Effects of Ursodeoxycholic and Cholic Acid Feeding on Hepatocellular Transporter Expression in Mouse...

Effects of Ursodeoxycholic and Cholic Acid Feeding onHepatocellular Transporter Expression in Mouse Liver

PETER FICKERT,* GERNOT ZOLLNER,* ANDREA FUCHSBICHLER,‡ CONNY STUMPTNER,‡

CHRISTINE POJER,* RAINER ZENZ,‡ FRANK LAMMERT,§ BRUNO STIEGER,i PETER J. MEIER,i

KURT ZATLOUKAL,‡ HELMUT DENK,‡ and MICHAEL TRAUNER**Departments of Medicine and ‡Pathology, Karl-Franzens University, Graz, Austria; §Department of Medicine, University of Aachen, Aachen,Germany; iDivision of Clinical Pharmacology and Toxicology, Department of Medicine, University Hospital Zurich, Zurich, Switzerland

Background & Aims: Cholestasis is associated with re-tention of potentially toxic bile acids and alterations inhepatocellular transporter expression. Conversely, non-toxic ursodeoxycholic acid (UDCA) stimulates bile secre-tion and counteracts cholestasis. This study aimed todetermine the effects of UDCA and cholic acid (CA) onthe expression of hepatocellular transporters for bileacids (Ntcp, Bsep), organic anions (Oatp1, Mrp2), or-ganic cations (Mdr1a/b), and phospholipids (Mdr2) inmouse liver. Methods: Bile flow/composition was ana-lyzed in UDCA- or CA-fed mice. Transporter expressionwas studied by reverse-transcription polymerase chainreaction, Western blotting, and immunofluorescence mi-croscopy. Results: UDCA had no effect on basolateralNtcp and down-regulated Oatp1, whereas canalicularBsep and Mrp2 were up-regulated. CA down-regulatedbasolateral Ntcp and Oatp1, whereas canalicular Bsep,Mrp2, and Mdr1a/b were up-regulated. Neither UDCAnor CA affected Mdr2 expression. Both UDCA and CAstimulated biliary bile acid and glutathione excretion,although only CA increased phospholipid and choles-terol excretion. Conclusions: Down-regulation of basolat-eral and up-regulation of canalicular transporters in re-sponse to CA may represent a defense mechanism, inan attempt to prevent hepatocellular accumulation ofpotentially toxic bile acids. The therapeutic effects ofUDCA may be caused in part by stimulation of canalic-ular transporter expression in the absence of hepatocel-lular toxicity.

Cholestasis results in systemic and intrahepatic reten-tion of potentially toxic bile acids that can cause

liver injury, ultimately leading to biliary fibrosis andcirrhosis.1,2 Down-regulation of hepatocellular transportsystems may contribute to impaired hepatobiliary excre-tion of bile acids and other biliary constituents (e.g.,conjugated bilirubin, glutathione) during cholestasis.3,4

Bile acids regulate the expression of various genes, in-cluding those involved in the synthesis and intestinaltransport of bile acids such as cholesterol 7a-hydroxy-

lase, intestinal bile acid–binding protein, and ileal so-dium-dependent bile acid transporter.5–8 However, it isunknown whether bile acids accumulating during cho-lestasis contribute to the changes in hepatocellular trans-porter expression. Such information may be relevant forthe development of new rational strategies for the treat-ment of cholestatic liver disorders.

In contrast to potentially toxic bile acids, nontoxic,hydrophilic ursodeoxycholic acid (UDCA) is used to treata variety of cholestatic liver diseases, including primarybiliary cirrhosis (PBC), primary sclerosing cholangitis,cystic fibrosis, and intrahepatic cholestasis of preg-nancy.2,9,10 The therapeutic benefit of UDCA in thetreatment of cholestasis may result from a combination ofcytoprotective, antiapoptotic, immunomodulatory, andcholeretic effects.1,2,9,10 UDCA stimulates hepatobiliaryexcretory function and, thereby, counteracts cholestasisin several ways, including stimulation of vesicular exo-cytosis and insertion of hepatobiliary transporters intothe canalicular membrane.2,10–14 In addition, the anti-cholestatic effects of UDCA could in part be mediated bystimulation of gene expression of transport systems thatare down-regulated in cholestatic liver diseases, as sug-gested by stimulation of Cl2/HCO3

2 exchanger expres-sion and function in UDCA-treated PBC patients.15–17

However, the influence of UDCA on the expression ofother hepatocellular transporters remains unclear.

Abbreviations used in this paper: AP, alkaline phosphatase; Bsep,bile salt export pump; CA, cholic acid; FITC, fluorescein isothiocyanate;GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSH, glutathi-one; Lst-1, liver-specific organic anion transporter; Mdr1, multidrugexport pump; Mdr2, phospholipid flippase; Mrp2, conjugate exportpump; Ntcp, Na1/taurocholate cotransporter; Oatp, organic aniontransporting polypeptide; PBC, primary biliary cirrhosis; PCR, polymer-ase chain reaction; Pgp, P-glycoprotein; RT-PCR, reverse-transcriptionpolymerase chain reaction; TRITC, tetramethylrhodamine isothiocyanate.

© 2001 by the American Gastroenterological Association0016-5085/01/$35.00

doi:10.1053/gast.2001.25542

GASTROENTEROLOGY 2001;121:170–183

Therefore, the present study aimed to determine theeffects of nontoxic UDCA and potentially toxic cholicacid (CA) on messenger RNA (mRNA) and proteinexpression of 7 major hepatocellular transport systems.This information could provide novel insights into thetherapeutic mechanisms of UDCA and the influence ofbile acids on transporter expression in cholestasis.

