by National Institutes of Health Grant DK64891. The Medical University of Graz has ﬁled the patent for the use of norUDCA in the treatment of liver diseases, and Michael Trauner, Alan Hofmann, and Peter Fickert are listed as inventors (publication number WO2006119803). Address reprint requests to: Michael Trauner, M.D., Laboratory of Experimental and Molecular Hepatology, Department of Gastroenterology and Hepatology, Medical University Graz, Auenbruggerplatz 16, 8036 Graz, Austria. E-mail: [email protected]. Copyright © 2009 by the American Association for the Study of Liver Diseases. Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/hep.22891 Potential conﬂict of interest: Nothing to report. Additional Supporting Information may be found in the online version of this article. 1974 HALILBASIC ET AL. HEPATOLOGY, June 2009

Synthesis of norUDCA (3␣,7␤-Dihydroxy-5␤- Routine Serum Biochemistry. Alanine aminotrans- cholan-23-oic Acid), tauro-norUDCA [3␣,7␤-Dihy- ferase and alkaline phosphatase were analyzed with a Hi- droxy-5␤-cholan-23-oic Acid N-(2-Sulfoethyl)amide, tachi 917 analyzer (Boehringer Mannheim, Mannheim, norUDC-tau], and dinorUDCA (3␣,7␤-Dihydroxy-5␤- Germany). cholan-22-oic Acid, bisnorUDCA). The compounds Liver Histology. For conventional light microscopy, studied (Supporting Fig. 1) were synthesized from formalin-fixed livers were embedded in paraffin, and UDCA as described.14Ϫ16 Unconjugated bile acids were 4-␮m thick sections were stained with hematoxylin-eosin pure by thin layer chromatography; tauro-norUDCA was and Sirius red staining. The sections were coded and ex- pure by high-pressure liquid chromatography. amined by a pathologist (C. L.) who was unaware of the Analysis of Bile Flow and Composition. Bile flow animals’ genotype and treatment. and composition were determined as described.8 Before Immunohistochemistry for Keratin 19. As de- harvesting, mice were anesthetized with tribromoethanol scribed, keratin 19 (K19) immunohistochemistry was (Avertin) 250 mg/kg body weight intraperitoneally. The performed on cryosections with monoclonal rat anti– common bile duct was ligated, and the gallbladder was Troma-III antibody (dilution 1:100, Developmental cannulated for collection of bile. After a 10-minute equil- Studies Hybridoma Bank) developed under the auspices ibration period, bile was collected for 1 hour in test tubes of the National Institute of Child Health and Human Ϫ under mineral oil for determination of HCO3 , total car- Development (NICHID) and maintained at the Univer- bon dioxide and pH using an automatic blood gas ana- sity of Iowa (Iowa City, IA).22 lyzer (AVL 995 hp, AVL, Graz, Austria; COBAS Mira Western Blotting. ␣-Smooth muscle actin (␣-SMA) plus, Roche). For bile flow measurements and further bile and K19 protein levels were determined in liver homog- analysis, bile was collected for the following 30 minutes in enates (30 ␮g protein) via western blotting as de- preweighed test tubes. Bile flow was determined gravi- scribed8,23 using monoclonal mouse antibody against metrically and normalized to liver weight as described.17 ␣-SMA (dilution, 1:1,500; Sigma-Aldrich, Vienna, Aus- Biliary glutathione concentration was determined after tria) and monoclonal rat K19 (dilution, 1:1,500; Devel- protein precipitation using 5% metaphosphoric acid opmental Studies Hybridoma Bank). Blots were reprobed (Glutathione Assay Kit; Calbiochem, La Jolla, CA) ac- with an anti–␤-actin antibody (dilution, 1:5,000; Sigma- cording to the manufacturer’s instructions. Biliary bile Aldrich, Vienna, Austria) to confirm the specificity of the acid concentration (of 3␣-hydroxy bile acids) was mea- observed changes in transporter protein levels. sured enzymatically using a colorimetric bile acid kit Determination of Hepatic Hydroxyproline Con- (Ecoline Sϩ, Holzheim, Germany). tent. To quantify liver fibrosis, hepatic hydroxyproline Intrahepatic Mouse Bile Duct Units and Assess- was measured as described.24 ment of Ductular Secretion via Video-Optical Messenger RNA Analysis. Total hepatic RNA was Planimetry. Isolated bile duct units (IBDUs)—sealed isolated and reverse-transcribed into complementary fragments of isolated bile ductules with retained polarity DNA as described.21 In brief, total RNA was isolated that can secrete into a closed lumen—were prepared, pu- using TRIzol Reagent (Invitrogen, Austria) and re- rified, and cultured as described.18Ϫ20 Secretion in IBDUs verse-transcribed into complementary DNA by using is determined by measuring expansion of their lumen over the GeneAmp Gold RNA PCR Core Kit (Applied Bio- time using video-optical planimetry as an indicator of the systems, Vienna, Austria) according to the manufactur- ductular secretion rate, as described.18Ϫ20 After a er’s instructions. Real-time PCR was performed on a 5-minute baseline period, IBDUs were exposed to bile GeneAmp 7900 Sequence Detection System with Ge- acids (at 250 ␮M concentration) for up to 30 minutes, neAmp 7900 SDS software (Applied Biosystems, CA). and the luminal area was measured every 5 minutes via Polymerase chain reaction was performed in a 20-␮L video-optical planimetry. Serial images of the IBDUs reaction mixture containing TaqMan or SYBR Univer- were acquired as described.12 To determine whether se- sal PCR Master Mix (Applied Biosystems, CA). Poly- Ϫ cretion depends on HCO3 , IBDUs were perfused with merase chain reaction products were checked via gel Ϫ either HCO3 -free 4-(2-hydroxyethyl)-1-piperazine eth- electrophoresis for correct size and quality. All data Ϫ anesulfonic acid (HEPES) buffer or HCO3 -containing were normalized to 18s ribosomal RNA. The TaqMan Krebs-Ringer buffer. oligonucleotides were as follows: Mrp4 forward primer Bile Acid Analysis. Bile acids were extracted from 5Ј-TTAGATGGGCCTCTGGTTC-3Ј, reverse liver homogenate, serum, and bile and analyzed using primer 5Ј-GCCCACAATTCCAACCTTT-3Ј, probe electrospray ionization mass spectrometry and gas chro- 5Ј-ACTGCGCTCATCAAGTCCAGGG-3Ј (Gen- matography mass spectrometry as described.8,21 Bank accession number W54702); Cyp2b10 forward HEPATOLOGY, Vol. 49, No. 6, 2009 HALILBASIC ET AL. 1975

