pharmacological inhibition of apical sodium‐dependent bile...

Pharmacological Inhibition of Apical Sodium-Dependent Bile Acid Transporter Changes Bile

Composition and Blocks Progression of SclerosingCholangitis in Multidrug Resistance 2 Knockout Mice

Alexander G. Miethke,1,5 Wujuan Zhang,2 Julia Simmons,1 Amy E. Taylor,1 Tiffany Shi,1

Shiva Kumar Shanmukhappa,2,5 Rebekah Karns,3 Shana White,3 Anil G. Jegga,3,5 Celine S. Lages,1

Stephenson Nkinin,2 Bradley T. Keller,4 and Kenneth D.R. Setchell2,5

Deficiency for multidrug resistance 2 (mdr2), a canalicular phospholipid floppase, leadsto excretion of low-phospholipid “toxic” bile causing progressive cholestasis. Wehypothesize that pharmacological inhibition of the ileal, apical sodium-dependent bileacid transporter (ASBT), blocks progression of sclerosing cholangitis in mdr2–/– mice.Thirty-day-old, female mdr2–/– mice were fed high-fat chow containing 0.006% SC-435,a minimally absorbed, potent inhibitor of ASBT, providing, on average, 11 mg/kg/dayof compound. Bile acids (BAs) and phospholipids were measured by mass spectrometry.Compared with untreated mdr2–/– mice, SC-435 treatment for 14 days increased fecalBA excretion by 8-fold, lowered total BA concentration in liver by 65%, reduced totalBA and individual hydrophobic BA concentrations in serum by >98%, and decreasedplasma alanine aminotransferase, total bilirubin, and serum alkaline phosphatase levelsby 86%, 93%, and 55%, respectively. Liver histology of sclerosing cholangitis improved,and extent of fibrosis decreased concomitant with reduction of hepatic profibrogenicgene expression. Biliary BA concentrations significantly decreased and phospholipidsremained low and unchanged with treatment. The phosphatidylcholine (PC)/BA ratioin treated mice corrected toward a ratio of 0.28 found in wild-type mice, indicatingdecreased bile toxicity. Hepatic RNA sequencing studies revealed up-regulation of puta-tive anti-inflammatory and antifibrogenic genes, including Ppara and Igf1, and down-regulation of several proinflammatory genes, including Ccl2 and Lcn2, implicated inleukocyte recruitment. Flow cytometric analysis revealed significant reduction of fre-quencies of hepatic CD11b1F4/801 Kupffer cells and CD11b1Gr11 neutrophils,accompanied by expansion of anti-inflammatory Ly6C– monocytes in treated mdr2–/–

mice. Conclusion: Inhibition of ASBT reduces BA pool size and retention of hydropho-bic BA, favorably alters the biliary PC/BA ratio, profoundly changes the hepatic tran-scriptome, attenuates recruitment of leukocytes, and abrogates progression of murinesclerosing cholangitis. (HEPATOLOGY 2015; 00:000-000)

Chronic fibrosing cholangiopathies, such as pro-gressive familial intrahepatic cholestasis type 3(PFIC3), biliary atresia, or primary sclerosing

cholangitis (PSC), carry high morbidity and mortality

owing to complications from progressive cholestasis andfibrosis. Cholestasis in these conditions often resultsfrom impaired hepatocellular and bile duct epithelialexcretion of bile constituents, primarily bile acids (BAs),

Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; ANOVA, analysis of variance; ASBT, apical sodium-dependent bile acid transporter;BA, bile acid; C4, 7a-hydroxy-4-cholesten-3-one; Cyp7A1, cholesterol 7a-hydroxylase; DOT, day of treatment; FCM, flow cytometry; FGF, fibroblast growth factor;FGFR, FGF receptor; FXR, farnesoid X receptor; Glp-1, glucagon-like peptide 1; H&E, hematoxylin and eosin; IHC, immunohistochemistry; KCs, Kupffer cells;KO, knockout; Mdr2, multidrug resistance 2; mRNA, messenger RNA; MS, mass spectrometry; PC, phosphatidylcholine; PFIC3, progressive familial intrahepaticcholestasis type 3; PLs, phospholipids; PSC, primary sclerosing cholangitis; qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing; TCA, tauro-cholic acid; TCDCA, taurochenodeoxycholate; TDCA, taurodeoxycholate; TMCA, tauro-b-muricholic acid; WT, wild type

1

phospholipids (PLs), and cholesterol, and from ductalobstruction of bile flow. Bile duct epithelial cells becomeinjured, which leads to up-regulation of adhesion mole-cules, secretion of proinflammatory cytokines and che-mokines, and, ultimately, recruitment of leukocytes tothe portal tract driving fibro-obliteration of the biliarytree, as previously reviewed for cholestatic disorders andtheir animal models.1,2 Formation and retention of“toxic” BA likely play important roles in perpetuatingthe disease process.3 For instance, hydrophobic BA spe-cies are known to trigger hepatobiliary inflammationand cause hepatocyte death by apoptosis.4 Further evi-dence for the pathogenic role of BAs in cholangiopathiesoriginates from genome-wide association studies onpatients with PSC, which identified not only geneslinked to the human leukocyte antigen complex, butalso GPBAR1 encoding the BA receptor, TGR5, as pos-sible disease genes.5 Among many functions, TGR5 hasbeen implicated in regulating production of proinflam-matory cytokines by macrophages.6

