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Am J Physiol Gastrointest Liver Physiol 290: G923-G932, 2006. First published December 15, 2005; doi:10.1152/ajpgi.00490.2005
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LIVER AND BILIARY TRACT

Coordinated induction of bile acid detoxification and alternative elimination in mice: role of FXR-regulated organic solute transporter-{alpha}/beta in the adaptive response to bile acids

Gernot Zollner,1 Martin Wagner,1 Tarek Moustafa,1 Peter Fickert,1 Dagmar Silbert,1 Judith Gumhold,1 Andrea Fuchsbichler,2 Emina Halilbasic,1 Helmut Denk,2 Hanns-Ulrich Marschall,3 and Michael Trauner1

1Laboratory of Experimental and Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine, and 2Department of Pathology, Medical University Graz, Graz, Austria; and 3Karolinska University Hospital Huddinge, Stockholm, Sweden.

Submitted 17 October 2005 ; accepted in final form 9 December 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The bile acid receptor farnesoid X receptor (FXR) is a key regulator of hepatic defense mechanisms against bile acids. A comprehensive study addressing the role of FXR in the coordinated regulation of adaptive mechanisms including biosynthesis, metabolism, and alternative export together with their functional significance is lacking. We therefore fed FXR knockout (FXR–/–) mice with cholic acid (CA) and ursodeoxycholic acid (UDCA). Bile acid synthesis and hydroxylation were assessed by real-time RT-PCR for cytochrome P-450 (Cyp)7a1, Cyp3a11, and Cyp2b10 and mass spectrometry-gas chromatography for determination of bile acid composition. Expression of the export systems multidrug resistance proteins (Mrp)4–6 in the liver and kidney and the recently identified basoalteral bile acid transporter, organic solute transporter (Ost-{alpha}/Ost-beta), in the liver, kidney, and intestine was also investigated. CA and UDCA repressed Cyp7a1 in FXR+/+ mice and to lesser extents in FXR–/– mice and induced Cyp3a11 and Cyp2b10 independent of FXR. CA and UDCA were hydroxylated in both genotypes. CA induced Ost-{alpha}/Ost-beta in the liver, kidney, and ileum in FXR+/+ but not FXR–/– mice, whereas UDCA had only minor effects. Mrp4 induction in the liver and kidney correlated with bile acid levels and was observed in UDCA-fed and CA-fed FXR–/– animals but not in CA-fed FXR+/+ animals. Mrp5/6 remained unaffected by bile acid treatment. In conclusion, we identified Ost-{alpha}/Ost-beta as a novel FXR target. Absent Ost-{alpha}/Ost-beta induction in CA-fed FXR–/– animals may contribute to increased liver injury in these animals. The induction of bile acid hydroxylation and Mrp4 was independent of FXR but could not counteract liver toxicity sufficiently. Limited effects of UDCA on Ost-{alpha}/Ost-beta may jeopardize its therapeutic efficacy.

nuclear receptors; cholestrasis; liver; kidney; intestine; farnesoid X receptor; cholic acid; ursodeoxycholic acid

BILE ACIDS are potentially hepatotoxic, and their intrahepatic accumulation in cholestasis causes liver injury, ultimately leading to liver fibrosis and cirrhosis (11). Therefore, bile acid homeostasis needs to be tightly regulated (3, 7, 34, 35). The bile acid receptor farnesoid X receptor (FXR; NR1H4) plays a critical role in the transcriptional regulation of various steps in bile acid homeostasis (7), and loss of FXR leads to increased susceptibility to bile acid-induced liver injury (26, 48).

Bile acid-induced liver damage may be counteracted by a coordinated reduction of basolateral bile acid uptake, decreased bile acid synthesis, and induction of bile acid metabolism/detoxification followed by alternative basolateral bile acid secretion (40). Bile acid detoxification can occur via phase I hydroxylation mediated by the cytochrome P-450 (Cyp) enzymes Cyp3a11 (2, 31, 44) (representing the rodent ortholog of human CYP3A4) and potentially Cyp2b10. After alternative secretion via the hepatocyte’s basolateral membrane, these water-soluble metabolites can be eliminated by the kidney. Alternative hepatic basolateral bile acid excretion during cholestasis is accomplished by the induction of multidrug resistance-associated protein (Mrp)3 (8, 10, 24, 38, 47) and Mrp4 (6, 21, 24, 38, 45). Mrp4 is localized to the basolateral membrane of hepatocytes and to apical membranes of proximal tubular cells in the kidney (6, 36) and may thus not only assist Mrp3 in alternative hepatic basolateral bile acid export but also renal Mrp2 in urinary bile acid elimination. The roles of Mrp5 and Mrp6 in bile acid transport remain elusive but possible because both are localized to basolateral membranes and are expressed in the liver and kidney (18, 23, 43).