Table 1. Bile Flow and Composition in Mdr2؊/؊ Mice under nor-Bile Acid Treatment

Control norUDCA T-norUDCA dinorUDCA (4 ؍ n) (8 ؍ n) (8 ؍ n) (8 ؍ Variable (n

Bile ﬂow (␮L/gLWϪ1/minϪ1) 2.1 Ϯ 0.2 3.4 Ϯ 0.4* 3.1 Ϯ 0.5* 1.9 Ϯ 0.2† Bile acid output (nmol/gLWϪ1/minϪ1) 10.1 Ϯ 2.5 21.7 Ϯ 7.4* 18.5 Ϯ 4.9* 10.8 Ϯ 2.0† Bicarbonate output (nmol/gLWϪ1/minϪ1) 56.4 Ϯ 4.0 104.4 Ϯ 22.3* 76.8 Ϯ 12.5*,† 50.8 Ϯ 6.7† Glutathione output (nmol/gLWϪ1/minϪ1) 3.9 Ϯ 1.2 6.5 Ϯ 1.0* 4.1 Ϯ 0.7*,† 4.7 Ϯ 1.1†

Values are expressed as the mean Ϯ standard deviation. Control, standard chow-fed animals; norUDCA, norUDCA–fed animals; T-norUDCA, tauro-norUDCA–fed animals; dinorUDCA, dinorUDCA–fed animals. *P Ͻ 0.05 versus control. †P Ͻ 0.05 versus norUDCA. primer 5Ј-CAATGGGGAACGTTGGAAGA-3Ј, re- (123.6 Ϯ 47.0 nmol/g liver weightϪ1/minuteϪ1) and un- verse primer 5Ј-TGATGCACTGGAAGAGGAAC-3Ј, treated controls (44.3 Ϯ 6.5 nmol/g liver weightϪ1/min probe 5Ј- TTCGTAGATTCTCTCTGGCCACCAT- Ϫ1). These differences between norUDCA and tauro- Ј Ϫ GAGA-3 (GenBank accession number NM_009998); norUDCA in their ability to induce a HCO3 -rich bile Sult2a1 forward primer 5Ј-GGAAGGACCACGACT- flow both in normal and cholestatic conditions could be CATAAC-3Ј, reverse primer 5Ј-GATTCTTCA- secondary to the potential of norUDCA to undergo chole- Ј Ј 10 Ϫ CAAGGTTTGTGTTACC-3 , probe 5 -CCC- hepatic shunting and/or stimulate HCO3 -rich ATCCATCTCTTCTCCAAGTCTTTCTTCAG-3Ј choleresis at the level of the bile ducts 11. (GenBank accession number L02335). 18sRNA was norUDCA Significantly Increases Fluid Secretion .determined using SYBR Green Mastermix and forward in IBDUs in a Partially HCO ؊-Dependent Manner Ј Ј 3 primer 5 -GTAACCCGTTGAACCCCATT-3 , re- To directly address the choleretic potential of side chain– Ј Ј verse primer 5 -CCATCCAATCGGTAGTAGCG-3 shortened derivatives of UDCA at the cholangiocyte level, (GenBank accession number X00686). we perfused mouse IBDUs in vitro with norUDCA, Statistical Analysis. Data are reported as the arith- tauro-norUDCA, or dinorUDCA and measured luminal Ϯ metic mean standard deviation of reported animals in expansion rates as a marker of cholangiocellular bile secre- each group. Statistical analysis was performed using SPSS tion.12 IBDUs perfused with UDCA were used as an in- software. The data were analyzed with a nonparametric 12 Ϫ Ͻ ternal control as reported. In HCO3 -containing Mann-Whitney U test. A P value 0.05 was considered media, norUDCA significantly increased cholangiocyte significant. fluid secretion in IBDUs above the levels obtained by Results perfusion with equimolar amounts of tauro-norUDCA and UDCA, while the stimulation of the secretion by Ϫ norUDCA and Its Side Chain Derivatives Have dinorUDCA was the lowest (Table 2). In HCO3 -free Differing Effects on Bile Flow and Composition. To conditions, bile acid–induced cholangiocyte secretion was drastically reduced, but not abolished. The stimula- test whether side chain modification of norUDCA influ- Ϫ enced bile flow and composition in a cholestatic mouse tion of cholangiocyte secretion in the absence of HCO3 Ϫ/Ϫ was similar for both norUDCA and tauro-norUDCA in model, we fed Mdr2 mice, a model for spontaneous Ϫ sclerosing cholangitis and biliary fibrosis,4 with nor-bile contrast to previously mentioned differences in HCO3 - acids. In contrast to the significant increase of bile flow containing medium (Table 2). These results strongly ar- Ϫ gue for the concept that norUDCA directly stimulates and biliary output of bile acids, HCO3 and glutathione Ϫ Ϫ Ϫ by norUDCA treatment in Mdr2 / mice8 (Table 1), fluid and HCO3 secretion in cholangiocytes in addition tauro-norUDCA had no effect on glutathione output, to stimulating canalicular bile flow. Ϫ ؊ and the effects on HCO3 output were much less pro- norUDCA-Induced Stimulation of Biliary HCO3 nounced (Table 1). dinorUDCA altered neither bile flow Output In Vivo Is Partly Cftr-Independent. To fur- Ϫ nor bile acid, HCO3 , or glutathione output (Table 1). ther understand norUDCA’s mechanisms of action and to Ϫ/Ϫ Ϫ Similar to Mdr2 mice, norUDCA also stimulated bile test whether norUDCA-induced HCO3 -rich hyper- flow and bicarbonate output in corresponding wild-type choleresis is Cftr-dependent, we fed wild-type and Cftr Ϫ Ϫ animals as reported.8 Of note, stimulation of biliary knockout (Cftr / ) mice with norUDCA for 1 week. In Ϫ HCO3 output in wild-type animals was significantly wild-type littermates, norUDCA significantly increased Ϫ higher under norUDCA (241.2 Ϯ 45.3 nmol/g liver HCO3 output (5.2-fold) and bile flow (3.2-fold) (Table Ϫ1 Ϫ1 Ϫ/Ϫ Ϫ weight /minute ) compared with tauro-norUDCA 3). In Cftr mice norUDCA also stimulated HCO3 1976 HALILBASIC ET AL. HEPATOLOGY, June 2009