The multidrug resistance gene 2 knockout (KO)mouse (mdr2–/–) represents an established animal modelfor chronic cholestatic disorders, especially PFIC3,7 andmay recapitulate some aspects of the pathogenesis ofPSC.8 These mice display absence of PL from bile,9

which disrupts formation of mixed micelles and subse-quently leads to “toxic bile,” triggering an inflammatorycascade.10 This model has been extensively used for pre-clinical studies of novel therapies for chronic cholestasis,targeting anticholestatic, -inflammatory, and fibrogenicpathways.11,12

BA homeostasis is tightly regulated through de novosynthesis from cholesterol in the liver, efficient reuptakeof BAs in the distal small intestine, and feedback regula-tion of hepatic de novo synthesis through the BA/farne-soid X receptor (FXR)/fibroblast growth factor (FGF)19(Fgf15 in mice)–signaling pathway. BAs act throughFXR in ileal enterocytes and induce expression of thehormone, FGF19/15, which subsequently binds to thereceptor complex, FGFR4-b-Klotho, on the surface ofhepatocytes, resulting in repression of cholesterol 7a-hydroxylase (Cyp7A1)-mediated BA synthesis. Less than5% of the circulating BA pool typically escapes reab-sorption and is eliminated in feces on a daily basis. Themajority of BAs are absorbed by active transport in theterminal ileum, mediated by the ABCB4-encoded apicalsodium-dependent bile acid transporter (ASBT). Thecompound, SC-435, is a specific, nonabsorbable inhibi-tor of ASBT and BA reuptake in the distal ileum.13

Consequently, we hypothesized that inhibiting theenterohepatic circulation of BAs by pharmacologicallyblocking ASBT with SC-435 would diminish the BApool size and attenuate progression of sclerosing cholan-gitis in the mdr2–/– mouse model of chronic cholestasis.

Material and Methods

Mice. Mdr2–/– mice in BALB/cJ background were agenerous gift from Prof. Lammert (Homburg Univer-sity, Homburg, Germany) and wild-type (WT) BALB/cJ mice were purchased from Charles River Laboratories(Wilmington, MA). All mice were bred in-house and

From the 1Division of Pediatric Gastroenterology, Hepatology, and Nutrition, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 2Division ofPathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH; 3Division of Biomedical Informatics, Cincinnati Children’sHospital Medical Center, Cincinnati, OH; 4Consultant to Shire plc, San Diego, CA; 5Department of Pediatrics, University of Cincinnati College of Medicine,Cincinnati, OH

Received March 25, 2015; accepted July 7, 2015.Additional Supporting Information may be found at onlinelibrary.wiley.com/doi/10.1002/hep.27973/suppinfo.The National Institute of Diabetes, Digestive and Kidney Diseases (NIDDK) grant DK095001 and the foundation “PSC Partners seeking a cure” supported

this study (to A.G.M.). Lumena Pharmaceutical Inc. (now Shire plc) provided research funds to A.G.M and K.D.R.S. The project was supported, in part, by theNational Institutes of Health (NIH; T32DK007727 [to A.T.]) and PHS Grant P30 DK078392 (Pathology Core, Gene Expression Core, Bioinformatics) of theDigestive Disease Research Core Center in Cincinnati and the Research Flow Cytometry Core in the Division of Rheumatology at Cincinnati Children’s HospitalMedical Center, supported, in part, by NIH AR-47363, NIH DK78392, and NIH DK90971.

Investigators having affiliations with Lumena Pharmaceutical Inc. or Shire plc were not involved in the collection of data, analysis, or interpretation of theresults.

This article was presented, in part, at the FALK symposium on “Bile Acids as Signal Integrators and Metabolic Modulators” in Freiburg, Germany, October 8-11, 2014, at the Biliary Atresia and Related Diseases Meeting in Berlin, Germany, 2014, and at the 65th AASLD Annual Meeting in Boston, MA, November 7-11, 2014.

Address reprint requests to: Alexander G. Miethke, M.D., Division of Pediatric Gastroenterology, Hepatology, and Nutrition, MLC 2010, Cincinnati Children’sHospital Medical Center, 3333 Burnet Avenue, Cincinnati, OH 45229. E-mail: [email protected]; fax: 11-513-636-5581.

Copyright VC 2015 by the American Association for the Study of Liver Diseases.View this article online at wileyonlinelibrary.com.DOI 10.1002/hep.27973Potential conflict of interest: Dr. Setchell owns stock and is a Board Member of Asklepion Pharmaceuticals, LLC. He consults for NGM Biopharmaceuticals Inc.

Dr. Keller consults for Shire plc. Dr. Miethke received grants from Lumena Pharmaceutical Inc.