Materials and MethodsAnimals

Male Swiss Albino mice (strain Him OF1 SPF; 25–30g body weight), obtained from the Institute of LaboratoryAnimal Research, University of Vienna, School of Medicine(Himberg, Austria), were housed with a 12:12-hour light-darkcycle and permitted ad libitum consumption of water and astandard mouse diet (Marek, Vienna, Austria). The experimen-tal protocols were approved by the local Animal Care and UseCommittee, according to criteria outlined in the Guide for theCare and Use of Laboratory Animals prepared by the NationalAcademy of Sciences, as published by the National Institutesof Health (NIH publication 86-23, revised 1985).

Materials

UDCA was kindly provided by the Falk Foundation(Freiburg, Germany). CA was obtained from Aldrich (Stein-heim, Germany). The following reagents were used: AMVreverse transcriptase, Klenow polymerase, Sp6 and T7 RNApolymerase and restriction enzymes (Boehringer Mannheim,Mannheim, Germany); AmpliTaq DNA Polymerase (Perkin-Elmer, Branchburg, NJ); Prime RNase Inhibitor (5 Prime33Prime Inc., Boulder, CO); deoxynucleoside triphosphates(Pharmacia, Uppsala, Sweden); pCR II vector and One Shotcompetent cells (Invitrogen, Groningen, The Netherlands);Quantum Prep Plasmid Miniprep and Maxiprep kits (Bio-RadLaboratories, Hercules, CA). All other chemicals used were ofhighest purity commercially available and purchased fromSigma (Steinheim, Germany).

Bile Acid Feeding

To study the effects of bile acids on hepatocellulartransporter expression, mice were fed a diet supplemented with0.5% or 1% (wt/wt) UDCA or CA for 7 days.18,19 Controlswere fed a standard mouse diet for 7 days. The livers wereexcised in general anesthesia with avertin (10 mg, intraperi-toneal), and aliquots were snap-frozen in liquid nitrogen (formolecular analysis and immunofluorescence) or fixed in 4%neutral buffered formaldehyde solution (for hematoxylin-eosinstaining) or 2.5% glutaraldehyde (for electron microscopy).

Routine Serum Biochemistry and Bile AcidMeasurements

Serum samples were stored at 270°C until analysis ofaspartate aminotransferase (AST), alanine aminotransferase(ALT), and alkaline phosphatase (AP) by routine clinical

chemistry testing performed on a Hitachi 717 analyzer (Boehr-inger Mannheim). For determination of total serum bile acidlevels, a commercially 3a-hydroxysteroid dehydrogenase assay(Merck, Darmstadt, Germany) was used.19 Tests were per-formed in duplicates.

Liver Histology

For conventional light microscopy, livers were fixed in4% neutral buffered formaldehyde and embedded in paraffin.Sections were cut from paraffin blocks and stained with hema-toxylin and eosin. The sections were coded and examined by apathologist (H.D.), who was blinded to the animals’ treat-ment.

Electron Microscopy

For electron microscopy, liver tissue was fixed in 2.5%glutaraldehyde in 0.1 mol/L cacodylate buffer, pH 7.3. Afterpostfixation in 1% OsO4, the tissue was dehydrated andembedded in Agar-100 resin (Agar Scientific, Essex, UK).Fifty-nanometer sections were contrasted with uranyl acetateand lead citrate and studied in a CM 100 electron microscope(Philips, Eindhoven, Netherlands). Morphometrical analysis ofthe canalicular cross-sectional luminal area was performed onimages recorded with a video camera (Gatan Wide Angle TVSystem; Munster, Germany) attached to the CM 100 electronmicroscope using AnalySiS software (Soft-Imaging, Munster,Germany).

Bile Flow Measurements and Analyses

To investigate functional implications of potentialchanges in transporter expression, bile flow and compositionwere analyzed in mice fed a UDCA- or CA- (1%, 7 days)supplemented diet or control diet (n 5 5 for each group). Micethat were fasted overnight but had free access to water wereanesthetized (10 mg avertin, intraperitoneal), and the gallblad-der was cannulated after common bile duct ligation for col-lection of bile as described previously.20 After a 5-minuteequilibration period, bile was collected in pretared tubes over1 hour, and bile flow was determined gravimetrically assuminga density of 1.0 g/mL. All samples were frozen and stored at220°C until analysis of biliary bile acid, phospholipid, cho-lesterol, and glutathione (GSH) concentrations, as describedpreviously.18,21 In addition, biliary bile acid composition wasassessed by high pressure liquid chromatography.21,22

RNA Extraction

Isolation of total RNA was performed according to aprocedure described by Krieg et al.23 RNA was quantifiedspectrophotometrically at 260 nm, and the quality of totalRNA was controlled by denaturing formaldehyde agarose gelelectrophoresis.

Determination of mRNA Copy Numbers

Steady-state mRNA levels of hepatobiliary trans-port systems and glyceraldehyde-3-phosphate dehydrogenase(GAPDH) were assessed by competitive reverse-transcription

July 2001 BILE ACIDS AND HEPATOCELLULAR TRANSPORTERS 171

polymerase chain reaction (RT-PCR) using internal RNA stan-dards with mutated restriction sites as described.24 This methodwas cross-validated with quantitative Northern blotting and hasbeen successfully applied to quantification of several liver mRNAtranscripts.24 In brief, defined amounts of RNA standards (differ-ing from the wild-type sequence only in a few bases and thusintroducing a new restriction site) and total wild-type RNA werereverse cotranscribed into complementary DNA (cDNA) in thesame reaction vial. An aliquot of the cDNA reaction product(containing both standard and wild-type cDNAs) was amplifiedby polymerase chain reaction (PCR). After a restriction digest,PCR products were separated by agarose gel electrophoresis,which allowed distinction of standard and wild-type derived PCRproducts. After video densitometry with an Intas Digit-Storesystem (Gottingen, Germany) and RFLP-Scan software (Scanalyt-ics, Billerica, MA), the mRNA copy number was calculated bycomparison of standard and wild-type band intensities. Correc-tions for relative band sizes and heterodimer formation wereperformed as described.24 In contrast to conventional RT-PCRtechniques, the use of internal RNA standards allows to controlfor potential variations in reverse transcription and PCR ampli-fication efficiencies.25