Table 2. Effect of nor-Bile Acids on Fluid Secretion in IBDUs with the production of less alkaline bile in Cftr-defective ؊ ؊ using HCO3 - Containing and HCO3 -Free Buffer animals. % Increase in norUDCA and tauro-norUDCA Feeding Increases Luminal Area after Variable 30 Minutes n P value Biliary Bile Acid Hydrophilicity. To investigate whether the replacement of hydrophobic (cytotoxic) bile HCO3 buffer Ϫ/Ϫ Control 12 Ϯ 59 acids in phospholipid-depleted bile of Mdr2 mice by UDCA 250 ␮M42Ϯ 13 9 Ͻ0.001* more hydrophilic ones may contribute to the therapeutic norUDCA 250 ␮M65Ϯ 21 33 Ͻ0.001* mechanisms of norUDCA, we next determined the bile Ͻ 0.01‡ Ϫ/Ϫ T-norUDCA 250 ␮M49Ϯ 18 18 Ͻ0.001* acid composition in Mdr2 mice treated with Ͻ0.05† norUDCA and its analogues (Table 4). Bile acid feeding NS‡ did not increase biliary bile acid concentrations compared dinorUDCA 250 ␮M29Ϯ 12 18 Ͻ0.01* Ͻ0.001† with controls. Bile acids were efficiently conjugated with Ϫ/Ϫ Ͻ0.05‡ taurine in untreated Mdr2 mice, as no unconjugated Ϫ HCO3 -free HEPES buffer bile acids were present. In contrast, unconjugated bile Control 15 Ϯ 58 acids were present in all three bile acid–fed groups. In norUDCA 250 ␮M31Ϯ 11 10 Ͻ0.01* T-norUDCA 250 ␮M33Ϯ 78Ͻ0.01* mice receiving norUDCA, about the half of bile acids NS† were present in unconjugated form. In animals receiving dinorUDCA 250 ␮M21Ϯ 12 8 NS* tauro-norUDCA, 14% were not taurine–conjugated, and NS† in animals receiving dinorUDCA only 7% of biliary bile Values are expressed as the mean Ϯ standard deviation. acids were unconjugated. The presence of taurine-conju- Abbreviations: HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; NS, not significant. gated bile acids in mice receiving norUDCA can be ex- *P Ͻ 0.05 versus control. plained by the ability of the ileal transport system to †P Ͻ 0.05 versus norUDCA 250 ␮M. absorb and thereby conserve the small fraction of Ͻ ␮ ‡P 0.05 versus UDCA 250 M. norUDCA that undergoes taurine conjugation. Con- versely, the presence of unconjugated bile acids in mice output, while the increase in bile flow failed to reach receiving tauro-norUDCA results from tauro-norUDCA statistical significance (P ϭ 0.053). Of note, the degree of undergoing deconjugation by intestinal bacteria with ab- Ϫ stimulation of HCO3 output (5.8-fold) and bile flow sorption of norUDCA. The failure of dinorUDCA to alter (3.3-fold) by norUDCA in CftrϪ/Ϫ mice was similar to biliary bile acid composition is remarkable. It suggests that achieved in wild-type mice. In line with these in vivo that dinorUDCA is poorly absorbed by the gastrointesti- findings, norUDCA also stimulated fluid secretion in IB- nal tract. The only major biotransformation of any of the DUs from CftrϪ/Ϫ mice, although to a lower extent com- bile acid was 6␤-hydroxylation to form ␤-muricholic or pared with IBDUs from wild-type animals (data not nor-␤-muricholic acid (nor-␤-MCA). Electrospray ion- shown). Collectively, these finding suggest that ization mass spectrometry could also confirm the excre- Ϫ norUDCA is able to generate a biliary HCO3 secretion tion of minor amounts of nonamidated norUDCA- and also in conditions characterized by defective Cftr. Of nor-␤-MCA-glucuronides and sulfates. interest, the stimulation of glutathione output by norUDCA Is Highly Enriched in Liver and Serum norUDCA in wild-type animals was abrogated in CftrϪ/Ϫ of Mdr2؊/؊ Mice. As shown in Table 4, concentrations mice (Table 3). Furthermore, the amount of bile acid of bile acids in serum and liver were 4 to 5 times higher in output was lower in CftrϪ/Ϫ mice, a finding consistent the norUDCA group compared with controls, whereas