2 MIETHKE ET AL. HEPATOLOGY, Month 2015

http://onlinelibrary.wiley.com/doi/10.1002/hep.27973/suppinfo

kept in conventional conditions. Thirty-day-old, femalemdr2–/– and WT mice were treated with 0.006% SC-435 admixed to high-fat breeder chow (Mouse diet5020 with 9% fat; Cincinnati Lab Supply, Cincinnati,OH) for 14 days until time of harvest, providingapproximately 11 mg/kg/day of the compound. Controlmice were treated under identical conditions, butreceived Lab 5020 chow without the compound. Allprotocols were approved by the animal care and usecommittee of the Cincinnati Children’s Research Foun-dation (Cincinnati, OH).

Fecal BA. Mice were single-housed on raised, wire-bottom cages for 48 hours before collection of all fecalmaterial, which was rehydrated with water equal to 2times the total fecal weight, overnight at 4�C. BAs wereextracted with 50% (v/v) t-butanol/water (v/v) for 45minutes at 37�C, then centrifuged at 2,000g for 15minutes. BA concentration was determined using theDiazyme Total Bile Acids Universal (enzymatic; Dia-zyme Laboratories, Poway, CA) kit and read on a BioTekSynergy H1 Plate Reader (Bio-Tek Instruments Inc,Winooski, VT).

Plasma and Serum Biochemistries. Plasma totalbilirubin and alanine aminotransferase (ALT) levels weredetermined as reported before.14 Serum alkaline phos-phatase (ALP) concentration was measured using DIS-CRETPAK reagents from Catachem (C174-0C;Catachem, Inc., Bridgeport, CT).

Analysis of BA, PL, Cholesterol and C4 Concen-trations by Liquid Chromatography/Mass Spectrom-etry. Quantitative analysis of BA, PL, and cholesterolwas carried out using a Waters API triple-quadruplemass spectrometer interfaced with an Aquity UPLC sys-tem (Aquity, Milford, MA). Serum and biliary BAs wereextracted on a reverse-phase/solid-phase cartridge. Livertissue was exhaustively extracted with organic solventsbefore aqueous dilution and solid-phase extraction. BAsin biological samples were quantified against curves con-structed for the 15 most common endogenous BAsbased on a stable-isotope dilution analysis with single-ion recording mass spectrometry (MS).15 PLs wereextracted by the Folch procedure and quantified usingphosphatidylcholine (PC) standards based on MS withmultiple reactions monitoring function. Total PL was cal-culated based on the summation of 13 major PC speciesdetected in serum or bile. Bile samples were treated withalkaline hydrolysis to measure the total cholesterol concen-trations by an isotope dilution atmospheric pressure chem-ical ionization liquid chromatography MS method. Plasma7a-hydroxy-4-cholesten-3-one (C4) concentration wasdetermined by a validated stable-isotope dilution tandemMS method using deuterium-labeled 7a-hydroxy-4-cho-

lesten-3-one ([2H7-25,26,26,26,27,27,27]-7a-hydroxy-4-cholesten-3-one; D7-C4) as the internal standard.

Quantitative Real-Time Polymerase Chain Reac-tion. Total RNA was isolated from liver tissue, reversetranscribed, and messenger RNA (mRNA) concentra-tion of TNFa was determined by SYBR green quantita-tive polymerase chain reaction (qPCR). SMA (Acta2)and TIMP1 genes were amplified using TaqMan Univer-sal Master Mix II, with the probes, Mm01546133_m1and Mm00441818_m1, respectively, and run on anApplied Biosystems 7900H Fast Real Time PCR System(Applied Biosystems, Foster City, CA). The DDCtmethod was used for computation of relative mRNAconcentrations, and data were normalized to mouseHPRT expression.

Liver Histology, Ultrastructure, and Ki67 Immu-nohistochemistry. Liver histology was blindly assessedby S.K.S. for inflammation, necrosis, bile duct prolifera-tion, and fibrosis on a 1-41 scale, as previously used ina model of obstructive cholangiopathy.16 Masson’strichrome-stained liver slides were scanned using aAperio AT2 Digital Whole Slide scanner (Leica Micro-systems Inc., Buffalo Grove, IL) at a resolution of50,000 pixels per inch (0.5 mm per pixel). Pixels wereclassified as fibrosis (blue), liver cells (red), or back-ground (white) by means of an image analysis algo-rithm, as described previously.17 For electronmicroscopy, liver tissue was fixed in 3% buffered glutar-aldehyde, subjected to ultrathin sectioning, and exam-ined with a Hitachi H7650 transmission electronmicroscope (Hitachi, Tokyo, Japan). Immunohisto-chemistry (IHC) for Ki67 was performed on paraffin-embedded liver sections, using anti-mKi67 (clone SP6)after heat antigen retrieval with 1 M of Na citrate(pH 5 6).

