1. Volume 125, Issue 3, Pages 825-838 (September 2003)
Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice 
Martin Wagner, Peter Fickert, Gernot Zollner, Andrea Fuchsbichler, Dagmar Silbert, Oleksiy Tsybrovskyy, Kurt Zatloukal, Grace L. Guo, John D. Schuetz, Frank J. Gonzalez, Hanns-Ulrich Marschall, Helmut Denk, Michael Trauner 
Gastroenterology 
Volume 125, Issue 3, Pages (September 2003)
DOI: /S (03)

2. Figure 1
FXR+/+ and FXR−/− develop different types of liver injury in response to CBDL. Representative macroscopic and histological features in 7-day CBDL FXR+/+ (A, C, and E) and FXR−/− (B, D, and F). (A) CBDL in FXR+/+ induces pronounced dilatation of the common bile duct (CBD; arrowheads). Note also the appearance of necrotic areas (arrows) on the liver surface. (B) Situs of a CBDL FXR−/−. Note that the CBD (arrowheads) is less dilated compared with the CBDL FXR+/+. The liver surface appears normal, without appearance of necrotic areas. (C) Typical liver histology in CBDL FXR+/+ showing bile duct elongation and a bile infarct (asterisk; acinar zones 1 and 2). (D) CBDL FXR−/− lack development of bile infarcts. (E) Bile infarct (asterisk) in FXR+/+ infiltrated with neutrophils (arrowheads). (F) Liver injury in CBDL FXR−/− comprises disseminated liver cell necroses (asterisks), mild to moderate steatosis, and an increased number of mitotic figures (arrowheads). Original magnification for (C) and (D), 10×; for E and F, 20×. cv, central vein; pv, portal vein; bd, bile duct.
Gastroenterology  , DOI: ( /S (03) )

3. Figure 2
Necrosis represents the main type of cell death in CBDL FXR+/+ and FXR−/−. Double-immunofluorescence labeling for activated caspase-3 (red) and cytokeratin (CK) 18 (green) in 7-day CBDL FXR+/+ (A and C) and FXR−/− (B and D). (A) Zone of an early bile infarct (asterisk) with an altered hepatocellular CK-IF network characterized by fuzzy and partially destroyed cell borders and diminution of the CK-IF network. Only a minority of cells is positive for activated caspase-3 (arrowhead). (C) Activated caspase-3 (yellow because of colocalization with its cleaved substrate CK 18) in cells with CK-IF network breakdown and granular cytoplasmic condensation (characteristic of apoptotic cell death) in a minority of cells (arrowheads), indicating that hepatocyte necrosis represents the main type of cell death in CBDL FXR+/+. (B and D) CBDL FXR−/− liver showing disseminated hepatocellular necroses (arrowheads). Apoptotic cell death is detected only in a minority of cells (arrow). pv, portal vein. Bar = 20 μm.
Gastroenterology  , DOI: ( /S (03) )

4. Figure 3
Bile duct proliferation and elongation is more pronounced in CBDL FXR+/+ than in CBDL FXR−/−. Representative immunohistochemistry for cytokeratin 19 (brown) in 7-day CBDL FXR+/+ (A) and FXR−/− (B). For morphometric analysis, the bile duct mass (yellow borders) was measured by a semiautomatic system and normalized to the size of portal veins (green borders) as described.15 (A and B) Note the higher degree of bile duct proliferation and elongation in CBDL FXR+/+ compared with CBDL FXR−/−. (C) Morphometric analysis showed significantly increased bile duct mass in CBDL mice compared with naı̈ve mice (P < 0.001) and, in addition, a significantly higher degree in CBDL FXR+/+ when compared with CBDL FXR−/− (P < 0.01). Data are presented as mean values with 95% confidence intervals for bile duct areas per portal field. (Original magnification for A and B, 20×.)
Gastroenterology  , DOI: ( /S (03) )

5. Figure 4
FXR−/− lack an increase in biliary pressure in response to common bile duct ligation (CBDL). Cholangiomanometry was performed after relaparotomy of CBDL (for 3 days) FXR+/+ and CBDL FXR−/− mice. After cannulation, a catheter was fixed in the common bile duct and connected to a low-pressure transducer. After equilibration to ambient air pressure, the system was occluded, and the increase in intrabiliary pressure was continuously measured for 6 minutes (the time point when a steady state was reached); n = 3 for each genotype. In CBDL FXR+/+, intrabiliary pressure rapidly increased after closure of the system (closed circles). In contrast, biliary pressure in CBDL FXR−/− (open circles) did not exceed the ambient pressure calibration line. BDL, bile duct ligation.
Gastroenterology  , DOI: ( /S (03) )

