Peter Fickert

CAR and PXR agonists stimulate hepatic bile acid and bilirubin detoxification and elimination pathways in mice

Induction of hepatic phase I/II detoxification enzymes and alternative excretory pumps may limit hepatocellular accumulation of toxic biliary compounds in cholestasis. Because the nuclear xenobiotic receptors constitutive androstane receptor (CAR) and pregnane X receptor (PXR) regulate involved enzymes and transporters, we aimed to induce adaptive alternative pathways with different CAR and PXR agonists in vivo. Mice were treated with the CAR agonists phenobarbital and 1,4‐bis‐[2‐(3,5‐dichlorpyridyloxy)]benzene, as well as the PXR agonists atorvastatin and pregnenolone‐16α‐carbonitrile. Hepatic bile acid and bilirubin‐metabolizing/detoxifying enzymes (Cyp2b10, Cyp3a11, Ugt1a1, Sult2a1), their regulatory nuclear receptors (CAR, PXR, farnesoid X receptor), and bile acid/organic anion and lipid transporters (Ntcp, Oatp1,2,4, Bsep, Mrp2‐4, Mdr2, Abcg5/8, Asbt) in the liver and kidney were analyzed via reverse‐transcriptase polymerase chain reaction and Western blotting. Potential functional relevance was tested in common bile duct ligation (CBDL). CAR agonists induced Mrp2‐4 and Oatp2; PXR agonists induced only Mrp3 and Oatp2. Both PXR and CAR agonists profoundly stimulated bile acid–hydroxylating/detoxifying enzymes Cyp3a11 and Cyp2b10. In addition, CAR agonists upregulated bile acid–sulfating Sult2a1 and bilirubin‐glucuronidating Ugt1a1. These changes were accompanied by reduced serum levels of bilirubin and bile acids in healthy and CBDL mice and by increased levels of polyhydroxylated bile acids in serum and urine of cholestatic mice. Atorvastatin significantly increased Oatp2, Mdr2, and Asbt, while other transporters and enzymes were moderately affected. In conclusion, administration of specific CAR or PXR ligands results in coordinated stimulation of major hepatic bile acid/bilirubin metabolizing and detoxifying enzymes and hepatic key alternative efflux systems, effects that are predicted to counteract cholestasis. (HEPATOLOGY 2005.)

Adaptive changes in hepatobiliary transporter expression in primary biliary cirrhosis

BACKGROUND/AIMS Information about alterations of hepatobiliary transporter expression in primary biliary cirrhosis (PBC) could provide important insights into the pathogenesis of cholestasis. This study aimed to determine the expression of hepatobiliary transport systems for bile salts (Na(+)/taurocholate cotransporter, NTCP; bile salt export pump, BSEP), organic anions (organic anion transporting protein, OATP2; canalicular conjugate export pump, MRP2; basolateral MRP homologue, MRP3), organic cations (canalicular multidrug export pump, MDR1), and phospholipids (canalicular phospholipid flippase MDR3) in livers from patients with advanced stages of PBC. METHODS Transporter mRNA and protein levels were assessed by reverse transcription polymerase chain reaction and Western blot analysis. Tissue distribution of transporters was investigated by immunohistochemistry and immunofluorescence microscopy. Hepatic bile acids were measured by gas chromatography-mass spectrometry. RESULTS Compared to controls, basolateral uptake systems (NTCP, OATP2) were reduced, canalicular export pumps for bile salts and bilirubin (BSEP, MRP2) were preserved, while canalicular MDR P-glycoproteins (MDR1, MDR3) and the basolateral efflux pump MRP3 were increased in PBC. Double immunofluorescence labeling with a canalicular marker (dipeptidyl peptidase IV) demonstrated proper canalicular localization of BSEP and MRP2 in PBC. OATP2 and MRP2 expression correlated inversely with hepatic levels of hydrophobic bile acids, while positively correlating with hepatic enrichment with ursodeoxycholic acid. CONCLUSIONS Down-regulation of basolateral uptake systems and maintenance/up-regulation of canalicular and basolateral efflux pumps may represent adaptive mechanisms limiting the accumulation of toxic biliary constituents.