RNA Sequences and ToppGene Unbiased PathwayAnalysis. Total hepatic RNA was isolated from thecaudate liver lobe. RNA sequencing (RNA-seq) librarieswere prepared with the Illumina TruSeq RNA prepara-tion kit and sequenced on the Illumina Hi-Seq 2000(Illumina, San Diego, CA). Sequences were aligned tothe reference genome with TopHat18 and processedwith Cufflinks,19 which quantifies each transcript ineach sample using reference annotations produced bythe University of California Santa Cruz UCSC. Differ-entially expressed genes with a fold change of >52.0and P< 0.05 between SC-435-treated and control ani-mals were submitted to pathway enrichment analysiswith ToppFun application from the ToppGene Suite,which uses unbiased methods to assess pathway enrich-ment.20 Raw and normalized data are accessible throughthe National Center for Biotechonology Information’s

HEPATOLOGY, Vol. 00, No. 00, 2015 MIETHKE ET AL. 3

Gene Expression Omnibus (accession no. GSE66231).RNA-seq data can be viewed online (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token5cjavskagdnopfmx&acc5GSE66231).

Flow Cytometry. Livers were perfused with 10 mLof of RPMI (Gibco, Grand Island, NY) with 1 lg/mL ofcollagenase D (Roche Diagnostics, Indianapolis, IN)before mechanical disruption with a Miltenyi Biotec dis-integrator (Miltenyi Biotec, Cambridge, MA). Single-cellpreparation and staining was performed, as describedbefore,14 using the following antibodies from BioLegend(anti-mCD3-Brilliant violet 421, anti-mCD19-Brilliantviolet 605; BioLegend, San Diego, CA), eBioscience(anti-mF4/80-PE, anti-mCD11b-PerCP-Cy5.5, anti-mGR1-APC-eFluor 780; eBioscience, San Diego, CA),and BD-Pharmingen (anti-mLy6C-PE-Cy7; BD-Pharmingen, Bedford, MA). Data were acquired on aBD Canto III and analyzed using FlowJo software (ver-sion 7.6.5; Tree Star, Inc., Ashland, OR).

Statistical Analysis. Values are expressed as mean-6 standard error of the mean and statistical significancewas determined by unpaired t test, with a significanceset at P< 0.05. One-way analysis of variance (ANOVA)with post-hoc Tukey‘s multiple comparison test wasused to assess statistical significance between more thantwo groups.

Results

SC-435 Increased Fecal BA Excretion and Low-ered Serum BA Levels. Compared to age- andgender-matched control mice receiving chow withoutthe compound, 48-hour fecal BA excretion after 14 daysof treatment with SC-435 was increased by 8.6-fold inmdr2–/– and by 3.2-fold in mdr21/1 mice (Fig. 1A).Increased fecal BA losses were associated with reductionof total BA concentration in liver tissue by 65% (Fig.1B) and a decrease in serum total BA levels by 98.9% inSC-435-treated compared to control mdr2–/– mice (Fig.1C). The major BA species, taurocholic (TCA) andtauro-b-muricholic acid (TMCA), which constituted95.9% of serum BA in control mdr2–/– mice, were bothsignificantly reduced after treatment with SC-435 (Fig.1D). Importantly, the more hydrophobic BAs, tauroche-nodeoxycholate (TCDCA) and taurodeoxycholate(TDCA), which were significantly elevated in controlmdr2–/– compared to mdr21/1 mice (1,260- and 13-fold, respectively) were significantly reduced by treat-ment with SC-435.

SC-435 Exerted Anticholestatic, Anti-Inflammatory,and Antifibrogenic Effects. Altered BA homeostasiswas associated with profound changes in the sclerosing

cholangitis phenotype in mdr2–/– mice. Whereas controlmdr2–/– mice started to lose weight after day 7 of thetreatment period, those treated with SC-435 maintainedweight in a similar manner to mdr21/1 mice (Fig. 2A).Furthermore, biomarkers of hepatocellular injury(plasma ALT concentration) and cholestasis (plasmatotal bilirubin and serum ALP concentrations), whichwere all increased in untreated mdr2–/– mice comparedto mdr21/1 controls, were reduced in SC-435-treatedmdr2–/– mice by 86%, 93%, and 55%, respectively(Fig. 2B).

Fig. 1. SC-435 increases fecal BA excretion and lowers liver andserum BA levels in mdr2–/– mice. Thirty-day-old, female mdr2–/– andage- and sex-matched mdr21/1 received chow (Lab 5020) containing0.006% SC-435 or the same chow without the compound (controls).Feces were collected for 48 hours before day of treatment (DOT) 14;BA were extracted and subjected to enzymatic quantification (A). Con-centrations of individual BA in liver and serum samples, collected onDOT 14, were determined by tandem MS and plotted as total liverand serum BA concentration in (B) and (C), respectively. Individualspecies were grouped as hydrophilic and hydrophobic BAs in (D). Sta-tistical analysis: one-way ANOVA with Tukey’s multiple comparison testwas applied to the data, with *, **, *** denoting P values of<0.05, <0.01, <0.001, respectively. Abbreviations: CA, cholic acid;DCA, deoxycholic acid; GCA, glycocholic acid; GCDCA, glycocheno-deoxycholic acid; MCA, muricholic acid; THDCA, taurohyodeoxycholicacid; TLCA, taurolithocholic acid; TUDCA, tauroursodeoxycholic acid;UDCA, ursodeoxycholic acid.


