Dagmar Silbert

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.

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)

Side Chain Structure Determines Unique Physiologic and Therapeutic Properties of norUrsodeoxycholic Acid in Mdr2−/− Mice

24‐norursodeoxycholic acid (norUDCA), a side chain–modified ursodeoxycholic acid derivative, has dramatic therapeutic effects in experimental cholestasis and may be a promising agent for the treatment of cholestatic liver diseases. We aimed to better understand the physiologic and therapeutic properties of norUDCA and to test if they are related to its side chain length and/or relative resistance to amidation. For this purpose, Mdr2−/− mice, a model for sclerosing cholangitis, received either a standard diet or a norUDCA‐, tauro norursodeoxycholic acid (tauro‐ norUDCA)‐, or di norursodeoxycholic acid (di norUDCA)‐enriched diet. Bile composition, serum biochemistry, liver histology, fibrosis, and expression of key detoxification and transport systems were investigated. Direct choleretic effects were addressed in isolated bile duct units. The role of Cftr for norUDCA‐induced choleresis was explored in Cftr−/− mice. norUDCA had pharmacologic features that were not shared by its derivatives, including the increase in hepatic and serum bile acid levels and a strong stimulation of biliary HCO3− ‐output. norUDCA directly stimulated fluid secretion in isolated bile duct units in a HCO3− ‐dependent fashion to a higher extent than the other bile acids. Notably, the norUDCA significantly stimulated HCO 3− ‐output also in Cftr−/− mice. In Mdr2−/− mice, cholangitis and fibrosis strongly improved with norUDCA, remained unchanged with tauro‐ norUDCA, and worsened with di norUDCA. Expression of Mrp4, Cyp2b10, and Sult2a1 was increased by norUDCA and di norUDCA, but was unaffected by tauro‐ norUDCA. Conclusion:The relative resistance of norUDCA to amidation may explain its unique physiologic and pharmacologic properties. These include the ability to undergo cholehepatic shunting and to directly stimulate cholangiocyte secretion, both resulting in a HCO3− ‐rich hypercholeresis that protects the liver from cholestatic injury. (HEPATOLOGY 2009;49:1972–1981.)

Fxr(-/-) mice adapt to biliary obstruction by enhanced phase I detoxification and renal elimination of bile acids.

Farnesoid X receptor knockout (Fxr−/−) mice cannot upregulate the bile salt export pump in bile acid loading or cholestatic conditions. To investigate whether Fxr−/− mice differ in bile acid detoxification compared with wild-type mice, we performed a comprehensive analysis of bile acids extracted from liver, bile, serum, and urine of naive and common bile duct-ligated wild-type and Fxr−/− mice using electrospray and gas chromatography mass spectrometry. In addition, hepatic and renal gene expression levels of Cyp2b10 and Cyp3a11, and protein expression levels of putative renal bile acid-transporting proteins, were investigated. We found significantly enhanced hepatic bile acid hydroxylation in Fxr−/− mice, in particular hydroxylations of cholic acid in the 1β, 2β, 4β, 6α, 6β, 22, or 23 position and a significantly enhanced excretion of these metabolites in urine. The gene expression level of Cyp3a11 was increased in the liver of Fxr−/− mice, whereas the protein expression levels of multidrug resistance-related protein 4 (Mrp4) were increased in kidneys of both genotypes during common bile duct ligation. In conclusion, Fxr−/− mice detoxify accumulating bile acids in the liver by enhanced hydroxylation reactions probably catalyzed by Cyp3a11. The metabolites formed were excreted into urine, most likely with the participation of Mrp4.

Hepatobiliary transporter expression in human hepatocellular carcinoma.

Abstract: Background/Aims: Treatment of hepatocellular carcinoma (HCC) is hampered by resistance to chemotherapy, which might be mediated by multidrug resistance P‐glycoproteins (MDR P‐gps) and MDR‐associated proteins (MRPs). The effectiveness of cytostatics could be further impeded by reduced hepatocellular drug uptake into HCCs. Therefore, we aimed to determine P‐gp, MRP and organic anion transporting protein OATP2 (SLC21A6) expression in HCC. Furthermore, we investigated expression of the major bile salt uptake system Na+/taurocholate cotransporter NTCP (SLC10A1), since bile salt‐coupled chemotherapeutics were proposed to increase therapeutic drug enrichment in HCC.