Role of farnesoid X receptor in determining hepatic ABC transporter expression and liver injury in bile duct-ligated mice

BACKGROUND & AIMS Cholestasis induces changes in hepatic adenosine triphosphate-binding cassette (ABC) transporter expression. We aimed to investigate the role of the nuclear bile acid receptor (farnesoid X receptor [FXR]) in mediating changes in ABC transporter expression and in determining liver injury. METHODS Hepatic ABC transporter (multidrug resistance-associated proteins [Mrp] 2-4 and bile salt export pump [Bsep]) expression and localization were studied in common bile duct-ligated (CBDL) FXR knockout (FXR(-/-)), wild-type (FXR(+/+)), and sham-operated mice. Serum alanine aminotransferase, alkaline phosphatase, bilirubin and bile acid levels, hepatic bile acid composition, and liver histology were investigated. Cholangiomanometry and bile duct morphometry were performed. RESULTS CBDL induced expression of Mrp 3 and Mrp 4 in FXR(+/+) and even more in FXR(-/-), whereas Mrp 2 expression remained unchanged. Bsep expression was maintained in CBDL FXR(+/+) but remained undetectable in CBDL FXR(-/-). Alanine aminotransferase levels and mortality rates did not differ between CBDL FXR(+/+) and FXR(-/-). CBDL increased biliary pressure and induced bile ductular proliferation and bile infarcts in FXR(+/+), whereas FXR(-/-) had lower biliary pressures, less ductular proliferation, and developed disseminated liver cell necroses. CONCLUSIONS Overexpression of Mrp 3 and Mrp 4 in CBDL mice is FXR independent and could play an important role in the adaptive hepatic ABC transporter response to cholestasis. Maintenance of Bsep expression strictly depends on FXR and is a critical determinant of the cholestatic phenotype. Lack of bile infarcts in CBDL FXR(-/-) suggests that development of bile infarcts is related to bile acid-dependent bile flow and biliary pressure. This information is relevant for the potential use of FXR modulators in the treatment of cholestatic liver diseases.

Inflammation-induced cholestasis.

Inflammatory cytokines produced in response to various infectious and non‐infectious stimuli are potent inducers of intrahepatic cholestasis (inflammation‐induced cholestasis). The cholestatic effect of cytokines results mainly from inhibition of expression and function of hepatocellular transport systems which normally mediate hepatic uptake and biliary excretion of bile salts and various non‐bile salt organic anions (e.g. bilirubin). These cytokine effects are reversible and bile secretory function is restored upon disappearance of the inflammatory injury. This review summarizes the clinical, pathophysiological and molecular aspects of inflammation‐induced cholestasis.

Bile Acids as Regulators of Hepatic Lipid and Glucose Metabolism

Besides their well-established roles in dietary lipid absorption and cholesterol homeostasis, bile acids (BA) also act as metabolically active signaling molecules. The flux of reabsorbed BA undergoing enterohepatic circulation, arriving in the liver with the co-absorbed nutrients (e.g. glucose, lipids), provides a signal that coordinates hepatic triglyceride (TG), glucose and energy homeostasis. As signaling molecules with systemic endocrine functions, BA can activate protein kinases A and C as well as mitogen-activated protein kinase pathways. Additionally, they are ligands for a G-protein-coupled BA receptor (TGR5/Gpbar-1) and activate nuclear receptors such as farnesoid X receptor (FXR; NR1H4). FXR and its downstream targets play a key role in the control of hepatic de novo lipogenesis, very-low-density lipoprotein-TG export and plasma TG turnover. BA-activated FXR and signal transduction pathways are also involved in the regulation of hepatic gluconeogenesis, glycogen synthesis and insulin sensitivity. Via TGR5, BA are able to stimulate glucagon-like peptide-1 secretion in the small intestine and energy expenditure in brown adipose tissue and skeletal muscle. Dysregulation of BA transport and impaired BA receptor signaling may contribute to the pathogenesis of non-alcoholic fatty liver disease (NAFLD). Thus, BA transport and BA-controlled nuclear receptors and signaling pathways are promising drug targets for treatment of NAFLD. As such, FXR and/or TGR5 ligands have shown promising results in animal models of NAFLD and clinical pilot studies. Despite being a poor FXR and TGR5 ligand, ursodeoxycholic acid (UDCA) improves hepatic ER stress and insulin sensitivity. Notably, norUDCA, a side chain-shortened homologue of UDCA, improves fatty liver and atherosclerosis in Western diet-fed ApoE–/– mice. Collectively, these findings suggest that BA and targeting their receptor/signaling pathways may represent a promising approach to treat NAFLD and closely linked disorders such as obesity, diabetes, dyslipidemia and arteriosclerosis.