In addition to the ATP-binding cassette (ABC) transporter family, the recently identified organic solute transporter (Ost)-{alpha}/Ost-beta represents a novel candidate basolateral bile salt export system in the liver. Both subunits ({alpha} and beta) of this heteromeric transporter are required for the transport of various bile acids and their conjugates (4, 25, 42). In the ileum, where bile acids are taken up by the apical bile salt transporter (Asbt), Ost-{alpha}/Ost-beta mediates their basolateral excretion, thus contributing to the enterohepatic bile acid circulation (4). Ost-{alpha}/Ost-beta is also expressed in the kidney and (although to a lower extent) in the liver, where it localizes to the basolateral membrane of hepatocytes and cholangiocytes (29). Although renal localization has not yet been investigated, it is conceivable to hypothesize that Ost-{alpha}/Ost-beta may be localized to the basolateral membrane as in the liver and intestine. It is attractive to speculate that this recently characterized basolateral bile acid exporter may also take part in the adaptive transporter response during cholestasis.

Although molecular changes of individual transporters and enzymes in response to bile acid challenge and cholestasis have been reported in the past, a study investigating the role of FXR in orchestrating bile acid effects on adaptive mechanisms together with their functional significance (i.e., bile acid composition) is still lacking. Therefore, the aims of this study were 1) to determine whether potentially toxic cholic acid (CA), a major bile acid accumulating during cholestasis, coordinately regulates expression of bile acid synthesis, detoxification enzymes, and alternative basolateral bile acid transporters (including Mrp4–6 and Ost-{alpha}/Ost-beta) in the liver and kidney in the presence and absence of FXR; 2) to compare these changes with those induced by therapeutically used ursodeoxycholic acid (UDCA); and 3) to assess the functional significance of the observed molecular changes by studying bile acid composition.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. C57BL/6J mice with targeted disruption of FXR (26) and wild-type littermates, kindly provided by Dr. Frank J. Gonzalez [National Institutes of Health (National Institutes of Health, Bethesda, MA)], were housed with a 12:12-h light-dark cycle and permitted ad libitum consumption of water and a standard mouse diet. The experimental protocols were approved by the local Animal Care and Use Committee according to criteria outlined in the NIH Guide for the Care and Use of Laboratory Animals (NIH Pub. No. 86-23, Revised 1985).

Bile acid feeding. To study the effects of CA and UDCA on transporter expression, male mice were fed a diet supplemented with either CA [1% (wt/wt), Aldrich, Steinheim, Germany] or UDCA [1% (wt/wt), kindly provided by the Falk Foundation, Freiburg, Germany] for 7 days (8, 37, 47). Controls were fed a standard mouse diet. Livers were excised and processed as described (8, 47). Parts of the animals were analyzed in a previous study (47).

Preparation of total RNA and determination of mRNA levels by real-time PCR. Total RNA was prepared, and mRNA levels of Mrp4, Cyp3a11, Cyp2b10, Asbt, and 28S rRNA were assessed by real-time TaqMan PCR as described previously (38, 39). TaqMan oligonucleotides and probes for Ost-{alpha}/Ost-beta, Mrp5, and Mrp6 were as follows: Mrp5 forward primer 5'-ctggatatctctgtgcattctcat-3', reverse primer 5'-gaaaaggtcatgtaggagaaaagt-3', and probe 5'-aagcccttccggaccactaccaagc-3' (GenBank Accession No. BC042581); Mrp6 forward primer 5'-ctggtgggtcttctggatg-3', reverse primer 5'- ctgggcctgtctggtctg-3', and probe 5'-cagtgacgacctcggaggctttcct-3' (GenBank Accession No. NM_018795); Ost-{alpha} forward primer 5'-gtctcaagtgatgaactgcca-3' and reverse primer 5'-ttgagtgctgagtccaggtc-3' (GenBank Accession No. NM_145932); and Ost-beta forward primer 5'-gtattttcgtgcagaagatgcg-3' and reverse primer 5'-tttctgtttgccaggatgctc-3' (GenBank Accession No. AY279396). Sybr green assays without the use of specific probes were performed for determination of Ost-{alpha} and Ost-beta mRNA.

Preparation of liver membranes and analysis of Mrp4 protein levels by Western blot analysis. Liver and kidney membranes were prepared as described previously (33, 39, 47). Mrp4 protein levels were determined using a polyclonal antibody against Mrp4 (dilution 1:1,000; kindly provided by Dr. John D. Schuetz, St. Jude’s Children’s Research Hospital, Memphis, TN) as previously described (38, 39). Blots were reprobed with an anti-beta-actin antibody (dilution 1:5,000; Sigma) to confirm the specificity of changes in transporter protein levels.

Bile acid measurements. Liver pieces (50–100 µg) and serum (50–100 µl) of 3 animals/group were pooled, extracted, and analyzed by electrospray- and gas chromatography-mass spectrometry as described previously (38).