Construction of internal RNA standards. Internalhomologous RNA standards with mutated restriction siteswere generated for the following hepatobiliary transport sys-tems (Table 1) by introducing a new restriction site using themegaprimer PCR technique: Na1/taurocholate cotransporter(Ntcp), organic anion transporting polypeptide 1 (Oatp1), andOatp4 also known as the liver-specific organic anion trans-porter (Lst-1), bile salt export pump (Bsep), multidrug exportpumps 1a and 1b (Mdr1a and Mdr1b), phospholipid flippase(Mdr2), and the conjugate export pump (Mrp2).25–27 Thestandard for GAPDH was generated by loop-out mutagenesis

resulting in a deletion of 30 base pairs.28 Mutated PCRproducts were cloned into pCRII vectors (Invitrogen), andtheir successful mutagenesis was checked by restriction digestand sequencing with an Abi Prism automatic sequencer (Per-kin-Elmer). To obtain RNA standards, the cloned DNA stan-dards were linearized and transcribed into sense RNA in vitrousing Sp6 or T7 RNA polymerase (Boehringer Mannheim)following a standard protocol.28

Reverse transcription and competitive PCR. Re-verse transcription of wild-type and standard RNA was per-formed simultaneously in one tube for all transporters andGAPDH following the protocol of the cDNA Cycle Kit forRT-PCR (Invitrogen) using 200 ng total RNA, defined copynumbers of standard RNAs for 7 hepatobiliary transportersand GAPDH in a 20-mL reaction mixture, containing 1mmol/L specific downstream primers (Table 1), 10 U AMVreverse transcriptase (Boehringer Mannheim), 13 RT buffer(50 mmol/L Tris-HCl, 8 mmol/L MgCl2 , 30 mmol/L KCl, 1mmol/L dithiothreitol, pH 8.5), 4 mmol/L sodium pyrophos-phate, 15 U PRIME RNase Inhibitor (5 Prime 3 3 PrimeInc.), and 1 mmol/L of each deoxynucleoside triphosphate(Pharmacia). Total RNA, RNA standards, and primers weremixed, heated to 65°C for 10 minutes, and quick-chilled onice before the other components were added. Reverse transcrip-tion was performed for 60 minutes at 42°C, followed byheating to 94°C for 2 minutes and chilling on ice. PCR wasperformed using one tenth of the cDNA products in a 50 mLreaction mixture containing 13 PCR buffer (60 mmol/LTrisHCl, 15 mmol/L (NH4)2SO4, 1.5 mmol/L MgCl2 , pH 8.5,for Ntcp, Oatp1, Oatp4, Bsep, and Mdr2; 60 mmol/LTrisHCl, 15 mmol/L (NH4)2SO4, 2 mmol/L MgCl2 , pH 9, forMdr1a, Mdr1b, Mrp2, and GAPDH), 0.25 mmol/L of eachdeoxynucleoside triphosphate (Pharmacia), 1 mmol/L of each

Table 1. Primers for Competitive RT-PCR and Generation of Internal RNA Standards With Mutated Restriction Site

Gene name(gene symbol)

Upperprimer

Lowerprimer

Restrictionsite Mutagenic primer

ReferenceAccession Nr

Ntcp 290–307 905–886 BamHI (470) 462–479; 59-ACAGCAAAGuGATCCTACG-39 Cattori et al.59

(Scl10a1) U95131Oatp1 1320–1341 1917–1900 XbaI (1728) 1732–1711; 59-GGCTTuCTAGAGAGAAGATGAAT-39 Hagenbuch et al.60

(Scl21a1) NM013797Oatp4 (Lst-1) 1419–1438 2013–1994 XbaI (1603) 1612–1593; 59-TTTTCCCCTuCTAGACAGGTTC-39 Ogura et al.61

(Scl21a6) AB031959Bsep 2094–2115 2517–2498 XbaI (2302) 2308–2289; 59-TCAACTTuCTAGAACAAGCAC-39 Green et al.62

(Abcb11) AF133903Mdr1a 2726–2749 3206–3184 XbaI (2969) 2979–2956; 59-GAAAAATACATuCTAGACCTGGGTG-39 Hsu et al.63

(Abcb1a) M33581Mdr1b 1834–1857 2397–2374 HindIII (2193) 2200–2180; 59-CCGCCAAAuAGCTTACCAGAGG-39 Gros et al.64

(Abcb1b) M14757Mdr2 990–1007 1405–1382 SacI (1213) 1225–1202; 59-CACACTGAGAGCTuCCGATGAGGAT-39 Gros et al.65

(Abcb4) J03398Mrp2 3661–3680 4478–4455 XbaI (4022) 4013–4036; 59-ATTCATCCCTuCTAGAACTGCCTCT-39 Fritz et al.66

(Abcc2) AF227274GAPDH 428–445 551–535 30-bp deletion Sabath et al.67

M32599

NOTE. Base pair numbering corresponds to published cDNA or mRNA sequences. Restriction enzymes and positions of restriction sites mutatedfor generation of standards. Mutagenic primers with exchanged nucleotides (written in bold letters) and position u of restriction site.bp, base pair.

172 FICKERT ET AL. GASTROENTEROLOGY Vol. 121, No. 1

primer, and 5 mL dimethyl sulfoxide. The vials were heated to94°C, then AmpliTaq DNA polymerase (Perkin-Elmer) wasadded, and subsequently cycled in an OmniGene system (Hy-baid, Middle-Essex, UK) for 45 cycles at 94°C, 1 minute,60°C (for Oatp1, Oatp4, Mrp2) or 55°C (for Ntcp, Bsep,Mdr1a, Mdr1b, Mdr2, GAPDH), 1 minute, and 72°C, 1minute. The program ended with a final extension step at72°C for 4 minutes. Control experiments were conducted toexclude contamination by genomic DNA or PCR products.