Table 3. Bile Flow and Composition in Wild-Type and Cftr؊/؊ Mice with or without norUDCA Treatment

Wild-Type Cftr؊/؊

Control norUDCA Control norUDCA (5 ؍ n) (3 ؍ n) (4 ؍ n) (4 ؍ Variable (n

Bile ﬂow (␮L/gLWϪ1/minϪ1) 0.8 Ϯ 0.2 2.6 Ϯ 0.6* 0.6 Ϯ 0.4 2.0 Ϯ 0.8 Bile acid output (nmol/gLWϪ1/minϪ1) 17.9 Ϯ 4.7 34.4 Ϯ 9.2 10.9 Ϯ 7.9 18.9 Ϯ 6.0† Bicarbonate output (nmol/gLWϪ1/minϪ1) 24.3 Ϯ 6.0 127.2 Ϯ 40.5* 14.3 Ϯ 6.5 83.2 Ϯ 39.3* Glutathione output (nmol/gLWϪ1/minϪ1) 3.1 Ϯ 0.7 5.3 Ϯ 1.2* 2.5 Ϯ 2.1 2.7 Ϯ 1.2†

Mice were fed a control diet or a diet containing norUDCA for 1 week. Values are expressed as the mean Ϯ standard deviation. *P Ͻ 0.05 wild-type norUDCA versus wild-type control, CftrϪ/Ϫ norUDCA versus CftrϪ/Ϫ control. †P Ͻ 0.05 CftrϪ/Ϫ control versus wild-type control, CftrϪ/Ϫ norUDCA versus wild-type norUDCA. HEPATOLOGY, Vol. 49, No. 6, 2009 HALILBASIC ET AL. 1977

Table 4. Bile Acid Concentration in Serum, Liver, and Bile: Biliary Bile Acid Composition In Mdr2؊/؊ Mice under nor-Bile Acid Treatment

Control norUDCA T-norUDCA dinorUDCA (4 ؍ n) (6 ؍ n) (6 ؍ n) (6 ؍ Variable (n

Serum (␮mol/L) 57 Ϯ 12 313 Ϯ 80* 95 Ϯ 41† 60 Ϯ 10† Liver (nmol/g) 69 Ϯ 27 307 Ϯ 81* 106 Ϯ 40† 109 Ϯ 45† Bile (mmol/L) 4.3 Ϯ 1.4 4.2 Ϯ 2.2 3.8 Ϯ 2.3 2.9 Ϯ 0.5 Biliary bile acids (rel. %) Taurine-amidated Cholic 36.9 Ϯ 0.6 8.1 Ϯ 0.9 12.1 Ϯ 2.1 35.4 Ϯ 0.9 Allocholic Ͻ0.1 Ͻ0.1 Ͻ0.1 2.3 Ϯ 0.9 Chenodeoxycholic 3.9 Ϯ 0.6 Ͻ0.1 Ͻ0.1 3.9 Ϯ 0.3 Ursodeoxycholic 0.6 Ϯ 0.3 0.8 Ϯ 0.4 1.1 Ϯ 0.6 0.6 Ϯ 0.3 ␤-Muricholic 40.2 Ϯ 1.7 4.4 Ϯ 0.7 3.8 Ϯ 0.6 34.0 Ϯ 0.9 ␣-Muricholic 3.4 Ϯ 0.6 1.3 Ϯ 1.1 0.3 Ϯ 0.2 8.4 Ϯ 0.9 ␻-Muricholic 6.2 Ϯ 1.6 0.6 Ϯ 0.4 0.6 Ϯ 0.4 5.7 Ϯ 0.3 norUDCA Ͻ0.1 23.1 Ϯ 1.2 56.2 Ϯ 5.5 Ͻ0.1 nor␤-Muricholic Ͻ0.1 11.0 Ϯ 1.1 11.7 Ϯ 1.9 Ͻ0.1 Glucuronidated, non-amidated nordiol Ͻ0.1 5.6 Ϯ 2.3 2.4 Ϯ 2.1 Ͻ0.1 nortriol Ͻ0.1 3.1 Ϯ 1.5 0.8 Ϯ 0.7 Ͻ0.1 Non-amidated, non-glucuronidated norUDCA Ͻ0.1 13.2 Ϯ 0.9 0.2 Ϯ 1.4 Ͻ0.1 nor␤-Muricholic Ͻ0.1 17.1 Ϯ 2.4 0.4 Ϯ 0.4 Ͻ0.1 norol-one Ͻ0.1 9.0 Ϯ 2.3 8.4 Ϯ 2.7 Ͻ0.1 nordiol-sulfate Ͻ0.1 1.5 Ϯ 1.1 0.4 Ϯ 0.4 Ͻ0.1 dinorUDCA Ͻ0.1 Ͻ0.1 Ͻ0.1 6.4 Ϯ 0.5 dinorol-one Ͻ0.1 Ͻ0.1 Ͻ0.1 0.4 Ϯ 0.3