Statistical analysis. In each group, three to five animals were studied. Data are reported as arithmetic means ± SD. For statistical analysis, one-way ANOVA was used with Bonferroni posttesting testing when appropriate. Differences between FXR+/+ and FXR–/– animals were evaluated by Student’s t-test. A P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
CA and UDCA both coordinately repressed Cyp7a1 and induced Cyp3a11 and Cyp2b10 in FXR+/+ and FXR–/– animals. The effect of CA feeding on Cyp7a1 expression in the presence and absence of FXR or short heterodimer partner (SHP) has been examined in the past (12, 26, 41) but has been repeated for the present study to compare the effects of CA with UDCA. Baseline Cyp7a1 mRNA expression was higher in FXR–/– than FXR+/+ animals (160 ± 6% of standard diet-fed FXR+/+ mice, P < 0.05). Cyp7a1 mRNA expression was markedly repressed by both CA and UDCA in FXR+/+ animals to 7 ± 4% and 7 ± 7% of standard diet-fed FXR+/+ animals, respectively (both P < 0.001; Fig. 1A). Repression of Cyp7a1 by both bile acids was attenuated but still evident in FXR–/– animals [42 ± 23% of standard diet-fed FXR+/+ mice for CA (P < 0.05) and 66 ± 62% for UDCA (P < 0.05); Fig. 1A].


Figure 1
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  		Fig. 1. Cholic acid (CA) and ursodeoxycholic acid (UDCA) repress cytochrome P-450 (Cyp)7a1 and induce Cyp3a11 and Cyp2b10 in farnesoid X receptor (FXR)+/+ and FXR–/– mice. Total RNA was isolated from control diet-fed mice (solid bars) and CA-fed [open bars; 1% (wt/wt), 7 days] and UDCA-fed [shaded bars; 1% (wt/wt), 7 days] FXR+/+ and FXR–/– mice and analyzed by real-time PCR as described in MATERIALS AND METHODS. A, B, and C: data are expressed as a percentage (means + SD) of standard diet-fed FXR+/+ mice. Values are averages obtained from 5 animals/group. A: CA and UDCA repressed Cyp7a1 mRNA in FXR+/+ mice and, to a lesser extent, in FXR–/– mice. Both bile acids induced Cyp3a11 (B) and Cyp2b10 (C) mRNA levels independent of FXR. *P < 0.05, CA- or UDCA-fed vs. control diet-fed animals; #P < 0.05, FXR+/+ vs. FXR–/– mice.

 
Baseline mRNA expression of the sterol hydroxylating enzymes Cyp2b10 and Cyp3a11 were higher in FXR–/– mice compared with FXR+/+ mice [285 ± 160%, P < 0.05, and 184 ± 85% of standard diet-fed FXR+/+ mice, not significant (NS), respectively; Fig. 1, B and C]. In wild-type mice, both CA and UDCA induced Cyp3a11 mRNA to 341 ± 200% and 486 ± 85% and Cyp2b10 mRNA to 1,882 ± 1,362% and 2,314 ± 1,323% of standard diet-fed FXR+/+ mice, respectively (Fig. 1, B and C). Both bile acids induced Cyp3a11 and Cyp2b10 mRNA to similar extents in FXR–/– mice as in FXR+/+ mice; only Cyp2b10 induction by UDCA was less pronounced than in FXR+/+ mice (Fig. 1, B and C).

Taken together, these findings suggest that the induction of Cyp3a11 and Cyp2b10 by CA and UDCA is independent of FXR and that repression of Cyp7a1 by both bile acids in part depends on FXR.

Bile acid composition in CA- and UDCA-fed FXR+/+ and FXR–/– mice. To evaluate the functional significance of the changes in gene expression of sterol hydroxylating enzymes, we assessed intrahepatic and serum bile acid levels and composition. Baseline intrahepatic and serum bile acid levels did not differ between naive FXR+/+ and FXR–/– mice, as previously described (38, 47). The relative bile acid composition in the liver and serum is shown in Tables 1 and 2. In line with previous findings (47), CA feeding led to significantly higher total intrahepatic bile acid concentrations in FXR–/– animals compared with FXR+/+ animals (442.7 ± 126.9 vs. 145.6 ± 40.4 µg/g liver, respectively, P < 0.05; Table 1) (26, 47). The intrahepatic enrichment of CA in FXR+/+ and FXR–/– mice fed with 1% CA was 60.2 ± 14% and 57.8 ± 10%, respectively (Table 1). UDCA treatment resulted in higher intrahepatic bile acid concentrations in FXR–/– mice compared with FXR+/+ mice as well (427.2 ± 59.8 vs. 282.9 ± 50.3 µg/g liver, respectively), and total intrahepatic bile acid levels were about twofold higher in UDCA- than in CA-fed FXR+/+ mice (Table 1). Intrahepatic UDCA enrichment was comparable between both genotypes (52.6 ± 5% for FXR+/+ mice and 42.2 ± 10% for FXR–/– mice; Table 1).