http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=cjavskagdnopfmx&acc=GSE66231





Consistent with the change in plasma biochemistries,liver histology of sclerosing cholangitis featuring peri-portal inflammation and fibrosis, as depicted in repre-sentative hematoxylin and eosin (H&E)- and trichrome-stained sections in Fig. 3A, was significantly improvedby SC-435 treatment. Review of histology, using a previ-ously validated sclerosing cholangitis scoring system,revealed reduced scores for periportal inflammation, bileduct proliferation, necrosis, and fibrosis in SC-435-treated KO mice compared to controls (Fig. 3B). Reduc-tion in fibrosis score was consistent with results fromAperio-based image analysis of trichrome-stained liversections. Percentage fibrosis of total liver tissue wasreduced from 4.7 to 1.6 with SC-435 treatment (Fig.3C). Histological data were corroborated by targetedgene expression studies for signature genes of sclerosing-cholangitis–associated hepatic inflammation (TNFa)and fibrosis (SMA and TIMP1). mRNA expressions forTNFa, SMA, and TIMP1 were significantly down-regulated in SC-435 versus control mdr2–/– mice(Fig. 3D).

SC-435 Treatment Upregulated Genes of BA Syn-thesis and Hepatoprotective Pathways, and Down-Regulated Proinflammatory Genes. To further probethe mechanism of action by which disruption of theenterohepatic circulation of BAs blocked progression ofsclerosing cholangitis in mdr2–/– mice, we performedRNA-seq studies on total liver RNA purified from SC-435-treated and control mice of both genotypes. Therewere 497 genes that were >2-fold up-regulated by SC-435 treatment in mdr2–/– mice, of which 32 (6.4%)were also up-regulated in SC-435-treated mdr21/1

mice, when compared to the corresponding untreatedgenotype. In contrast, 332 genes were down-regulatedin mdr2–/– mice, of which only three (0.9%) were alsodown-regulated in mdr21/1 mice (Fig. 4A). In anunbiased analysis, many genes related to cholesterol andBA metabolism were significantly up-regulated by SC-435 treatment, whereas genes in pathways related to leu-kocyte recruitment and function were generally down-regulated when compared to untreated mdr2–/– mice(Fig. 4B). Most notably, Cyp7a1 and Hmgcr, encodingthe rate-limiting enzymes of BA and cholesterol synthe-sis, respectively, were up-regulated by SC-435 in mice ofboth genotypes. Several genes encoding proteins previ-ously implicated in protection from cholestatic injurywere also up-regulated in SC-435-treated mdr2–/– mice.For instance, these included Ppara encoding the tran-scription factor Ppara, Car1/Car3/Car5a encoding iso-forms of carbonic anhydrase, Aqp8 encoding the waterchannel aquaporin 8, and Igf1 encoding insulin growthfactor 1 (Fig 4C). Among the down-regulated genes intreated mdr2–/– mice, a few were related to BA and cho-lesterol metabolism, including Nr0b2/Shp, the down-stream messenger molecule of Fxr, and Abcb1a/Mdr1and Abcg5/Abcg5 encoding canalicular transporters forBA and cholesterol, respectively. The majority of genesdown-regulated after SC-435 treatment were proinflam-matory and -fibrogenic. The highest fold changes in dif-ferential gene expression were observed for the genesCcl2, Cxcl1, and Lcn2 (Fig. 4C). Tgfb1 mRNA expres-sion was reduced by 97% in SC-435-treated versus con-trol mdr2–/– mice (P 5 0.001). Expression data forcandidate genes, which are transcriptionally regulated byBAs through Fxr-signaling, are listed in the SupportingTable 1. For instance, SC-435 treatment was associatedwith increased hepatic expression of Fgf15, but did notsignificantly alter expression of Abcb11/Bsep or Abcc2/Mrp2 in mdr2–/– mice.

SC-435 Treatment Profoundly Altered Bile Com-position. Consistent with hepatic up-regulation ofCyp7a1, the rate-limiting enzyme of BA synthesis, upontreatment with SC-435, plasma levels of the sterol

Fig. 2. SC-435 prevents wasting and reduces biomarkers of hepato-cellular injury and cholestasis in mdr2–/– mice. Body weights wererecorded for mice of the four groups during the 14 days of intervention(A). ALT, total bilirubin, and ALP levels were determined on bloodsampled by cardiocentesis on DOT 14. Statistical analysis: Unpaired ttest was applied to the data from treated and control mdr2–/– mice in(A), one-way ANOVA with Tukey’s multiple comparison test in (B).