Control, standard chow-fed animals. T-norUDCA, T-norUDCA-fed animals. Values are expressed as the mean Ϯ standard deviation. Values do not add up to 100% because trace bile acids (1%) are not shown. *P Ͻ 0.05 versus control. †P Ͻ 0.05 versus norUDCA. bile acid levels did not change after tauro-norUDCA and ␣-SMA expression levels (Fig. 1B). Increased hepatic hy- dinorUDCA feeding (Table 4). Similar to bile, 43% to droxyproline content (205 Ϯ 61 ␮g/g liver) in Mdr2Ϫ/Ϫ 50% of liver and serum norUDCA and its metabolites mice, reflecting liver fibrosis, was significantly reduced by were nonamidated. The composition of serum and liver norUDCA treatment (129 Ϯ 25 ␮g/g liver), whereas bile acids in the other groups did not differ significantly tauro-norUDCA and dinorUDCA did not have a signifi- from that found in the bile (data not shown). cant effect on liver hydroxyproline levels (172 Ϯ 47 and norUDCA Is Superior to tauro-norUDCA and di- 187 Ϯ 96 ␮g/g liver, respectively). norUDCA feeding norUDCA in Improving Cholestatic Liver Injury in reversed the ductular reaction, as evidenced by signifi- -Mdr2؊/؊ Mice. We next determined the impact of these cantly lower hepatic K19 protein levels, serving as a spe differential pharmacologic mechanisms on the therapeu- cific cholangiocyte marker (Fig. 2A). On the other hand, tic efficacy of the nor-bile acids on cholestatic liver injury tauro-norUDCA had no effect on the ductular reaction in Mdr2Ϫ/Ϫ mice. norUDCA feeding significantly im- (Fig. 2A), and dinorUDCA increased ductular mass, most proved serum alanine aminotransferase and alkaline phosphatase levels (Table 5) and liver histology (Fig. 1 and Supporting Fig. 2) as described.8 In contrast to Table 5. Effects of nor-Bile Acids on Liver Enzymes in ؊ ؊ norUDCA, the reduction of alanine aminotransferase and Mdr2 / Mice alkaline phosphatase levels in response to tauro- Control norUDCA T-norUDCA dinorUDCA ؍ ؍ ؍ ؍ norUDCA feeding was less pronounced and dinorUDCA Variable (n 10) (n 8) (n 9) (n 7) treatment worsened liver biochemistry (Table 5). Peri- ALT (U/L) 395 Ϯ 101 272 Ϯ 35* 331 Ϯ 100 903 Ϯ 177*,† Ϯ Ϯ Ϯ Ϯ , ductal fibrosis on hematoxlin-eosin and Sirius red stain- AP (U/L) 212 33 170 22* 188 34 940 175* † ing was markedly reduced by norUDCA treatment (Fig. Control, standard chow-fed animals; norUDCA, norUDCA–fed animals; 1A and Supporting Fig. 2); in agreement, ␣-SMA protein T-norUDCA, tauro-norUDCA–fed animals; dinorUDCA, dinorUDCA–fed animals. Values are expressed as the mean Ϯ standard deviation. levels were reduced as well (Fig. 1B). In contrast, tauro- *P Ͻ 0.05 versus control. norUDCA and dinorUDCA had only negligible effects on †P Ͻ 0.05 versus norUDCA. 1978 HALILBASIC ET AL. HEPATOLOGY, June 2009

Fig. 1. norUDCA is more effective than tauro-norUDCA and dinorUDCA in reducing biliary fibrosis in Mdr2Ϫ/Ϫ mice. (A) Sirius red staining of liver in standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2Ϫ/Ϫ mice. Compared with striking reduction of fibrosis with periductal collagen fibers (red) in norUDCA-treated Mdr2Ϫ/Ϫ mice, the effects of tauro-norUDCA and dinorUDCA were much weaker (magnification ϫ20). bd, bile duct; pv, portal vein. (B) Western blot analysis of hepatic ␣-SMA protein levels (marker for activated myofibroblasts) in liver homogenates of standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2Ϫ/Ϫ mice. Densitometry data are expressed as n-fold change relative to standard chow-fed Mdr2Ϫ/Ϫ mice. Values are expressed as the mean Ϯ standard deviation. Note a significant decrease in ␣-SMA protein levels by norUDCA treatment. *P Ͻ 0.05 versus control.

Fig. 2. norUDCA, but not tauro-norUDCA and dinorUDCA, reduce ductular proliferation in Mdr2Ϫ/Ϫ mice. (A) Immunohistochemistry of cholangiocytes using an anti-K19 antibody in standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2Ϫ/Ϫ mice (magnification ϫ20). Ductular proliferation in standard chow-fed Mdr2Ϫ/Ϫ mice was reduced by norUDCA and remained unchanged after tauro-norUDCA. dinorUDCA even increased ductular proliferation in Mdr2Ϫ/Ϫ mice. (B) Western blot analysis of hepatic K19 protein levels in liver homogenates of standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2Ϫ/Ϫ mice. Densitometry data are expressed as n-fold change relative to standard chow-fed Mdr2Ϫ/Ϫ mice. Values are expressed as the mean Ϯ standard deviation. Note the significant decrease in K19 protein levels by norUDCA and significant increase by dinorUDCA. *P Ͻ 0.05 versus control. #P Ͻ 0.05 versus norUDCA. HEPATOLOGY, Vol. 49, No. 6, 2009 HALILBASIC ET AL. 1979