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  		Table 1. Intrahepatic bile acid levels and composition

 

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  		Table 2. Serum bile acid levels and composition

 
The induction of phase I metabolism during CA and UDCA feeding in both genotypes was reflected by the occurrence of hydroxylation products of CA and UDCA, respectively, in the liver and serum (Tables 1 and 2). In the serum of CA-fed FXR–/– mice, hydroxylation products of CA at 1beta-, 2beta-, 4beta-, and 6{alpha}-positions were identified, totally consisting of 7.4% of total serum bile acids, which was higher than in FXR+/+ mice, where 4.8% hydroxylations of CA at C1 or C6 positions were found (Table 2). Deoxycholic acid (DCA), which is formed in the intestine by bacterial 7-dehydrogenation of CA, contributed to ~20% of serum bile acids in both genotypes. Also, this hydrophobic bile acid underwent extensive hydroxylation as reflected by 20% and 13% of 1beta-hydroxy-DCA in the serum of FXR+/+ and FXR–/– mice, respectively (Table 2). During UDCA feeding, this hydrophilic bile acid became hydroxylated as well, at 1beta-, 4beta-, 5beta-, and 22-positions, but was also oxidized at C3 (Tables 1 and 2). In total, phase I products of UDCA contributed to ~20% of all hepatic and serum bile acids during UDCA feeding in both genotypes. Electrospray-mass spectrometry did not indicate the abundance of significant amounts of glycine amidates, sulfates, or glucosidic conjugates in the liver or serum during CA or UDCA feeding.

Taken together, these data indicate that the induction of sterol hydroxylases Cyp3a11 and Cyp2b10 results in the formation of hydroxylation products of CA and UDCA. Moreover, the high amount of hydroxylated bile acids in serum suggests alternative basolateral efflux of these compounds.

Effects of CA and UDCA feeding on hepatic and renal Mrp4 expression in FXR+/+ and FXR–/– mice. Baseline Mrp4 mRNA and protein levels were 22- and 10-fold higher in the kidney compared with the liver (both P < 0.01; data not shown). Baseline Mrp4 mRNA and protein levels in the liver and kidney were not significantly different between FXR+/+ and FXR–/– mice (Figs. 2 and 3) (38). CA feeding in FXR+/+ mice had no significant effects on hepatic Mrp4 mRNA (200 ± 131%, NS; Fig. 2A) and protein levels (123 ± 62% of standard diet-fed controls, NS; Fig. 2B). In FXR–/– livers, however, CA induced Mrp4 mRNA to 1,334 ± 411% (Fig. 2A) and protein levels to 384 ± 37% of standard diet-fed FXR+/+ livers (both P < 0.05; Fig. 2B). In contrast, UDCA feeding in FXR+/+ livers significantly increased hepatic Mrp4 mRNA to 484 ± 203% (Fig. 2C) and protein to 360 ± 58% of standard diet-fed FXR+/+ livers (both P < 0.05; Fig. 2D). Mrp4 induction was more pronounced in FXR–/– livers (712 ± 108% for mRNA and 1,068 ± 65% for protein, both P < 0.05; Fig. 2, C and D). Mrp4 mRNA and protein levels of Mrp4 correlated with total amounts of intrahepatic bile acid levels irrespective of the type of the fed bile acid (r = 0.9, r2 = 0.8, and P < 0.05 for mRNA and r = 0.8, r2 = 0.6, and P = 0.06 for protein).


Figure 2
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  		Fig. 2. Effects of CA and UDCA feeding on hepatic multidrug resistance protein (Mrp)4 expression in FXR+/+ and FXR–/– mice. Total RNA and liver membranes were isolated from control diet-fed mice (solid bars) and CA-fed [1% (wt/wt), 7 days] and UDCA-fed [1% (wt/wt), 7 days] FXR+/+ and FXR–/– mice (shaded bars) and analyzed by real-time PCR (A and C) and Western blot analysis (B and D) as described in MATERIALS AND METHODS. A and C: mRNA values; B and D: representative immunoblots. Data are expressed as percentage (means + SD) of standard diet-fed FXR+/+ mice. Values are averages obtained from 3–5 animals/group. CA induced Mrp4 mRNA (A) and protein (B) levels in FXR–/– mice but not in FXR+/+ mice. In contrast, UDCA significantly induced hepatic mRNA Mrp4 (C) and protein (D) levels in both genotypes. *P < 0.05, CA- or UDCA-fed vs. control diet-fed animals; #P < 0.05, FXR+/+ vs. FXR–/– mice.