6. Figure 5
Alterations of tight junctions in CBDL FXR+/+ and FXR−/−. Immunofluorescence labeling of the tight junction protein ZO-1 (red) was performed in naı̈ve FXR+/+ (A) and naı̈ve FXR−/− (C), 7-day CBDL FXR+/+ (B), and 7-day CBDL FXR−/− (D). (A and C) Immunofluorescence in naı̈ve FXR+/+ and FXR−/− shows a regular ZO-1 pattern. (B) ZO-1 staining in CBDL FXR+/+ shows a strikingly altered tight junction morphology characterized by an elongated and partially discontinuous pattern along the canalicular membrane. In addition, the canalicular system is apparently collapsed, suggesting disruption of tight junctions (arrowheads). (D) ZO-1 staining in CBDL FXR−/− shows preserved tight junction morphology with continuous ZO-1 staining. Bar = 20 μm.
Gastroenterology  , DOI: ( /S (03) )

7. Figure 6
Effects of CBDL on Mrp3 expression in FXR+/+ and FXR−/−. Total RNA and liver membranes were isolated from control and CBDL (1, 3, and 7 days) FXR+/+ and FXR−/− and analyzed by reverse-transcription PCR (A) and Western blotting (B). In addition, Mrp3 tissue localization was assessed by immunofluorescence staining (C-F) as described in Materials and Methods. (A) Mrp3 steady-state mRNA levels were increased in both genotypes after CBDL (n = 5 in each group; mean ± SEM; ∗CBDL vs. naı̈ve, P < 0.05). Values are expressed as percentage of controls (100%). (B) Baseline Mrp3 protein levels were compared in naı̈ve FXR+/+ and FXR−/− samples, which were run on identical gels. Mrp3 protein amounts were equal in naı̈ve FXR+/+ and FXR−/−. CBDL led to significantly increased Mrp3 protein levels in 7-day CBDL FXR+/+ and to an even higher extent in 3- and 7-day CBDL FXR−/−. Data are expressed as the -fold change compared with controls (n = 3 to 4 in each group; ∗P < 0.05 vs. control). (C and E) Weak immunostaining for Mrp3 of basolateral hepatocellular membranes and bile duct epithelial cells in both naı̈ve genotypes. (D and F) Enhanced basolateral staining 7 days after CBDL. Canaliculi (arrowheads) stain negative for Mrp3. (F) Note also that Mrp3 immunostaining appears even more pronounced in CBDL FXR−/−. bd, bile duct. Bar = 20 μm.
Gastroenterology  , DOI: ( /S (03) )

8. Figure 7
Effects of bile duct ligation on Mrp4 expression in FXR+/+ and FXR−/−. Total RNA and liver membranes were isolated from control and CBDL (1, 3, and 7 days) FXR+/+ and FXR−/− and analyzed by reverse-transcription PCR (A) and Western blotting (B). In addition, Mrp4 tissue localization was assessed by immunofluorescence staining (C-F) as described in Materials and Methods. (A) Steady-state mRNA levels were significantly induced after CBDL in both genotypes and to an even higher degree in CBDL FXR−/− compared with CBDL FXR+/+ (n = 5 in each group; means ± SEM; ∗CBDL vs. naı̈ve, P < 0.05; #FXR+/+ vs. FXR −/−, P < 0.05). Values are expressed as percentage of controls (100%). (B) Baseline Mrp4 protein levels were compared in naı̈ve FXR+/+ and FXR−/− samples, which were run on identical gels. Mrp4 protein amounts were equal in naı̈ve FXR+/+ and FXR−/−. CBDL led to significantly increased Mrp4 protein levels in 7-day CBDL FXR+/+ and to an even higher extent in 3- and 7-day CBDL FXR−/−. Data are expressed as the -fold change compared with controls (n = 3 to 4 in each group; ∗P < 0.05 vs. control). (C and E) In control conditions, only a sinusoidal immunostaining was observed in both genotypes. (D and F) In response to CBDL, a specific basolateral Mrp4 staining was seen in both genotypes. Canaliculi stained negative for Mrp4 (arrowheads). Bar = 20 μm.
Gastroenterology  , DOI: ( /S (03) )