Role of nuclear bile acid receptor, FXR, in adaptive ABC transporter regulation by cholic and ursodeoxycholic acid in mouse liver, kidney and intestine

BACKGROUND/AIMS Adaptive changes in transporter expression in liver and kidney provide alternative excretory pathways for biliary constituents during cholestasis and may thus attenuate liver injury. Whether adaptive changes in ATP-binding cassette (ABC) transporter expression are stimulated by bile acids and their nuclear receptor FXR is unknown. METHODS Hepatic, renal and intestinal ABC transporter expression was compared in cholic acid (CA)- and ursodeoxycholic acid (UDCA)-fed wild-type (FXR(+/+)) and FXR knock-out mice (FXR(-/-)). Expression was assessed by reverse transcription-polymerase chain reaction, immunoblotting and immunofluorescence microscopy. RESULTS CA feeding stimulated hepatic Mrp2, Mrp3, Bsep and renal Mrp2 as well as intestinal Mrp2 and Mrp3 expression. Lack of Bsep induction by CA in FXR(-/-) was associated with disseminated hepatocyte necrosis which was not prevented by compensatory induction of Mrp2 and Mrp3. With the exception of Bsep, UDCA stimulated expression of hepatic, renal and intestinal ABC transporters independent of FXR without inducing liver toxicity. CONCLUSIONS Toxic CA and non-toxic UDCA induce adaptive ABC transporter expression, independent of FXR with the exception of Bsep. Stimulation of hepatic Mrp3 as well as intestinal and renal Mrp2 by UDCA may contribute to its therapeutic effects by inducing alternative excretory routes for bile acids and other cholephiles.

Dual farnesoid X receptor/TGR5 agonist INT-767 reduces liver injury in the Mdr2-/- (Abcb4-/-) mouse cholangiopathy model by promoting biliary HCO⁻₃ output

Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. The nuclear farnesoid X receptor (FXR) and the membrane G protein‐coupled receptor, TGR5, regulate bile acid (BA) homeostasis and inflammation. Therefore, we hypothesized that activation of FXR and/or TGR5 could ameliorate liver injury in Mdr2−/− (Abcb4−/−) mice, a model of chronic cholangiopathy. Hepatic inflammation, fibrosis, as well as bile secretion and key genes of BA homeostasis were addressed in Mdr2−/− mice fed either a chow diet or a diet supplemented with the FXR agonist, INT‐747, the TGR5 agonist, INT‐777, or the dual FXR/TGR5 agonist, INT‐767 (0.03% w/w). Only the dual FXR/TGR5 agonist, INT‐767, significantly improved serum liver enzymes, hepatic inflammation, and biliary fibrosis in Mdr2−/− mice, whereas INT‐747 and INT‐777 had no hepatoprotective effects. In line with this, INT‐767 significantly induced bile flow and biliary HCO  3− output, as well as gene expression of carbonic anhydrase 14, an important enzyme able to enhance HCO  3− transport, in an Fxr‐dependent manner. In addition, INT‐767 dramatically reduced bile acid synthesis via the induction of ileal Fgf15 and hepatic Shp gene expression, thus resulting in significantly reduced biliary bile acid output in Mdr2−/− mice. Conclusion: This study shows that FXR activation improves liver injury in a mouse model of chronic cholangiopathy by reduction of biliary BA output and promotion of HCO  3− ‐rich bile secretion. (HEPATOLOGY 2011;54:1303–1312)

Mechanisms of disease: mechanisms and clinical implications of cholestasis in sepsis.