Specificity was confirmed by the product of predicted size andby sequence analysis and comparison with published sequences.To distinguish standard and wild-type amplicons, equal amountsof PCR products were digested with the corresponding restrictionenzyme (Table 1) for at least 4 hours, and restriction fragmentswere run on ethidium bromide–stained 2% agarose gels.

Preparation of Liver Membranes

Liver membranes were prepared as described previ-ously with a minor modification (single 100,000g centrifuga-tion step of gauze-filtered liver homogenates).29 Protein con-centrations in whole liver homogenates and the obtained livermembranes were determined using a Bradford Kit (Bio-Rad,Richmond, CA). Protein yields in the membrane fractionsobtained from bile acid–fed mice were similar to that incontrol animals (data not shown).

Analysis of Transporter Protein Levels byWestern Blotting

Similar amounts of protein (150 mg for Ntcp, Oatp1,P-glycoproteins [Pgps]; 300 mg for Bsep, Mrp2) were loaded

Figure 2. Effects of bile acid feeding on bile canalicular ultrastructure. To study the effects of bile acid feeding on the canalicular ultrastructure,electron microscopy of mouse livers was performed as described in Materials and Methods after feeding of a control diet (A, D), UDCA (1%, 7days; B, E), and CA (1%, 7 days; C, F ). Note dilatation of canaliculi without loss of microvilli in UDCA-fed mice (B, E) and to a higher degree inCA-fed animals (C, F ). Bar (A–F ) is 500 nm. n, nucleus; m, mitochondria; *canaliculi.

Table 2. Serum Biochemistry of Bile Acid–Fed Mice

Experimentalgroup AST (U/L) ALT (U/L) AP (U/L)

BA(mmol/L)

Control 203 6 53 32 6 7 43 6 15 4 6 0,8CA 0.5% 268 6 83 287 6 87a 387 6 2a n.d.CA 1% 360 6 168a 313 6 9a 402 6 61a 58 6 32a

UDCA 0.5% 180 6 40 40 6 10 120 6 64 n.d.UDCA 1% 200 6 30 45 6 15 140 6 47a 91 6 35a

NOTE. Values are means 6 SD.BA, bile acids; n.d., not determined.aP , 0.05 compared with controls (standard diet–fed mice).


onto a 7.5% (Mrp2, Pgps) or 10% (Ntcp, Oatp1) sodiumdodecyl sulfate–polyacrylamide gel, without boiling, and sub-jected to electrophoresis.30,31 Samples from controls, andUDCA- and CA-fed animals were always run together onidentical gels. Equal protein loading was confirmed by Coo-massie staining of gels and Ponceau S staining of membranesafter transfer (not shown). After electrotransfer onto nitrocel-lulose membranes (BioRad), the blots were blocked with Tris-buffered saline containing 0.1% Tween and 5% dry milk for 1hour at room temperature and incubated overnight at 4°Cwith polyclonal antibodies against Ntcp (dilution, 1:2500),Oatp1 (dilution, 1:2500), Mrp2 (dilution, 1:2000), and Bsep(dilution, 1:2000).32–35 For detection of Pgps, the commer-cially available C219 antibody (dilution, 1:2500; Signet, Ded-ham, MA) recognizing all 3 Mdr Pgp–isoforms (Mdr1a/b,Mdr2) and Bsep was used.36,37 Blots were reprobed with ananti-b-actin antibody (dilution, 1:5000, Sigma) to confirmequal protein loading and the specificity of the changes intransporter protein levels. Immune complexes were detectedusing horseradish-conjugated goat antirabbit or sheep anti-mouse IgG F(ab9)2 fragments (dilution, 1:1000; Dako, Glas-trup, Denmark) according to the ECL Western blotting de-tection system (Amersham, Buckinghamshire, UK) oriodinated protein A in conjunction with autoradiography.After exposing the blots to Trimax XDA Plus (3M Imation,Rochester, NY), band intensities were determined with anIntas system densitometer and RFLP-Scan Software (Scanalyt-ics) or scanning of the autoradiographs with a CAMAG TLC IIScanner (CMAG AG, Muttenz, Switzerland).

Immunofluorescence Microscopy

Immunofluorescence staining was performed on ace-tone-fixed (220°C for 10 minutes) cryosections of liver tissueusing polyclonal antibodies against Mrp2 (dilution, 1:50),Bsep (dilution, 1:100), and the monoclonal C219 antibody(dilution, 1:30; Signet). For detection of Ntcp, cryosections ofliver tissue were fixed in 4% paraformaldehyde solution inphosphate-buffered saline (PBS) for 20 minutes, rinsed 3 timesin 50 mmol/L NH4Cl in PBS, and treated with 5% TritonX-100 in 50 mmol/L NH4Cl in PBS for 5 minutes, followedby incubation with a polyclonal antibody against Ntcp (dilu-tion, 1:25). For better visualization of the hepatic architecture,counterstaining of the cytokeratin network was performedusing a monoclonal or polyclonal antibody recognizing cyto-keratin 8 and 18.38 As secondary antibodies, fluorescein iso-thiocyanate (FITC)–conjugated goat antimouse IgG (Zymed,San Francisco, CA) or tetramethylrhodamine isothiocyanate(TRITC)–conjugated swine antirabbit IgG (Dako) were used.Negative controls were performed by omitting the primaryantibodies. Fluorescent staining was visualized using an MRC600 laser scanning confocal device (Bio-Rad) attached to aZeiss Axiophot (Zeiss, Oberkochen, Germany). The fluorescentimages were collected using the confocal photomultiplier tubeas full frame (768 3 512 pixels). For dual labeling, separateexcitation wavelengths (488 nm for FITC; 568 nm for TRITC)from a krypton/argon ion laser were used. Separate filter tubesallowed acquisition and storage of the images of the iden-tical optical focal plane. Overlapping of green (FITC) and red

Table 3. Bile Secretion and Composition in Bile Acid–Fed Mice

DietBile flow

(mL z min21 z g liver21)

Biliary output (nmol z min21 z g liver21)Bile acid composition(main bile acids in %)Bile acids Phospholipids Cholesterol Glutathione