intermediate, C4, a surrogate marker of BA synthesis,21

were increased by 3-fold in mdr2–/– and by 10-fold inmdr21/1 mice (Fig. 5A). We next examined whetherthis increased synthesis resulted in higher concentrationsof BA in bile, the compartment in which low-phospholipid, nonmicellar bile directly injures bile ductepithelium.9,10 As expected, biliary PC concentration inmdr2–/– mice was reduced to 0.29% of concentrationsfound in bile of mdr21/1 controls and did not signifi-cantly change upon treatment with SC-435 (Fig. 5B).However, SC-435 treatment reduced significantly thebiliary concentrations of BA and cholesterol by 98.8 and97.6%, respectively, in these mice. Therefore, despitepersistence of low biliary phospholipid levels, the biliaryPC/BA ratio was markedly increased in SC-435-treatedmdr2–/– mice (mean, 0.05 6 0.02), although still lowerthan WT control mice (mean, 0.28 6 0.07), and theaverage coefficient of 0.30 reported for most mammals

under physiological conditions.22 Using the Wang andCarey phase diagram to describe the influence of biliarycomposition on cholesterol crystallization,23,24 controlmdr2–/– mice plot in the 2 phase (liquid crystal) zone A(predicting precipitation of cholesterol), whereas SC-435-treated mdr2–/– mice, as with WT animals, plot inthe 1 phase micellar zone, predicting micellar solubiliza-tion of cholesterol. Collectively, these findings suggestthat SC-435 attenuates progression of sclerosing cholan-gitis in mdr2–/– mice by altering bile composition toreduce the “toxicity” of nonmicellar bile. Therefore, wenext examined evidence for diminished injury to cholan-giocytes in SC-435-treated mdr2–/– mice.

SC-435 Treatment Reduced CholangiocyteInjury. Typically, acute injury to cholangiocytes is fol-lowed by regeneration, which can be labeled by the pro-liferation marker, Ki67.25 Proliferation of cholangiocyteswas significantly increased in control mdr2–/–, compared

Fig. 3. SC-435 treatment is associated with reduced periportal inflammation and fibrosis in mdr2–/– mice. Sections from paraffin-embeddedliver samples obtained from mdr2–/– mice on DOT 14 were subjected to H&E and trichrome staining. Representative photomicrographs of sec-tions are shown in (A), with arrowheads denoting bile duct profiles. Components of sclerosing cholangitis score (inflammation, ductal prolifera-tion, necrosis, and fibrosis) were analyzed on a 1-41 scale (B). Trichrome-stained slides from livers of SC-435-treated and control mdr2–/– micewere subjected to automated Aperio-based tissue image analysis to quantify the percentage of liver fibrosis (C). Total hepatic RNA from 44-day-old mice of the four groups was subjected to qPCR and relative expression of the pro-inflammatory gene TNFa and of the profibrogenic genesSMA and TIMP1 was computed by DDCT analysis using HPRT as the housekeeping gene (D). Statistical analysis: Unpaired t test was applied tothe data of the two groups (SC-435 vs. control) in mdr2–/– mice in (B), with *, **, *** denoting P values of <0.05, <0.01, <0.001, respec-tively, and one-way ANOVA in (C) and (D).


to mdr21/1, mice by IHC for Ki67. Reduced frequencyof Ki671 cells among cholangiocytes of interlobular bileducts in SC-435-treated versus control mdr2–/– micesuggested reduced bile duct epithelial injury in these ani-mals (Fig. 6A). Previous studies in mdr2–/– mice revealedthat injury to cholangiocytes allows leaking of BA intothe periductal space, which was associated with disrup-tion in basement membranes surrounding the interlobu-lar bile ducts.10 Marked improvement in ultrastructureof basement membrane of the interlobular bile duct wasobserved in SC-435-treated mdr2–/– mice. In treatedmice, 70% of interlobular bile ducts had continuous andintact basement membrane, as opposed to only 40% in

untreated mdr2–/– mice. In diseased bile ducts, the base-ment membrane was discontinuous and irregular tobeing absent in some instances (n 5 10 bile ducts/group;Fig. 6B).

SC-435 Treatment Reduced Recruitment of KupfferCells and Neutrophils to the Liver and Promoted Expan-sion of Anti-Inflammatory Monocytes. Diminishedleukocyte recruitment and function was an additionalmechanism of action of SC-435, indicated by the globalhepatic gene expression studies. A significant reductionin frequencies of F4/801CD11b1 Kupffer cells (KCs)and Gr11CD11b1 neutrophils, respectively, wasobserved by flow cytometry (FCM) in SC-435-treated

Fig. 4. SC-435 treatment is associated with hepatic up-regulation of genes controlling BA pool size and putative hepatoprotective genes anddown-regulation of proinflammatory genes. RNA-seq studies were performed on total hepatic RNA from mice of the four treatment groups. Distri-bution of up- and down-regulated genes (>2-fold difference) compared to untreated mice of the respective genotype (KO and WT across the dif-ferent treatment groups is depicted in a Venn diagram; A). Based on pathway enrichment analysis, 12 pathways comprising significantly up- ordown-regulated genes in SC-435-treated mdr2–/– compared to mdr2–/– control mice are depicted with P values for these pathways plotted as –log10 (B). Twenty-three genes from the up- and down-regulated pathways, respectively, are displayed in the heatmaps with expression levels nor-malized to control mdr21/1 mice (C).


versus control mdr2–/– mice. Frequency of F4/80-CD11b1 monocytes in mdr2–/– mice was increasedupon SC-435 treatment, attributable to a rise in theanti-inflammatory subset of Ly6C– monocytes in liver,

whereas the proportion of proinflammatory and -fibro-genic Ly6C1 monocytes was reduced (Fig. 7).