 

Figure 3
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  		Fig. 3. Effects of CA and UDCA feeding on renal Mrp4 expression in FXR+/+ and FXR–/– mice. Total RNA and kidney membranes were isolated from control diet-fed mice (solid bars) and CA-fed [1% (wt/wt), 7 days] and UDCA-fed [1% (wt/wt), 7 days] FXR+/+ and FXR–/– mice (shaded bars) and analyzed by real-time PCR (A and C) and Western blot analysis (B and D) as described in MATERIALS AND METHODS. A and C: mRNA values; B and D: representative immunoblots. Data are expressed as percentages (means + SD) of standard diet-fed FXR+/+ mice. Values are averages obtained from 3–5 animals/group. CA induced Mrp4 mRNA (A) and protein (B) levels in FXR–/– mice but not in FXR+/+ mice. In contrast, UDCA significantly induced renal mRNA Mrp4 (C) and protein (D) levels in both genotypes. *P < 0.05, CA- or UDCA-fed vs. control diet-fed animals. #P < 0.05, FXR+/+ vs. FXR–/–

 
Similar to the liver, CA had no effects on Mrp4 mRNA and protein levels in the kidney of FXR+/+ mice (138 ± 86% and 121 ± 72% of controls, respectively, both NS; Fig. 3, A and B). CA feeding in FXR–/– mice, however, again increased renal Mrp4 mRNA and protein expression to 333 ± 124% (P < 0.05) and 208 ± 54% (NS) of standard diet-fed FXR+/+ mice, respectively (Fig. 3, A and B). The induction of Mrp4 in the kidney was less pronounced compared with the liver. UDCA upregulated renal Mrp4 mRNA and protein levels in wild-type animals to 330 ± 72% and 893 ± 39% of standard diet-fed FXR+/+ animals, respectively (both P < 0.05; Fig. 3, C and D). In FXR–/– animals, Mrp4 induction was slightly more pronounced (513 ± 147% for mRNA and 977 ± 40% for protein expression of standard diet-fed FXR+/+ animals, both P < 0.05; Fig. 3, C and D). Renal Mrp4 protein levels also correlated with serum bile acid levels (r = 0.92, r2 = 0.86, and P < 0.01).

Taken together, the magnitude of Mrp4 induction depends on the amount of bile acid levels and is independent of FXR.

Hepatic and renal Mrp5 and Mrp6 mRNA expression levels were not affected by CA and UDCA feeding. Baseline Mrp5 mRNA levels were 10-fold higher in the kidney than in the liver, whereas Mrp6 mRNA expression in the kidney was only 14% of the liver, in line with previous results (18). Mrp5 and Mrp6 mRNA expression did not differ significantly between naive FXR+/+ and FXR–/– animals in the liver and kidney (Table 3). Neither CA nor UDCA feeding significantly affected Mrp5 and Mrp6 mRNA levels in livers and kidneys of FXR+/+ and FXR–/– animals (Table 3).


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  		Table 3. Mrp5 and Mrp6 mRNA expression in the liver and kidney of CA- and UDCA-fed FXR+/+ and FXR–/– mice

 
CA induced Ost-{alpha}/Ost-beta mRNA in the liver, kidney, and intestine in an FXR-dependent fashion, whereas UDCA had only minor effects on Ost-{alpha}/Ost-beta expression. Baseline Ost-{alpha} mRNA expression was similar in FXR+/+ and FXR–/– livers (Fig 4A), whereas hepatic Ost-beta mRNA was not detectable in naive FXR–/– livers (Fig. 4B). In the kidney and ileum, Ost-{alpha} and Ost-beta mRNA levels were comparable between naive FXR+/+ and FXR–/– animals (Fig. 4, C–F).


Figure 4
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  		Fig. 4. Effects of CA and UDCA feeding on hepatic, renal, and ileal organic soute transporter (Ost)-{alpha}/Ost-beta expression in FXR+/+ and FXR–/– mice. Total RNA was isolated from control diet-fed mice (solid bars) and CA-fed [open bars; 1% (wt/wt), 7 days] and UDCA-fed [shaded bars; 1% (wt/wt), 7 days] FXR+/+ and FXR–/– mice and analyzed by real-time PCR as described in MATERIALS AND METHODS. Data are expressed as percentages (means + SD) of standard diet-fed FXR+/+ mice. Values are averages obtained from 5 animals/group. CA induced Ost-{alpha} mRNA in the liver (A), kidney (C), and ileum (E) in a strictly FXR-dependent fashion, whereas UDCA increased Ost-{alpha} expression only in the ileum (E) and had no effect in the liver (A) and kidney (C). CA led to a massive induction of Ost-beta in the liver (B) and had only moderate effects in the kidney (D) and ileum (F). UDCA led to a slight upregulation of Ost-beta in the liver (B), kidney (D), and ileum (F). Both bile acids had no effect on Ost-beta expression in FXR–/– mice. *P < 0.05, CA- or VDCA-fed vs. control diet-fed animals.