Controls 2.5 6 1 112 6 59 17 6 1 1.67 6 0.3 7 6 1.4 TCA 45%, T-b-MCA 30%UDCA 1% 4.5 6 0.8a 296 6 90a 26 6 21 3.15 6 1.6 22.9 6 2.3c TUDCA 97%, T-b-MCA 2%CA 1% 5.6 6 1.2b 539 6 196b 53 6 14a 19.4 6 4.3c 16.8 6 2.1c TCA 98%, T-b-MCA 2%

NOTE. Bile was sampled over 1 hour from UDCA- and CA-fed and control animals on standard chow (n 5 5 each group) and analyzed for bile acid,phospholipid, cholesterol, and glutathione content as described in Materials and Methods. In addition, biliary bile acid composition wasassessed by high pressure liquid chromatography. Results are means 6 SD.TUDCA, tauroursodeoxycholic acid; TCA, taurocholic acid; T-b-MCA, tauro-b-muricholicacid.aP , 0.05, bP , 0.01, cP , 0.001 compared with controls (standard chow).

‹

Figure 1. Liver histology in bile acid–fed mice. (A) Feeding of hydrophilic, nontoxic UDCA (1%, 7 days) did not result in significant changes ofliver histology in comparison to controls. (B) Feeding of CA (1%, 7 days) led to disseminated hepatocyte necroses, increases of cell size, andnumber of mitotic figures (arrows), as well as dilatation of interlobular bile ducts. Original magnifications 103. cv, central vein; pv, portal vein.Figure 3. Effects of UDCA and CA feeding on Ntcp expression. Total RNA and liver membrane fractions were isolated from controls and UDCA-or CA- (0.5 and 1%, 7 days) fed mice and analyzed by competitive RT-PCR (A) and Western blotting (B). In addition, Ntcp tissue localization wasassessed by immunofluorescence staining (C–E), as described in Materials and Methods. (A) Compared with controls (gray bar), Ntcpsteady-state mRNA levels remained unchanged after UDCA (hatched bars) or CA feeding (black bars). Data (means 1 SD) are expressed as copynumbers/200 ng total RNA (n 5 5 in each group). (B) Representative immunoblot. Compared with controls (gray bar), UDCA (hatched bar) hadno significant effect on Ntcp protein levels, whereas CA (black bar) decreased Ntcp protein levels significantly. Data (means 1 SD) are expressedas percentages of controls (n 5 5 in each group). *P , 0.05 vs. controls; #P , 0.05 CA vs. UDCA. Specificity of the changes for Ntcp wasconfirmed by unchanged b-actin protein levels. Equal protein loading was confirmed by Coomassie (Coom) staining of gels. (C) Regularbasolateral fluorescent staining pattern for Ntcp in control animals and (D) UDCA-fed mice. (E) Reduced and disrupted Ntcp staining pattern inCA-fed mice. Note the increase in cell size in CA-fed mice. Bar 5 20 mm.


Figure 1.

Figure 3.

(TRITC) dyes resulted in a yellow signal. Only samples thatwere prepared in parallel in all steps were compared.

Statistical Analysis

In each group, 5 animals were studied for each giventime point. Data are reported as arithmetic means 6 SD unlessstated otherwise. Statistical analysis was conducted using aStudent t test as appropriate, or analysis of variance withposttesting (Bonferroni) when 3 groups were compared usingthe Sigmastat statistic program (Jandel Scientific, San Rafael,CA). A P value , 0.05 was considered significant.

ResultsEffects of Bile Acid Feeding on SerumBiochemistry, Liver Histology,and Ultrastructure

UDCA feeding had no significant effects on serumlevels of AST and ALT, except for a mild increase in APlevels in mice fed a 1% UDCA-supplemented diet (Table2). Feeding of UDCA was not followed by significanthistologic changes (Figure 1A ), except for a mild dila-tation of medium-sized and larger interlobular bile ducts(not shown). In contrast, CA feeding resulted in a dose-dependent, significant increase of AST, ALT, and APlevels (Table 2). Furthermore, CA feeding led to dissem-inated hepatocyte necroses, increased liver cell size, anincreased number of mitotic figures, and dilatation ofinterlobular bile ducts (Figure 1B). Total serum bile acidlevels were significantly elevated in both UDCA- andCA-fed mice (Table 2). Both UDCA and CA feedingresulted in marked canalicular dilatation as revealed byassessment of cross-sectional canalicular areas in the elec-tron micrographs, which was more pronounced in CA-fed (375% 6 38% of controls, P , 0.005) than inUDCA-fed animals (260% 6 66%, P , 0.01; Figure2A–C ). However, shortening or loss of microvilli was notobserved in bile acid–fed mice, which could reflect anincrease of the canalicular surface as the result of en-hanced vesicular targeting to the canalicular membranein response to bile acid feeding.39

Effects of Bile Acid Feeding on Bile Flowand Composition

UDCA and CA feeding significantly stimulatedbile flow to 180% (P , 0.05) and 224%, respectively, ofcontrols (P , 0.01; for details see Table 3). CA increasedbile acid excretion to a higher extent (480% of controls;P , 0.01) than UDCA (260%; P , 0.05). Consistentwith previous reports, UDCA and CA feeding of miceresulted in replacement of endogenous bile acids by thefed bile acid (i.e., UDCA or CA), which represented.95% of biliary bile acids (Table 3), also indicating

changes in bile acid pool composition.18,19 Moreover, CAbut not UDCA significantly increased biliary phospho-lipid and cholesterol excretion threefold (P , 0.05) and12-fold (P , 0.0001), respectively (Table 3). UDCAincreased GSH excretion threefold (P , 0.001) and CAtwofold (P , 0.001) in comparison to controls, indicat-ing that bile acid–independent bile flow was also stim-ulated by bile acid feeding.