Fig. 3. Effects of nor-bile acids on hepatic messenger RNA expression of phase I and II detoxification enzymes and alternative bile acid transporter. Relative messenger RNA expression of Cyp2b10, Sult2a1, and Mrp4 of standard chow-fed (control), norUDCA-fed, tauro-norUDCA-fed (T-norUDCA), and dinorUDCA-fed Mdr2Ϫ/Ϫ mice. Values are expressed as n-fold change compared with the control group (mean Ϯ standard deviation of five animals). norUDCA and dinorUDCA induced both detoxification enzymes Cyp2b10 and Sult2a1 as well as alternative export pump Mrp4. The effects of T-norUDCA were less pronounced. *P Ͻ 0.05 versus control. #P Ͻ 0.05 versus norUDCA. likely as a result of aggravation of liver injury. Western dinorUDCA) resulted in a marked reduction or even blotting for K19 (Fig. 2B) demonstrated a significantly complete abrogation of its therapeutic properties, respec- reduced expression by norUDCA, no effects by tauro- tively. Our data indicate that the effectiveness of norUDCA, and an increase in K19 protein mass by di- norUDCA in comparison with the other UDCA deriva- norUDCA (Fig. 2B). tives correlates with its ability to induce a hydrophilic, Ϫ Effects of norUDCA and Its Side Chain Derivatives HCO3 -rich choleresis of both canalicular and ductal or- on Bile Acid Detoxification and Alternative Export. igin that may flush the injured bile ducts.8 Previous find- To study the possible role of induction of bile acid detox- ings by Yoon et. al. on the choleretic properties of ifying enzymes and adaptive transporter expression, we norUDCA in three rodent species10 indicate that determined messenger RNA expression levels of key phase norUDCA may undergo cholehepatic shunting. This I (Cyp2b10) and phase II (Sult2a1) detoxifying enzymes multistep process includes reabsorption of the unconju- as well as phase III export system Mrp4, because these gated protonated, lipophilic bile acid by cholangiocytes, gene products have been identified as key targets of and transport into the periductal capillary plexus, which norUDCA.8 Cyp2b10, Sult2a1, and Mrp4 were highly drains into sinusoids leading to hepatocellular reuptake up-regulated in response to norUDCA treatment (Fig. 3), and canalicular re-excretion into bile. A key physicochem- whereas induction by tauro-norUDCA was less pronounced. In contrast to its toxic effects on cholestatic liver ical property of bile acids that undergo cholehepatic injury, dinorUDCA feeding resulted in a robust increase shunting is that they are weak organic acids that are easily of Cyp2b10, Sult2a1, and Mrp4 (Fig. 3). protonated and are able to cross cell membranes rapidly, presumably by a passive mechanism. Taurine conjugation Discussion results in a bile acid being present as a charged anion that can only undergo cholehepatic shunting via apical sodi- norUDCA has been shown to cure liver injury in um-dependent bile acid transporter Asbt,25 as taurine Mdr2Ϫ/Ϫ mice.8 Comparing different norUDCA ana- conjugation renders bile acids membrane-impermeable, logues, we now show that the therapeutic properties of thus limiting the HCO Ϫ generation in bile. Cholehe- norUDCA in the Mdr2Ϫ/Ϫ mouse model for sclerosing 3 patic shunting of bile acids may therefore ensue when cholangitis and portal fibrosis are unique and not shared taurine stores are depleted or conjugation-resistant bile by its analogues tauro-norUDCA and dinorUDCA. Most 10 importantly, we demonstrate that the unique pharmaco- acids are administered. In our experimental conditions, dynamic effects of norUDCA depend on its relative resis- the concept of cholehepatic shunting is further supported tance to taurine conjugation, its ability to undergo by the abundance of unconjugated norUDCA in the liver. cholehepatic shunting with stimulation of canalicular bile These data therefore underline the concept that the ther- flow, and its direct stimulation of cholangiocyte secretion. apeutic superiority of norUDCA may indeed be related to The therapeutic effects of norUDCA are highly spe- its relative resistance to conjugation, as well as its capacity cific. Conjugation with taurine (resulting in tauro- to undergo cholehepatic shunting and to stimulate cana- Ϫ norUDCA), as well as further side chain shortening (i.e., licular bile flow and HCO3 -rich hypercholeresis. 1980 HALILBASIC ET AL. HEPATOLOGY, June 2009