 
CA induced Ost-{alpha} mRNA to 286 ± 91%, 278 ± 58%, and 442 ± 109% of controls in the FXR+/+ liver, kidney, and ileum, respectively (P < 0.05 for all differences), whereas UDCA feeding upregulated Ost{alpha} only in the FXR+/+ ileum (365 ± 116%, P < 0.05; Fig. 4, A, C, and E). In FXR–/– animals, neither CA nor UDCA caused Ost-{alpha} induction in the liver, kidney, or ileum (Fig. 4, A, C, and E). CA resulted in a robust induction of Ost-beta mRNA to 2,816 ± 922% of controls in the liver (P < 0.05) but had only moderate effects on renal (224 ± 72% of controls, NS) and ileal Ost-beta expression (217 ± 24% of controls, P < 0.05; Fig. 4, B, D, and F). UDCA slightly increased hepatic, renal, and ileal Ost-beta expression to 158 ± 103%, 229 ± 28%, and 174 ± 45% of controls without reaching statistical significance (Figs. 4, B, D, and F). In FXR–/– mice, neither CA nor UDCA had any significant effect on Ost-beta expression in all tissues investigated (Figs. 4, B, D, and F).

Taken together, these findings clearly demonstrate that CA induces Ost-{alpha}/Ost-beta in an FXR-dependent fashion.

Expression of Asbt in the ileum and kidney of FXR+/+ and FXR–/– mice. Because ileal and proximal renal tubular bile acid uptake by Asbt may determine systemic bile acid levels and thus contribute to the observed differences in hepatic and serum bile acid levels after CA and UDCA feeding, we also investigated Asbt expression in the ileum and kidney. CA feeding in FXR+/+ mice slightly reduced ileal Asbt expression to 62 ± 17% of controls (NS), whereas UDCA induced ileal Asbt to 179 ± 33% of controls (P < 0.05). In the FXR–/– ileum, CA and UDCA slightly induced Asbt expression from baseline levels of 138 ± 115% of naive FXR+/+ ileum to 189 ± 97% and 187 ± 114% of naive FXR+/+ ileum, respectively (NS). In the kidney, CA and UDCA moderately repressed Asbt mRNA expression to 65 ± 13% and 70 ± 18% of controls in FXR+/+ animals, respectively, and in FXR–/– to 26 ± 13% and 63 ± 9% of naive FXR+/+ animals (NS).

Taken together, Asbt repression after CA or induction after UDCA feeding in the ileum may account for the observed differences in bile acid levels.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study demonstrates a coordinated activation of an adaptive program in response to CA and UDCA challenge. The findings demonstrate repression of Cyp7a1, catalyzing the rate-limiting step in bile formation, and induction of various genes involved in bile acid detoxification and basolateral export. Most importantly, we provide evidence that the recently identified bile acid export system Ost-{alpha}/Ost-beta is induced by CA in an FXR-dependent fashion. Lack of Ost-{alpha}/Ost-beta induction in FXR–/– animals might contribute to increased susceptibility to CA-induced liver injury in these animals. Notably, with the exception of Ost-{alpha}/Ost-beta, both toxic CA and nontoxic UDCA induced a similar adaptive response pattern in both genotypes, which may have important therapeutic implications for UDCA.

Repression of Cyp7a1, the key enzyme mediating the rate-limiting step in bile acid biosynthesis, may limit intrahepatic bile acid accumulation during cholestasis. As expected, CA repressed Cyp7a1 in FXR+/+ animals and to a lesser extent in FXR–/– animals (12, 26, 41). Downregulation of Cyp7a1 by CA in the absence of FXR has been reported previously (26) but was repeated to compare the effects of CA with those of UDCA for the present study. Surprisingly, UDCA also reduced Cyp7a1 expression in both genotypes; however, similar to CA, Cyp7a1 repression in the absence of FXR was attenuated. This in vivo finding is in line with results from a previous in vitro study (15) where UDCA was shown to be a partial agonist in FXR transactivation assays and to differentially regulate the expression of individual FXR targets such as Cyp7a1. Many studies have demonstrated multiple redundant pathways for bile acid feedback suppression of the Cyp7a1 gene (7). As such, bile acids also seem to have negative effects on hepatocyte nuclear factor (HNF)-4{alpha}-mediated activation of the Cyp7a1 promoter that are independent of FXR (5). Furthermore, a recent study (16) has indicated that rifampicin-activated pregnane X receptor (PXR; NR1I2) interacts with HNF-4{alpha} and reduces interaction of peroxisome proliferator-activated receptor-{gamma} coactivator (PGC-1{alpha}) with HNF-4{alpha}, thus leading to inhibition of human CYP7A1 gene transcription (16). Because PXR is a receptor for certain bile acids (31, 44), one might speculate that CA and UDCA or their metabolites are able to activate PXR and thus inhibit Cyp7a1 transcription. However, the detailed molecular mechanism of Cyp7a1 repression in mice still has to be determined. Nevertheless, suppression of bile acid synthesis via Cyp7a1 by UDCA may contribute to its therapeutic properties in the therapy of cholestatic disorders.