Cholestasis is a common complication in patients with extrahepatic bacterial infection and sepsis. This article gives a comprehensive overview of the molecular and cellular mechanisms of sepsis-associated cholestasis. Recent advances in the understanding of intrahepatic cholestasis have allowed us to delineate the molecular mechanisms that underlie sepsis-associated cholestasis and to describe their potential clinical and therapeutic applications. The mechanisms and clinical presentation of sepsis-associated liver injury vary according to the severity of the bacterial infection. Proinflammatory cytokines and nitric oxide cause cholestasis by impairing hepatocellular and ductal bile formation. Ischemic liver injury and, rarely, progressive sclerosing cholangitis can also be found in patients with septic shock, or major trauma with systemic inflammatory response syndrome. Treatment is mainly focused on eradication of the underlying infection and managing the sepsis. The use of ursodeoxycholic acid or extracorporeal liver support as treatments for sepsis-associated cholestasis is under investigation, but neither can be recommended in routine clinical practice at present. Patients with progressive sclerosing cholangitis should be considered for orthotopic liver transplantation.

Spontaneous cholecysto‐ and hepatolithiasis in Mdr2−/− mice: A model for low phospholipid‐associated cholelithiasis

Previously, we identified needle‐like and filamentous, putatively “anhydrous” cholesterol crystallization in vitro at very low phospholipid concentrations in model and native biles. Our aim now was to address whether spontaneous gallstone formation occurs in Mdr2 (Abcb4) knockout mice that are characterized by phospholipid‐deficient bile. Biliary phenotypes and cholesterol crystallization sequences in fresh gallbladder biles and non‐fixed liver sections were determined by direct and polarizing light microscopy. The physical chemical nature and composition of crystals and stones were determined by sucrose density centrifugation and before mass and infrared spectroscopy. Gallbladder biles of Mdr2−/− mice precipitate needle‐like cholesterol crystals at 12 weeks of age on chow. After 15 weeks, more than 50% of Mdr2−/− mice develop gallbladder stones, with female mice displaying a markedly higher gallstone‐susceptibility. Although gallbladder biles of Mdr2−/− mice contain only traces (≤ 1.1 mM) of phospholipid and cholesterol, they become supersaturated with cholesterol and plot in the left 2‐phase zone of the ternary phase diagram, consistent with “anhydrous” cholesterol crystallization. Furthermore, more than 40% of adult female Mdr2−/− mice show intra‐ and extrahepatic bile duct stones. In conclusion, spontaneous gallstone formation is a new consistent feature of the Mdr2−/− phenotype. The Mdr2−/− mouse is therefore a model for low phospholipid‐associated cholelithiasis recently described in humans with a dysfunctional mutation in the orthologous ABCB4 gene. The mouse model supports the concept that this gene is a monogenic risk factor for cholesterol gallstones and a target for novel therapeutic strategies. (HEPATOLOGY 2004;39:117–128.)

The keratin cytoskeleton in liver diseases.

The keratin intermediate filament (IF) cytoskeleton of hepatocytes has continuously gained medical relevance over the last two decades. Originally it was mainly recognized as a differentiation marker for diagnostic purposes in pathology. However, keratin IFs were soon identified as major cellular structures to be affected in a variety of chronic liver diseases, such as alcoholic and non‐alcoholic steatohepatitis (ASH, NASH), copper toxicosis, and cholestasis. Based on observations in keratin gene knock‐out mice, the insight into the functional role of keratins was extended from a mere structural role providing mechanical stability to hepatocytes, to an additional role as target and modulator of toxic stress and apoptosis. The functional relevance of keratins in human diseases has recently been underlined by the identification of mutations in keratin genes in patients with liver cirrhosis. Copyright