Discussion

We have demonstrated that SC-435, a small-moleculeinhibitor of ASBT, increased fecal BA excretion, leadingto a dramatic reduction of liver and serum concentra-tions of BAs and biomarkers of hepatocellular and cho-lestatic injury in female mdr2–/– mice. This responsewas rapid, occurring within 14 days of treatment. Com-pared to control mice, body mass wasting was pre-vented, and progression of hepatic inflammation andfibrosis was attenuated. The mechanisms of action ofSC-435 may be pleiotropic, and some are supported byour data, including a (1) reduction of the total BA poolsize and circulating hydrophobic BA, (2) decrease of bil-iary BA and cholesterol concentrations rendering bileless hepatotoxic, and (3) reduction of cholestasis-associated inflammatory responses.

SC-435 has been reported to enhance fecal BA excre-tion in noncholestatic rodents.26 Although ASBT isexpressed in the terminal ileum of mdr2–/– in similarconcentration to that of mdr21/1 mice,27 it wasunknown whether enterohepatic reuptake of BA isdepressed under cholestatic conditions in these mice,thus conferring resistance to treatment with an ASBTinhibitor. Under our experimental conditions, SC-435treatment resulted in an 8-fold increase in fecal BAexcretion, with a concomitant 90-fold reduction ofserum BA concentrations and reduction of plasma ALT,total bilirubin, and serum ALP levels. Furthermore,fibrosis was diminished, as assessed by image analysis oftrichrome-stained liver sections.

We propose several mechanisms of action by whichtreatment with SC-435 exerts its anticholestatic, -inflam-matory, and fibrogenic effects in the mdr2–/– mousemodel of chronic cholestasis. Based on the physicochem-ical properties of SC-435 having minimal intestinalabsorption, we focused our investigations on SC-435-mediated regulation of BA synthesis and composition ofthe hepatic and biliary pool and its downstream conse-quences. SC-435 treatment rapidly lowered serum totaland hydrophobic BA concentrations. Hydrophobic BAshave previously been linked to pathogenesis of sclerosingcholangitis, especially in female mdr2–/– mice,28 andwere shown to stimulate hepatocyte apoptosis throughnonspecific detergent effects,29 activation of death recep-tors,30 and induction of oxidative damage causingmitochondrial dysfunction.31 Which of these pathwaysis predominant in mdr2–/– mice is unknown.

Fig. 5. Despite increase in hepatic BA synthesis rate, SC-435 treat-ment is associated with reduced biliary BA concentration. Concentra-tions for the biological precursor C4, which correlates with BAsynthesis, were determined by tandem MS in plasma samplesobtained on day of treatment (DOT) 14 (A). Bile was aspirated fromgallbladders on DOT 14 and subjected to MS-based quantification ofPC, cholesterol, and BA concentrations. Biliary molar ratios of PC/BA(linkage coefficient) were calculated based on these measurements,and mean values of bile constituents were plotted in a ternary phasediagram. Control mdr2–/– mice plot in region A, which is a 2 phasezone of the Wang and Carey diagram, characterized by very low PC/BAratio, cholesterol supersaturation, and formation of cholesterol crystals.SC-435-treated mdr2–/– and both groups of WT mice plot in the micel-lar 1 phase zone of the diagram (B). Statistical analysis: Unpaired ttest was applied to compare the data between the two groups (SC-435 vs. control) in mdr2–/– and mdr21/1 mice.


Global hepatic gene expression studies provided fur-ther insights into effects of SC-435 treatment on puta-tive mechanisms of action. Consistent with reduction ofthe BA pool size resulting in a state of low transhepaticBA flow, differential gene expression suggested decreasedactivation of Fxr, that is, the expression of Shp, arepressor of BA synthesis pathways, was diminishedresulting in increased expression of the rate-limitingenzyme (Cyp7a1) for BA synthesis. Importantly, genesencoding proteins with potentially protective propertiesduring cholestasis were also up-regulated in SC-435-treated mdr2–/– mice, including Ppara, which may pro-mote alternative BA elimination/detoxification andreduce inflammation,3 Car3 and Aqp8, involved inbicarbonate-rich hydrocholeresis reducing the toxiceffects of hydrophobic BA on biliary epithelium,32,33

and Igf1, reported to exert antifibrogenic properties incholestasis.12 In addition, of the 332 down-regulatedgenes in SC-435-treated mdr2–/– mice, 95 play a role ininflammation and fibrosis, most prominently Ccl2/Mcp1 and Lcn2/lipocalin 2. Mcp1 is expressed by bileduct epithelial cells upon exposure to BA34 and has beenlinked to activation of hepatic stellate cells in biliary

atresia and cystic fibrosis.35 Lipocalin 2 is secretedby hepatocytes and involved in leucocyte recruitmentand was found to be a sensitive biomarker of liverinjury.36