Effects of Bile Acid Feeding onHepatocellular Transporter Expression

CA but not UDCA down-regulates expression ofthe basolateral Ntcp. Bile acid feeding had no signifi-cant effect on Ntcp (Figure 3A ) and GAPDH steady-state mRNA levels (data not shown). UDCA slightlyinduced Ntcp protein expression to 120% 6 22% ofcontrols without reaching statistical significance, whileCA significantly reduced Ntcp protein levels to 30% 618% of controls (P , 0.01 vs. controls; P , 0.01 vs.UDCA; Figure 3B). Specificity of these findings wasconfirmed by unchanged protein levels for b-actin (Fig-ure 3B). The reduction of Ntcp expression in CA-fedanimals was also supported by the reduced and disruptedbasolateral immunofluorescence staining for Ntcp (Fig-ure 3E ). Taken together, these findings indicate post-transcriptional effects of CA on Ntcp expression in themouse liver.

UDCA and CA down-regulate expression of thebasolateral Oatp1 and Oatp4 (Lst-1). Oatp1 steady-statemRNA levels in UDCA- and CA-fed mice were reducedto 72% 6 23% and 75% 6 18%, respectively, ofcontrols (P , 0.05; Figure 4A ), accompanied by a re-duction of protein levels to 45% 6 18% in UDCA-fed(P , 0.01 vs. controls) and to 10% 6 9% in CA-fedanimals (P , 0.001 vs. controls; P , 0.01 vs. UDCA;Figure 4B). Steady-state mRNA levels of Oatp4 (whichmay be more relevant than Oatp1 for bile formation) inUDCA- and CA-fed mice were significantly reduced to46% 6 10% and 49% 6 12%, respectively, of controls(P , 0.001). No specific Oatp4 signal was obtained forWestern blotting and immunofluorescence in mouseliver, using an antirat Oatp4 (Lst-1) antibody (generatedby Bruno Stieger, unpublished results) and, therefore, isnot shown.

UDCA and CA induce expression of the canalicularBsep. CA induced Bsep mRNA levels to 250% 6 25% incomparison to controls (P , 0.05; Figure 5A), whereasUDCA had no effect. However, UDCA feeding increasedBsep protein levels to 250% 6 40% of controls (P , 0.01),suggesting post-transcriptional effects of UDCA, whereasCA increased Bsep to 150% 6 25% (P , 0.05; Figure 5B).A trend for higher Bsep protein levels was observed in


UDCA- compared with CA-fed animals that, however, didnot reach statistical significance. The observed increase ofprotein levels was further confirmed by an increased cana-licular immunofluorescence staining for Bsep in UDCA-and CA-fed animals in comparison to controls (Figure 5C–H ). Induction of Bsep expression may in part explainincreased bile acid excretion rates in bile acid–fed mice(Table 3). The relative incongruity between the increases inBsep protein levels and bile acid excretion rates in bileacid–fed mice suggests additional effects of bile acids oncanalicular transport activity.37,40

UDCA and CA induce expression of the canalicularMdr1a/b. Mdr1a and 1b steady-state mRNA levels wereincreased in a dose-dependent manner to fourfold and eight-fold, respectively, in CA-fed mice (P , 0.05). In contrast,only the highest (1%) UDCA dose slightly increased Mdr1asteady-state mRNA levels to twofold (P , 0.05; Figure6A). Neither CA nor UDCA feeding had a significant effecton Mdr2 steady-state mRNA levels (not shown). At theprotein level (assessed by Western blotting using the C219antibody recognizing all 3 Mdr Pgp–isoforms [Mdr1a/b,Mdr2]36,37), both UDCA and CA resulted in a dose-depen-dent increase up to 220% 6 30% and 230% 6 20%,respectively (P , 0.05), of controls (Figure 6B), againsuggesting a post-transcriptional effect of UDCA as ob-served for Bsep. Immunostaining with C219 revealed anincrease in the canalicular signal in UDCA- and CA-fedanimals when compared with controls (Figure 6C–H ).However, increased Bsep levels may also contribute to in-creased C219 signal in immunofluorescence and Westernblotting, because the C219 antibody also recognizes Bsep inaddition to all 3 Mdr isoforms.36,37

UDCA and CA induce expression of the canalicu-lar Mrp2. Feeding with bile acids resulted in a threefoldincrease in Mrp2 steady-state mRNA levels in compar-ison with controls (P , 0.05; Figure 7A ). At the proteinlevel, Mrp2 increased to 350% 6 22% in UDCA-fedmice (P , 0.01) and 220% 6 18% in CA-fed animals(P , 0.05; Figure 7B). A trend for higher Mrp2 proteinlevels was observed in UDCA- compared with CA-fedanimals without reaching statistical significance. Induc-tion of Mrp2 protein expression was also visible as in-creased canalicular Mrp2 staining in UDCA- and CA-fedmice compared with controls (Figure 7C–H ). Stimula-tion of Mrp2 expression may contribute to the increasedbiliary excretion of GSH, which is an Mrp2 substrate(Table 3).41

DiscussionOur aim was to investigate the effects of nontoxic

UDCA and potentially toxic CA on the expression ofhepatocellular transport systems, to obtain new insightsinto the anticholestatic mechanisms of UDCA and thepotential role of bile acids for transporter alterationsduring cholestasis. This study shows for the first timedifferential regulation of mRNA and protein levels ofbasolateral and canalicular transport systems in responseto bile acids (Figure 8).