Expression of bile acid synthesis and detoxification enzymes and the alternative bile acid efflux pump MRP4 in patients with primary biliary cirrhosis.

Background: Bile acid synthesis, transport and metabolism are markedly altered in experimental cholestasis. Whether such coordinated regulation exists in human cholestatic diseases is unclear. We therefore investigated expression of genes for bile acid synthesis, detoxification and alternative basolateral export and regulatory nuclear factors in primary biliary cirrhosis (PBC).

Alterations in Lipid Metabolism Mediate Inflammation, Fibrosis, and Proliferation in a Mouse Model of Chronic Cholestatic Liver Injury

BACKGROUND & AIMS The liver controls central processes of lipid and bile acid homeostasis. We aimed to investigate whether alterations in lipid metabolism contribute to the pathogenesis of chronic cholestatic liver disease in mice. METHODS We used microarray and metabolic profiling analyses to identify alterations in systemic and hepatic lipid metabolism in mice with disruption of the gene ATP-binding cassette sub-family B member 4 (Abcb4(-/-) mice), a model of inflammation-induced cholestatic liver injury, fibrosis, and cancer. RESULTS Alterations in Abcb4(-/-) mice, compared with wild-type mice, included deregulation of genes that control lipid synthesis, storage, and oxidation; decreased serum levels of cholesterol and phospholipids; and reduced hepatic long-chain fatty acyl-CoAs (LCA-CoA). Feeding Abcb4(-/-) mice the side chain-modified bile acid 24-norursodeoxycholic acid (norUDCA) reversed their liver injury and fibrosis, increased serum levels of lipids, lowered phospholipase and triglyceride hydrolase activities, and restored hepatic LCA-CoA and triglyceride levels. Additional genetic and nutritional studies indicated that lipid metabolism contributed to chronic cholestatic liver injury; crossing peroxisome proliferator-activated receptor (PPAR)-α-deficient mice with Abcb4(-/-) mice (to create double knockouts) or placing Abcb4(-/-) mice on a high-fat diet protected against liver injury, with features similar to those involved in the response to norUDCA. Placing pregnant Abcb4(-/-) mice on high-fat diets prevented liver injury in their offspring. However, fenofibrate, an activator of PPARα, aggravated liver injury in Abcb4(-/-) mice. CONCLUSIONS Alterations in lipid metabolism contribute to the pathogenesis and progression of cholestatic liver disease in mice.

Curcumin improves sclerosing cholangitis in Mdr2 / mice by inhibition of cholangiocyte inflammatory response and portal myofibroblast proliferation

Background and aim Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. Curcumin, the yellow pigment of the spice turmeric, has pleiotropic actions and attenuates hepatic damage in animal models of chemically-induced liver injury. Whether curcumin has beneficial effects in cholangiopathies is unknown. Methods Potential anticholestatic, anti-inflammatory and antifibrotic mechanisms of curcumin were explored in vivo in Mdr2−/− mice as a murine model of chronic cholangiopathy; as well as in vitro in a cholangiocyte cell line (HuCCT1) and portal myofibroblasts (MFBs) isolated from Mdr2−/− mice. Results Liver damage, cholestasis and fibrosis were reduced in Mdr2−/− mice after curcumin feeding. Moreover, curcumin inhibited cholangiocyte proliferation and expression of activation marker vascular cell adhesion molecule-1 in Mdr2−/− mice. Curcumin—similar to PPARγ synthetic agonist troglitazone—directly inhibited TNF-α-induced inflammatory activation of cholangiocytes in vitro, whereas these beneficial effects of curcumin were largely blocked by a PPARγ synthetic antagonist. In addition, curcumin blocked proliferation and activation of portal MFBs by inhibiting ERK1/2 phosphorylation, thus contributing to reduced fibrogenesis. Conclusions These results show that curcumin may have multiple targets in liver including activation of PPARγ in cholangiocytes and inhibition of ERK1/2 signalling in MFBs, thereby modulating several central cellular events in a mouse model of cholangiopathy. Targeting these pathways may be a promising therapeutic approach to cholangiopathies.