Many changes in the hepatic gene expression profileappear consistent with what would be expected to occurwhen severe cholestasis is alleviated, including Fxr inac-tivation and reduction of proinflammatory gene expres-sion, and might be operative in any cholestatic disorderin which enterohepatic BA uptake is disrupted. Otherresults of our studies may have more specific bearing forthe pathogenesis of sclerosing cholangitis in mdr2–/–

mice, in particular, our findings on the changes in bilecomposition. We propose that reduction of biliary BAand cholesterol concentrations, which are determinantsof bile solubility and cholangiocyte injury from precipi-tated bile microcrystals in mdr2–/– mice,9,10 were criticalfor attenuation of the sclerosing cholangitis phenotype.We speculate that increased cholehepatic shunting,reported to be the result of bile duct proliferation inmdr2–/– mice,27 and down-regulation of genes encodingthe canalicular BA and cholesterol transporters, Mdr1and Abcg5, respectively, contribute to this change in bile

Fig. 6. SC-435 treatment is associated with decreased bile duct epithelial injury and partial restoration of ultrastructural integrity. Paraffin-embedded liver sections from SC-435-treated and control mice (n 5 2-3/group) were subjected to Ki67 IHC. Ki67-positive cells were enumer-ated in large interlobular bile ducts (10 bile duct profiles/liver). Statistical analysis: One-way ANOVA was applied to compare the data betweenthe groups (A). In (B), glutaraldehyde-fixed liver sections were analyzed by electron microscopy. Left panel depicts a bile duct profile in controlmdr21/1 mice in low magnification, with asterisk (*) denoting the lumen. The other images show higher magnification of the basement mem-brane (denoted by arrowheads) in mdr21/1 mice, which is disrupted in control mdr2–/– mice; integrity is restored in SC-435-treated mdr2–/–

mice. Ten bile duct profiles were examined per group (n 5 2 mice/group), of which four were surrounded by intact basement membrane in con-trol and seven in SC-435-treated mice.


composition despite activation of de novo hepatic BAsynthesis after treatment with SC-435. Our findings ofdecreased frequency of Ki67-expressing cholangiocytesand improved liver ultrastructure with intact basementmembranes surrounding interlobular bile ducts mayindicate reduced BA leakage and support this mecha-nism of action of SC-435 in mdr2–/– mice.

Finally, we validated results of gene expression stud-ies, which revealed decreased hepatic expression of che-mokines recruiting leukocytes to the liver by showingthat SC-435 treatment decreased the frequency of KCs,neutrophils, and inflammatory monocytes accompaniedby expansion of anti-inflammatory Ly6C– monocytes.

Interestingly, this pattern of changes in hepatic leuko-cyte composition has previously been reported duringtreatment with a Tgr5 receptor agonist in a model ofsteatohepatitis.37 This raises the possibility that some ofthe effects of SC-435 on hepatic inflammation are medi-ated by Tgr5, as previously described for metaboliceffects observed in treatment with anionic resins, analternative method of disrupting enterohepatic circula-tion of BA. These studies suggested that binding of BAby resins resulted in spillover of BA into the colon,which led to Tgr5-dependent intestinal release ofglucagon-like peptide 1 (Glp-1) exerting glycemic con-trol in the liver.38 Whether ASBT inhibitors affect Tgr5

Fig. 7. SC-435 treatmentis associated with decreasedpopulation of liver with mac-rophages, neutrophils, and ashift toward anti-inflammatorymonocytes. Single-cell sus-pensions were prepared fromlivers of mice from the fourtreatment groups and sub-jected to FCM. The two toppanels delineate the gatingstrategies: After outgating ofcell duplets, debris, andlineage-positive CD31 orCD191 cells, macrophages(KCs) were identified as F4/801CD11b1 and neutrophilsas Gr1highCD11b1 cells. F4/80– and Gr1– CD11b1 mye-loid cells were further distin-guished into inflammatory(Ly6C1) and anti-inflammatory (Ly6C–) mono-cytes. Frequencies of thesecell populations were enumer-ated as denoted in thegraphs, and differences inmeans between groups weretested for statistical signifi-cance with one-way ANOVA.Abbreviation: SSC, sidescatter.


signaling in the colon and modulate secretion of Glp-1requires further investigations.

In summary, this preclinical study demonstrates thepotential of pharmacological inhibition of ASBT inhalting progression of fibrosing cholangiopathies duringthe early phase of the disease process and identifies sev-eral mechanisms of action, which may operatesynergistically.

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Author names in bold designate shared co-firstauthorship.

Supporting Information

Additional Supporting Information may be found atonlinelibrary.wiley.com/doi/10.1002/hep.27973/suppinfo.



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