As shown previously, UDCA, the parent compound of potential detrimental norUDCA effects in the context of norUDCA, directly stimulates fluid secretion in isolated frank biliary obstruction. cholangiocytes through a Cftr-dependent secretion of The effects of different nor-bile acids on liver fibrosis ATP that, in turn, interacts with apical purinergic recep- were closely linked to their effects on ductular reaction, ϩ Ϫ tors, which then stimulate Ca2 -dependent Cl channels which has been postulated to trigger biliary fibrosis in Ϫ Ϫ to secrete Cl . The secreted Cl is subsequently ex- mice and humans.22,26,27 Also in our studies the amount Ϫ changed with HCO3 by the apical anion exchanger of liver fibrosis in treated animals correlated with the AE2.12 To study whether norUDCA has direct choleretic amount of ductular reaction. norUDCA reduced both effects on cholangiocytes, we measured the secretory ef- ductular proliferation and biliary fibrosis. Although fects of the three bile acids by quantifying luminal expan- norUDCA reversed only the pathologically enhanced sion in IBDUs. In line with the choleretic effect observed ductular hyperproliferation in Mdr2Ϫ/Ϫ mice and never in vivo, norUDCA had the strongest effect on ductular induced ductopenia in wild-type animals, we cannot def- secretion in IBDUs. Secretory effects of nor-bile acids Ϫ initely rule out the theoretical risk for development of were for the most part HCO3 -dependent. Incubation of Ϫ ductopenia in humans with vanishing bile duct syn- IBDUs with a HCO3 -free buffer reduced norUDCA- dromes. Future clinical pilot trials should therefore con- stimulated fluid secretion by more than half, by one third sider only patients without advanced ductopenia. In for tauro-norUDCA and minimally for dinorUDCA. In- Ϫ/Ϫ Ϫ comparison to norUDCA, dinorUDCA-treated Mdr2 vestigating the mechanisms of these additional HCO3 - mice showed a brisk induction of the ductular reaction independent mechanisms was, however, beyond the that likely reflects a regenerative response to increased scope of the present study. Ϫ/Ϫ liver injury, as suggested by significantly increased serum Further experiments with norUDCA in Cftr mice Ϫ/Ϫ Ϫ aminotransferases. In contrast to the findings in Mdr2 now indicate that norUDCA-induced HCO3 secretion mice, wild-type mice fed with the same dosage of di- is partly Cftr-independent. This stimulation of biliary norUDCA with similar hepatic enrichment had induced HCO Ϫ excretion by norUDCA via an at least partly 3 bile flow, but did not show any alterations in liver en- Cftr-independent mechanism should encourage the use zymes or liver histology excluding a general toxic effect of norUDCA as a potential therapeutic strategy in cystic (data not shown). fibrosis-associated hepatobiliary disease. Reduced bile Ϫ Ϫ Bile acids are able to directly stimulate their metabo- acid output in norUDCA-treated Cftr / mice is likely lism and export by the induction of hepatic phase I and due to the lower biliary pH that promotes protonation phase II detoxification systems as well as by induction of and reabsorption of norUDCA. This observation also 28 provides an explanation as to why hypercholeretic bile basolateral and apical bile acid transporters. These Ϫ mechanisms, promoting detoxification of bile acids to acids directly stimulate HCO3 secretion in cholangiocytes. It is attractive to speculate that the mechanisms more water-soluble derivatives and facilitating their excre- could have evolved to improve the biliary disposal of po- tion via the kidney, could counteract the effects of cho- 28 8 tentially toxic weak organic acid compounds that other- lestasis and liver injury. In line with a previous study, wise would continue to recirculate in the liver. In fact, norUDCA feeding resulted in a robust induction of phase Ϫ I and phase II detoxification enzymes, explaining the in- HCO3 secretion by bile ducts would be predicted to reduce their protonation and increase the biliary disposal creased appearance of polyhydroxylated, sulfated, and of cholephiles. glucuronidated norUDCA metabolites in bile, serum, and 8,9 The unique combination of cholehepatic shunting urine in mice and in humans. Similar biotransforma- (which induces canalicular bile flow) and direct induction tion patterns seem to be common to other nor-bile acids as Ϫ 8 of a HCO3 -dependent secretion from cholangiocytes shown previously for norchenodeoxycholic and dinorch- may exert a flushing effect on the injured biliary tree, enodeoxycholic acid, both being amidation-resistant and likely removing and inactivating toxic compounds. In glucuronidated in the hamster and the rat.29 Taurine con- contrast to the beneficial choleretic effects of norUDCA, jugation abrogated the effects of norUDCA on induction UDCA-induced choleresis has been shown to result in of Cyp2b10, Sult2a1, and the basolateral export pump disruption of cholangioles and aggravation of liver injury Mrp4. Astonishingly, dinorUDCA effects on expression in the Mdr2Ϫ/Ϫ model for sclerosing cholangitis.4 The of detoxifying and export systems were comparable with Ϫ Ϫ underlying mechanism for these puzzling differences re- those of norUDCA, despite its toxic effect in Mdr2 / mains to be resolved, but could include differences in the mice. Therefore, the observed induction of detoxifying capability of directly stimulating ductular secretion. Nev- and alternative export systems is not likely to represent a ertheless, future clinical trials should take into account central therapeutic mechanism, and could rather support HEPATOLOGY, Vol. 49, No. 6, 2009 HALILBASIC ET AL. 1981 a secondary response to injury or to the accumulation of acids: hepatic transport and effect on biliary secretion of 23-nor-ursode- nor-bile acids. oxycholate in rodents. Gastroenterology 1986;90:837-852. 