Phase I hydroxylation renders bile acids more hydrophilic and thus less toxic and represents an important mechanism counteracting bile acid-induced liver damage (2, 9, 24, 27, 28, 30–32, 39, 44, 46). In the present work, expression of the sterol hydroxylases Cyp3a11 and Cyp2b10 was markedly induced by CA and UDCA administration independent of FXR. Other bile acid receptors such as PXR or the vitamin D receptor (VDR; NR1I1) may account for bile acid-mediated induction of Cyp2b10 and Cyp3a11. Administration of PXR ligands leads to the induction of Cyp3a11 and Cyp2b10 (31, 39, 44), and activation of VDR stimulates Cyp3a11 transcription (19). Therefore, it is attractive to speculate that the observed effects of CA and UDCA (or their metabolites) may be accounted, at least in part, for the activation of PXR and/or VDR. The role of the constitutive androstane receptor (CAR; NR1I3) in mediating the induction of phase I hydroxylation by bile acids remains to be determined, because CAR has not yet been shown to be a bile acid receptor.

For the human homolog of Cyp3a11, i.e., CYP3A4, hydroxylation of bile acids at the 1beta- and 6{alpha}/6beta-positions has been demonstrated in vitro (2, 44); thus at least the overexpression of Cyp3a11 is reflected by the appearance of hydroxylation products of CA and UDCA in the liver and serum. In total, phase I hydroxylation products of CA and DCA (formed by intestinal bacterial 7-dehydrogenation of CA) and UDCA contributed to ~20–25% of all hepatic and serum bile acids during CA and UDCA feeding in both genotypes. However, increased hydroxylation in CA-fed FXR–/– animals could not sufficiently prevent liver injury in these animals (26, 50).

Increased hepatocellular bile acid export might limit hepatic bile acid toxicity under cholestatic conditions. We and others (8, 22, 38, 47) have reported the induction of canalicular Mrp2 and basolateral Mrp3 after CA and UDCA feeding and after common bile duct ligation, which is considered to counteract intrahepatic bile acid accumulation. In addition, recent work suggests that Mrp4 may protect from liver injury in common bile duct-ligated mice (20). In the present work, hepatic and renal Mrp4 was markedly upregulated in FXR–/– mice but hardly affected in FXR+/+ mice fed CA. In contrast, administration of the therapeutic bile acid UDCA induced Mrp4 expression in the liver and kidney already in FXR+/+ mice and to an even greater extent in FXR–/– mice. Lack of Mrp4 induction by CA in FXR+/+ animals can be explained by lower total bile acid levels in these animals compared with those fed UDCA. Moreover, Mrp4 expression demonstrated a strong correlation with bile acid levels irrespective of bile acid composition. Repression of ileal bile acid uptake mediated by Asbt by CA and Asbt induction by UDCA may account for the observed differences in total bile acid levels between UDCA- and CA-fed FXR+/+ animals. However, this remains speculative because only mRNA levels but not ileal bile acid uptake or bile acid pool size were determined in the present study.

The detailed molecular mechanisms leading to Mrp4 induction by CA and UDCA remain to be determined. While previous studies in mice lacking PXR make the involvement of PXR unlikely (49) and a VDR response element in the Mrp4 gene promoter has not yet been described, CAR could represent a potential mediator of Mrp4 induction. Administration of a specific CAR ligand to mice resulted in Mrp4 upregulation (1, 39); however, the involvement of CAR remains speculative, because CAR activation by bile acids remains to be demonstrated.

Nevertheless, the even more pronounced Mrp4 induction in FXR–/– animals compared to FXR+/+ animals was unable to prevent CA-induced liver injury in these animals. These findings argue against a major role for hepatic basolateral Mrp4 in counteracting liver toxicity caused by intrahepatic accumulation of CA. In the kidney, active bile acid excretion into proximal renal tubuli by overexpressed apical Mrp4 may contribute to urinary bile acid elimination. However, the relative contribution of active bile acid secretion to passive glomerular filtration is unclear at the moment, but markedly elevated serum bile acid levels together with Mrp4 induction again question the functional relevance of this transporter in the kidney. Inhibition of renal bile acid reuptake might also lead to increased urinary bile acid elimination because Asbt expression was slightly repressed by CA and UDCA in both genotypes.