9. Figure 8
Effects of bile duct ligation on Mrp2 expression in FXR+/+ and FXR−/−. Total RNA and liver membranes were isolated from control and CBDL (1, 3, and 7 days) FXR+/+ and FXR−/− and analyzed by RT-PCR (A) and Western blotting (B). In addition, Mrp2 tissue localization was assessed by immunofluorescence staining (C-F) as described in Materials and Methods. (A) Mrp2 steady-state mRNA levels were unchanged in response to CBDL in FXR+/+ and FXR−/− compared with corresponding controls, but Mrp2 mRNA levels differed significantly between genotypes after 3-day CBDL (n = 5 in each group; means ± SEM; ∗FXR+/+ vs. FXR −/−, P < 0.05). Values are expressed as percentage of controls (100%). (B) Baseline Mrp2 protein levels were compared in naı̈ve FXR+/+ and FXR−/− samples, which were run on identical gels. Mrp2 protein amounts were equal in naı̈ve FXR+/+ and FXR−/−. CBDL had no effect on Mrp2 protein levels in either genotype. Data are expressed as the fold change compared with controls (n = 3 to 4 in each group). (C and E) Regular canalicular immunostaining for Mrp2 in both naı̈ve genotypes. (D and F) Increased canalicular surface in response to CBDL in both genotypes. Inserts show a proper canalicular localization of Mrp2 (red, yellow due to colocalization with cytokeratin 18 in green; original magnification, 100×). bd, bile duct; pv, portal vein. Bar = 20 μm.
Gastroenterology  , DOI: ( /S (03) )

10. Figure 9
Effects of bile duct ligation on Bsep expression in FXR+/+ and FXR−/−. Total RNA and liver membranes were isolated from control and CBDL (1, 3, and 7 days) FXR+/+ and FXR−/− and analyzed by reverse-transcription PCR (A) and Western blotting (B). In addition, Bsep tissue localization was assessed by immunofluorescence staining (C-F) as described in Materials and Methods. (A) Bsep steady-state mRNA levels were significantly lower in naı̈ve FXR−/− compared with FXR+/+. CBDL in FXR+/+ significantly increased Bsep expression on day 1, returning to baseline levels in CBDL until day 7, whereas Bsep expression remained at low levels in CBDL FXR−/− (n = 5 in each group; means ± SEM; ∗CBDL vs. naı̈ve, P < 0.05; #FXR+/+ vs. FXR−/−, P < 0.05). Values are expressed as percentage of controls (100%). (B) Baseline Bsep protein levels were compared in naı̈ve FXR+/+ and FXR−/− samples, which were run on identical gels. Naı̈ve FXR−/− had significantly lower Bsep protein levels compared with FXR+/+. CBDL FXR+/+ showed a trend for higher Bsep protein levels, without reaching statistical significance, whereas Bsep protein was virtually undetectable in CBDL FXR−/−. Data are expressed as the fold change compared with controls (n = 3 to 4 in each group; #FXR+/+ vs. FXR−/−, P < 0.05). (C) Regular canalicular immunostaining for Bsep in naı̈ve FXR+/+ (red, yellow due to colocalization with cytokeratin 18 in green). (E) Reduced Bsep staining in FXR−/−. (D) Increased Bsep staining in CBDL FXR+/+, whereas (F) Bsep is hardly detectable in CBDL FXR−/−. bd, bile duct; pv, portal vein. Bar = 20 μm.
Gastroenterology  , DOI: ( /S (03) )

11. Figure 10
Suggested sequence of events in CBDL FXR+/+ and FXR−/− mice. (A) CBDL in FXR+/+ induces (1) adaptive overexpression of basolateral Mrp3 and Mrp4. Note, also, that canalicular Bsep expression is preserved. CBDL results in (2) increased canalicular pressure, (3) alterations of tight junction structure, and (4) increased serum bile acid levels by regurgitation of bile via a paracellular pathway. (B) (1) CBDL in FXR−/− induces adaptive basolateral Mrp3 and Mrp4 expression to an even higher extent compared with CBDL FXR+/+. Bsep expression strictly depends on FXR and therefore is reduced in FXR−/−. CBDL in FXR−/− therefore results in (2) less pronounced increase in biliary pressure, (3) preserved tight junctions, and (4) less pronounced increase in serum and, conversely, intrahepatic bile acid levels. BA, bile acids.
Gastroenterology  , DOI: ( /S (03) )