UDCA stimulates the biliary excretion of bile acidsand other organic anions (e.g., bilirubin, bromosulfoph-thalein) under clinical and experimental conditions.2,9,10

These choleretic effects may in part be a result of stim-ulation of vesicular exocytosis through activation of pro-

Figure 4. Effects of UDCA and CA feeding on Oatp1 expression. Total RNA and liver membrane fractions were isolated from controls and UDCA-or CA- (0.5 and 1%, 7 days) fed mice and analyzed by competitive RT-PCR (A) and Western blotting (B). (A) Compared with controls (gray bar),Oatp1 steady-state mRNA levels were reduced after UDCA (hatched bars) and CA feeding (black bars). Data (means 1 SD) are expressed as copynumbers/200 ng total RNA (n 5 5 in each group). *P , 0.05 vs. controls. (B) Representative immunoblot. Compared with controls (gray bar),UDCA (hatched bar) moderately reduced Oatp1 protein levels, whereas CA (black bar) profoundly decreased Oatp1 protein levels. Data (means 1SD) are expressed as percentages of controls (n 5 5 in each group). *P , 0.05 vs. controls; #P , 0.05 UDCA vs. CA.


tein kinase C and mitogen-activated protein kinases,resulting in the insertion of transporters into the cana-licular membrane and consequently increased canaliculartransport capacity.13,14,42 In addition, UDCA stimulatesexpression and function of a Cl2/HCO3

2 exchanger inPBC patients.15–17 These observations prompted the hy-

pothesis that UDCA stimulates bile formation and,thereby, counteracts cholestasis by inducing gene expres-sion of hepatocellular transport systems. Our findingssupport this concept. Induction of Bsep protein levels byUDCA, as shown in the present study, is also consistentwith improved bile acid excretion in UDCA-treated

Figure 5. Effects of UDCA and CA feeding on Bsep expression. Total RNA and liver membrane fractions were isolated from controls and UDCA-or CA- (0.5 and 1%, 7 days) fed mice and analyzed by competitive RT-PCR (A) and Western blotting (B). In addition, Bsep tissue localization wasassessed by immunofluorescence staining (single labeling for Bsep [C–E]; corresponding doublelabeling with cytokeratin 8/18 [F–H ]). Overlap-ping of pericanalicular cytokeratin sheet (in green) with canalicular Bsep (in red ) immunolabeling results in a yellow signal. (A) Compared withcontrols (gray bar), Bsep steady-state mRNA levels remained unchanged after UDCA feeding (hatched bars), but increased after CA feeding (blackbars). Data (means 1 SD) are expressed as copy numbers/200 ng total RNA (n 5 5 in each group). *P , 0.05 vs. controls. (B) Representativeimmunoblot. Compared with controls (gray bar), UDCA (hatched bar) and CA (black bar) increased Bsep protein levels. Data (means 1 SD) areexpressed as percentages of controls (n 5 5 in each group). *P , 0.05 vs. controls. (C, F ) Regular canalicular fluorescent staining pattern forBsep in control animals. Enhanced canalicular staining pattern in UDCA-fed (D, G) and CA-fed mice (E, H ). Also note increase in cell size in bileacid–fed mice (G, H ), compared with controls (F ). Bar 5 20 mm.


Figure 6. Effects of UDCA and CA feeding on Mdr Pgp expression. Total RNA and liver membrane fractions were isolated from controls and UDCA-or CA- (0.5 and 1%, 7 days) fed mice and analyzed by competitive RT-PCR (A) and Western blotting (B). In addition, Pgp tissue localization wasassessed by immunofluorescence staining (single labeling for P-gps [C–E]; corresponding doublelabeling with cytokeratin 8/18 [F –H ]).Overlapping of pericanalicular cytokeratin sheet (in red ) with canalicular Pgp (in green) immunolabeling results in a yellow signal. (A) Comparedwith controls (gray bar), Mdr1a steady-state mRNA levels (upper panel ) increased mildly after UDCA feeding (hatched bars), but increasedmarkedly after CA feeding (black bars). Mdr1b steady-state mRNA levels (lower panel ) remained unchanged after UDCA feeding (hatched bars),but increased after CA feeding (black bars). Data (means 1 SD) are expressed as copy numbers/200 ng total RNA (n 5 5 in each group). *P ,0.05 vs. controls. (B) Representative immunoblot. Compared with controls (gray bar), UDCA (hatched bar) and CA (black bar) increased Pgp levels(as assessed by the C219 antibody recognizing all 3 mdr Pgp isoforms [Mdr1a, Mdr1b, Mdr2], as well as Bsep). *P , 0.05 vs. controls. Data(means 1 SD) are expressed as percentages of controls (n 5 5 in each group). (C, F ) Normal canalicular fluorescent staining pattern for Pgps(C219 antibody) in control animals. Enhanced canalicular staining pattern in UDCA-fed (D, G) and to a higher degree in CA-fed mice (E, H ). Alsonote increase in cell size in bile acid–fed mice (G, H ), compared with controls (F ). Bar 5 20 mm.


PBC, primary sclerosing cholangitis, and cystic fibrosispatients.43–45 Furthermore, increased Mrp2 levels afterUDCA feeding are in line with the bilirubin-loweringeffect of UDCA observed clinically.2,9 Stimulation ofbiliary GSH excretion by UDCA feeding, as shown inthe present study, may also be explained by elevated

Mrp2 protein levels.41,46 Increased synthesis of canalicu-lar transport systems, such as Bsep and Mrp2, could be arequirement for bile acid–induced insertion of thesetransport proteins into the canalicular membrane as pre-viously shown.13,37 The marked increase of the canalicu-lar area without loss of microvilli in the presence of

Figure 7. Effects of UDCA and CA feeding on Mrp2 expression. Total RNA and liver membrane fractions were isolated from controls and UDCA-or CA- (0.5 and 1%, 7 days) fed mice and analyzed by competitive RT-PCR (A) and Western blotting (B), respectively. In addition, Mrp2 tissuelocalization was assessed by immunofluorescence staining (single labeling for Mrp2 [C–E]; corresponding doublelabeling with cytokeratin 8/18[F –H ]). Overlapping of pericanalicular cytokeratin sheet (in green) with canalicular Mrp2 (in red ) immunolabeling results in a yellow signal. (A)Compared with controls (gray bar), Mrp2 steady-state mRNA levels increased after UDCA (hatched bars) and CA feeding (black bars). Data(means 1 SD) are expressed as copy numbers/200 ng total RNA (n 5 5 in each group). *P , 0.05 vs. controls. (B) Representative immunoblot.Compared with controls (gray bar), UDCA (hatched bar) and CA (black bar) increased Mrp2 protein levels. Data (means 1 SD) are expressed aspercentages of controls (n 5 5 in each group). *P , 0.05 vs. controls. (C, F ) Normal canalicular fluorescent staining pattern for Mrp2 in controlanimals. Enhanced canalicular staining pattern in UDCA-fed (D, G) and CA-fed mice (E, H ). Also note increase in cell size in bile acid–fed mice(G, H ), compared with controls (F ). Bar 5 20 mm.