11. Strazzabosco M, Sakisaka S, Hayakawa T, Boyer JL. Effect of UDCA on In conclusion, this comprehensive analysis of the dif- intracellular and biliary pH in isolated rat hepatocyte couplets and perfused ferential physiologic and therapeutic mechanisms of livers. Am J Physiol 1991;260:G58-G69. norUDCA and its derivatives tauro-norUDCA and di- 12. Fiorotto R, Spirli C, Fabris L, Cadamuro M, Okolicsanyi L, Strazzabosco M. Ursodeoxycholic acid stimulates cholangiocyte fluid secretion in mice norUDCA clearly demonstrates the therapeutic superior- via CFTR-dependent ATP secretion. Gastroenterology 2007;133:1603- ity of norUDCA in an animal model for sclerosing 1613. cholangitis and biliary fibrosis (Supporting Table 1). The 13. Snouwaert JN, Brigman KK, Latour AM, Malouf NN, Boucher RC, differential effects of the various nor-bile acids depend on Smithies O, et al. An animal model for cystic fibrosis made by gene target- ing. Science 1992;257:1083-1088. specific properties that affect their ability to undergo 14. Schteingart CD, Hofmann AF. Synthesis of 24-nor-5 beta-cholan-23-oic cholehepatic shunting, thereby stimulating canalicular acid derivatives: a convenient and efficient one-carbon degradation of the bile flow as well as by directly stimulating fluid secretion side chain of natural bile acids. J Lipid Res 1988;29:1387-1395. 15. Tserng KY, Hachey DL, Klein PD. An improved procedure for the syn- by cholangiocytes. These data may have high relevance for thesis of glycine and taurine conjugates of bile acids. J Lipid Res 1977;18: the design of novel pharmacologic treatments for cholan- 404-407. giopathies and liver diseases in general. 16. Huffman JW, Sobti RR. Synthetic approaches to steroidal alkaloids II (1). Preparation and reactions of 12-oxo-dinorcholanic acids. Steroids 1970; Acknowledgment: The authors thank Dr. W. Erwa 16:755-770. (Graz, Austria) and colleagues for performing liver func- 17. Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Pojer C, Zenz R, et al. tion tests. The authors also thank Dr. T. Claudel (Graz, Effects of ursodeoxycholic and cholic acid feeding on hepatocellular transporter expression in mouse liver. Gastroenterology 2001;121:170-183. Austria) for stimulating discussions and helpful sugges- 18. Spirli C, Nathanson MH, Fiorotto R, Duner E, Denson LA, Sanz JM, et al. tions and P. De and P. Komala (Bangalore, India) for Proinflammatory cytokines inhibit secretion in rat bile duct epithelium. synthesizing the UDCA derivatives. The rat anti-Troma- Gastroenterology 2001;121:156-169. III antibody developed by Dr. R. Kemler, Max-Planck 19. Spirli C, Fabris L, Duner E, Fiorotto R, Ballardini G, Roskams T, et al. Institute, Freiburg, Germany, was obtained from the De- Cytokine-stimulated nitric oxide production inhibits adenylyl cyclase and velopmental Studies Hybridoma Bank, developed under cAMP-dependent secretion in cholangiocytes. Gastroenterology 2003;124:737-753. the auspices of the NICHID, and maintained by the Uni- 20. Mennone A, Alvaro D, Cho W, Boyer JL. Isolation of small polarized bile versity of Iowa (Iowa City, IA). duct units. Proc Natl Acad SciUSA1995;92:6527-6531. 21. Wagner M, Fickert P, Zollner G, Fuchsbichler A, Silbert D, Tsybrovskyy References O, et al. Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice. Gastroenterol- 1. Ishibashi H, Komori A, Shimoda S, Gershwin ME. Guidelines for therapy ogy 2003;125:825-838. of autoimmune liver disease. Semin Liver Dis 2007;27:214-226. 22. Fickert P, Stoger U, Fuchsbichler A, Moustafa T, Marschall HU, Weiglein 2. Lazaridis KN, Strazzabosco M, Larusso NF. The cholangiopathies: disor- AH, et al. A new xenobiotic-induced mouse model of sclerosing cholangitis ders of biliary epithelia. Gastroenterology 2004;127:1565-1577. and biliary fibrosis. Am J Pathol 2007;171:525-536. 3. Fickert P, Zollner G, Fuchsbichler A, Stumptner C, Weiglein AH, Lam- 23. Trauner M, Arrese M, Soroka CJ, Ananthanarayanan M, Koeppel TA, mert F, et al. Ursodeoxycholic acid aggravates bile infarcts in bile duct- Schlosser SF, et al. The rat canalicular conjugate export pump (Mrp2) is ligated and Mdr2 knockout mice via disruption of cholangioles. down-regulated in intrahepatic and obstructive cholestasis. Gastroenterol- Gastroenterology 2002;123:1238-1251. ogy 1997;113:255-264. 4. Fickert P, Fuchsbichler A, Wagner M, Zollner G, Kaser A, Tilg H, et al. 24. Jamall IS, Finelli VN, Que Hee SS. A simple method to determine nano- Regurgitation of bile acids from leaky bile ducts causes sclerosing cholangitis in Mdr2 (Abcb4) knockout mice. Gastroenterology 2004;127:261- gram levels of 4-hydroxyproline in biological tissues. Anal Biochem 1981; 274. 112:70-75. 5. Trauner M, Fickert P, Wagner M. MDR3 (ABCB4) defects: a paradigm 25. Alpini G, Glaser S, Baiocchi L, Francis H, Xia X, Lesage G. Secretin ϩ for the genetics of adult cholestatic syndromes. Semin Liver Dis 2007;27: activation of the apical Na -dependent bile acid transporter is associated 77-98. with cholehepatic shunting in rats. HEPATOLOGY 2005;41:1037-1045. 6. Melum E, Boberg KM, Franke A, Bergquist A, Hampe J, Schreiber S, et al. 26. Sanchez-Munoz D, Castellano-Megias VM, Romero-Gomez M. Expres- Variation in the MDR3 gene influences disease progression in PSC pa- sion of bcl-2 in ductular proliferation is related to periportal hepatic stellate tients and disease susceptibility in epistatic interaction with polymorphism cell activation and fibrosis progression in patients with autoimmune cho- in OST-alpha gene. HEPATOLOGY 2007;46:265A. lestasis. Dig Liver Dis 2007;39:262-266. 7. Ziol M, Barbu V, Rosmorduc O, Frassati-Biaggi A, Barget N, Hermelin B, 27. Clouston AD, Powell EE, Walsh MJ, Richardson MM, Demetris AJ, et al. ABCB4 heterozygous gene mutations associated with fibrosing cho- Jonsson JR. Fibrosis correlates with a ductular reaction in hepatitis C: roles lestatic liver disease in adults. Gastroenterology 2008;135:131-141. of impaired replication, progenitor cells and steatosis. HEPATOLOGY 2005; 8. Fickert P, Wagner M, Marschall HU, Fuchsbichler A, Zollner G, 41:809-818. Tsybrovskyy O, et al. 24-norUrsodeoxycholic acid is superior to ursode- 28. Zollner G, Marschall HU, Wagner M, Trauner M. Role of nuclear recep- oxycholic acid in the treatment of sclerosing cholangitis in Mdr2 (Abcb4) tors in the adaptive response to bile acids and cholestasis: pathogenetic and knockout mice. Gastroenterology 2006;130:465-481. therapeutic considerations. Mol Pharm 2006;3:231-251. 9. Hofmann AF, Zakko SF, Lira M, Clerici C, Hagey LR, Lambert KK, et al. 29. Yeh HZ, Schteingart CD, Hagey LR, Ton-Nu HT, Bolder U, Gavrilkina Novel biotransformation and physiological properties of norursodeoxy- MA, et al. Effect of side chain length on biotransformation, hepatic trans- cholic acid in humans. HEPATOLOGY 2005;42:1391-1398. port, and choleretic properties of chenodeoxycholyl homologues in the 10. Yoon YB, Hagey LR, Hofmann AF, Gurantz D, Michelotti EL, Steinbach rodent: studies with dinorchenodeoxycholic acid, norchenodeoxycholic JH. Effect of side-chain shortening on the physiologic properties of bile acid, and chenodeoxycholic acid. HEPATOLOGY 1997;26:374-385.