A novel and attractive candidate for mediating alternative basolateral bile acid efflux is Ost-{alpha}/Ost-beta. CA markedly induced Ost-{alpha}/Ost-beta mRNA in FXR+/+ animals but not in FXR–/– animals in the present study, indicating an FXR-dependent regulation. This is in line with recently published studies (13, 14) demonstrating an FXR binding site within the gene promoters of Ost-{alpha} and Ost-beta. This suggests that increased intrahepatic bile acid accumulation and subsequent liver toxicity in CA-fed FXR–/– animals may not only be due to reduced bile salt export protein (Bsep) expression and impaired Na+/taurocholate cotransporter (Ntcp) and Cyp7a1 repression but also the consequence of absent Ost-{alpha}/Ost-beta induction. Overexpression of both Mrp3 and Mrp4 without Ost-{alpha}/Ost-beta induction was unable to prevent liver toxicity in FXR–/– mice, suggesting that Ost-{alpha}/Ost-beta may be a key efflux system for alternative basolateral bile acid export. The lack of Ost-{alpha}/Ost-beta induction by UDCA may jeopardize its therapeutic efficacy. The lack of Ost-{alpha}/Ost-beta induction may contribute to liver toxicity in FXR–/– animals fed CA, which cannot be prevented by the induction of bile acid hydroxylation and Mrp4. Because protein levels of Ost-{alpha}/Ost-beta could not be assessed due to lack of antibodies against murine Ost-{alpha}/Ost-beta and the functional significance of these findings remains hypothetical, the relative importance of Ost-{alpha}/Ost-beta to the adaptive transporter response has to be determined in future studies (including those using Ost-{alpha}/Ost-beta knockout mice).

Because both Mrp5 and Mrp6 are expressed in the liver and kidney (23), we speculated that Mrp5 and Mrp6 could possibly contribute to the alternative bile acid elimination in cholestasis. Although a role for in bile acid transport was questioned by a previous study (17), the role of Mrp5 in bile acid transport is currently under investigation. In the present study, neither CA nor UDCA were able to increase Mrp5 and Mrp6 expression, which makes their involvement in these adaptive excretory pathways less likely.

In summary, we demonstrated FXR-dependent and -independent adaptive changes including repression of bile acid synthesis and induction of bile acid hydroxylation and alternative basolateral efflux in response to bile acid challenge (Fig. 5). With the exception of Ost-{alpha}/Ost-beta, CA and UDCA caused similar changes; whether these adaptive mechanisms can be activated by other bile acid species remains to be determined. This study identifies Ost-{alpha}/Ost-beta as a novel, strictly FXR-dependent regulated transport system. One might speculate that the lack of Ost-{alpha}/Ost-beta induction may contribute to liver toxicity in FXR–/– animals fed CA, which cannot be prevented by the induction of bile acid hydroxylation and Mrp4. Stimulating Ost-{alpha}/Ost-beta expression during cholestasis might represent an attractive future target to counteract cholestatic liver injury.


Figure 5
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  		Fig. 5. Coordinated adaptive changes in bile acid synthesis, metabolism, and alternative export in CA- and UDCA-fed mice: effects of bile acid feeding in FXR+/+ (A) and FXR–/– (B) mice. In the liver, CA and UDCA repressed Cyp7a1 in FXR+/+ mice (A) and to a lesser extent in FXR–/– mice (B), suggesting partially FXR-dependent Cyp7a1 regulation, whereas both bile acids induced Cyp3a11 and Cyp2b10 to similar extents in both genotypes. A: CA upregulated hepatic Ost-{alpha}/Ost-beta, whereas UDCA had no effect on Ost-{alpha}/Ost-beta, in FXR+/+ mice. Absent Ost-{alpha}/Ost-beta induction in FXR–/– mice (B) indicates a strictly FXR-dependent regulation of this transporter and may contribute to the increased liver injury in CA-fed FXR–/– mice reported previously (26, 47). Mrp4 induction was independent of FXR and even more pronounced in FXR–/– mice, correlating with bile acid levels irrespective of bile acid treatment, but could not sufficiently counteract liver injury. Relatively low bile acid levels in CA-fed FXR+/+ mice were unable to induce Mrp4 expression (A). Neither CA nor UDCA affected Mrp5 and Mrp6 expression, arguing against a functional relevance of these transporters in adaptive bile acid excretion. In the ileum and kidney, regulation of Ost-{alpha}/Ost-beta by CA and UDCA in the ileum and kidney was similar but attenuated compared with the liver and Mrp4 induction in the kidney by CA or UDCA was comparable to the liver. The renal apical bile salt transporter (Asbt), mediating bile acid reuptake in proximal renal tubuli, was repressed by CA and UDCA in both genotypes, possibly facilitating urinary bile acid elimination. In the ileum, repression of Asbt in CA-fed FXR+/+ mice and the induction in UCDA-fed FXR+/+ mice may explain the higher bile acid levels in UDCA-treated animals.

 

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This work was supported by Austrian Science Foundation Grant P15502 [GenBank] and P18613 [GenBank] -BO5 (to M. Trauner).


   ACKNOWLEDGMENTS
 
The antibody against Mrp4 was kindly provided by Dr. John D. Schuetz (Memphis, TN), and FXR knockout breeding pairs were a generous gift from Frank J. Gonzalez (Bethesda, MD).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Trauner, Laboratory of Experimental and Molecular Hepatology, Div. of Gastroenterology and Hepatology, Dept. of Internal Medicine, Medical Univ. Graz, Auenbruggerplatz 15, Graz A-8036, Austria (e-mail: michael.trauner{at}meduni-graz.at)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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