significantly induced bile flow in bile acid–fed micecould be the result of enhanced vesicular targeting oftransporters to the canalicular membrane.39 Effects ofbile acids on transporter mRNA and protein levels asobserved in the present study may be more important forlong-term regulation of transport capacity, whereas in-sertion/retrieval into/from the canalicular membrane maybe more relevant for its short-term regulation.47

Because CA is a major primary bile acid that is re-placed during UDCA treatment,1,2 the present study alsoaimed at clarifying the effects of potentially toxic CA incomparison to UDCA. Down-regulation of Ntcp undervarious experimental and clinical cholestatic conditionsmay be caused by retention of toxic bile acids in line withreduced Ntcp and Oatp1 protein levels in CA-fedmice.48,49 Steady-state mRNA levels remained un-changed for Ntcp and were only moderately reduced forOatp1, suggesting that the effects of CA on expression ofthese transporters may be mediated predominantly at apost-transcriptional level. These results are consistentwith previous findings in Mdr2(2/2) mice and in a mousemodel of erythropoietic protoporphyria, which are bothcharacterized by elevated bile acid levels.50,51 Down-regulation of Ntcp, Oatp1, and Oatp4 (Lst-1), togetherwith increased Bsep expression (see below), as observedin CA-fed mice, may protect hepatocytes from bile acidoverload.48,50 Interestingly, bile flow and bile acid excre-tion were increased in CA-fed mice despite reduced Ntcpprotein levels in the present study. This is consistentwith the assumption that bile acid uptake is not the ratelimiting step (even under conditions when Ntcp proteinlevels are reduced to 30%), because of the larger baso-lateral surface area and the higher concentration gradi-ents across the canalicular membrane.52

Induction of Bsep by CA may be mediated at the tran-scriptional level as suggested by the stimulatory effect ofCA on Bsep promoter activity via interaction with thefarnesoid X receptor.53,54 Stimulation of Bsep expressionmay at least in part explain increased bile acid excretion

rates in bile acid–fed mice. However, the relative discrep-ancy between the increases in Bsep protein levels and bileacid excretion rates among UDCA- and CA-fed mice mayindicate additional bile acid effects such as stimulation oftransporter trafficking and activity (e.g., through activationof phosphoinositide 3-kinase).37,40 Up-regulation of canalic-ular transport systems might be an initial response of cho-lestatic hepatocytes in an attempt to overcome bile acidoverload. Indeed, transient up-regulation of Mrp2 and Bsephas been observed during the first 24 hours in variousexperimental animal models of cholestasis.29,55 Bsep induc-tion by CA is also consistent with partial recovery of Bsepexpression in prolonged bile duct ligation in rats and pres-ervation of Bsep expression in Mdr2(2/2) mice.55,56 Further-more, Mdr1a/b were also up-regulated by CA at the mRNAand protein levels. Up-regulation of Mdr1 could protectagainst bile acid–induced oxidative stress and could belinked to transcriptional activation of Mdr1b via activationof nuclear factor kB.36,57,58

In summary, we provide the first evidence that bothpotentially toxic CA and nontoxic UDCA increase proteinlevels of canalicular transport systems for bile acids andother organic anions (e.g., GSH, bilirubin diglucuronide).Down-regulation of basolateral and up-regulation of cana-licular transporters may represent a hepatocellular defensemechanism against potentially toxic bile acids. Therapeu-tics effective in maintaining/up-regulating transporter ex-pression during cholestasis should be expected to amelioratecholestatic liver injury. Maintenance/up-regulation of trans-porter protein levels by UDCA in the absence of bileacid–induced cytotoxicity may at least in part explain thebeneficial effects of UDCA in cholestasis.

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Figure 8. Schematic representation of alterations in hepatocellular transporter expression induced by bile acid feeding. Feeding of cholic acid(CA) resulted in down-regulation of basolateral Ntcp and Oatp1, but up-regulation of canalicular Bsep, Mdr1, and Mrp2. In contrast, feeding ofnontoxic UDCA resulted in up-regulation of Ntcp and down-regulation of Oatp1, whereas Bsep and Mrp2 were up-regulated. Neither UDCA nor CAhad an effect on Mdr2 expression (not shown).


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Received October 26, 2000. Accepted March 15, 2001.Address requests for reprints to: Michael Trauner, M.D., Division of

Gastroenterology and Hepatology, Department of Medicine, Karl-Franzens University, Auenbruggerplatz 15, A-8036 Graz, Austria.e-mail: [email protected]; fax: 43-316-385-3062.

Supported by grants 7171 and 8522 from the Jubilee Funds of theAustrian National Bank (to M.T.), the Joseph Skoda Prize from theAustrian Society of Internal Medicine (to M.T.), grant Lla997/3-1 fromDeutsche Forschungsgemeinschaft and a career development grantfrom Ministerium fur Schule und Weiterbildung, Wissenschaft undForschung des Landes Nordrhein-Westfalen (to F.L.), grant 3200-052190 from the Swiss National Foundation (to P.J.M.), and grant7401-MOB from the Austrian Science Foundation (to K.Z.).

Presented in part at the annual meetings of the European Associa-tion for the Study of the Liver, April 2000, Rotterdam, Netherlands andthe American Gastroenterological Association, May 2000, San Diego,California, and published in abstract form (J Hepatol 2000;32[suppl2]:A39; Gastroenterology 2000;118[suppl 2]:A934).


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