Emina Halilbasic

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.)

Bile acid transporters and regulatory nuclear receptors in the liver and beyond

Summary Bile acid (BA) transporters are critical for maintenance of the enterohepatic BA circulation where BAs exert their multiple physiological functions including stimulation of bile flow, intestinal absorption of lipophilic nutrients, solubilization and excretion of cholesterol, as well as antimicrobial and metabolic effects. Tight regulation of BA transporters via nuclear receptors is necessary to maintain proper BA homeostasis. Hereditary and acquired defects of BA transporters are involved in the pathogenesis of several hepatobiliary disorders including cholestasis, gallstones, fatty liver disease and liver cancer, but also play a role in intestinal and metabolic disorders beyond the liver. Thus, pharmacological modification of BA transporters and their regulatory nuclear receptors opens novel treatment strategies for a wide range of disorders.

Selective Activation of Nuclear Bile Acid Receptor FXR in the Intestine Protects Mice Against Cholestasis

BACKGROUND & AIMS Cholestasis is a liver disorder characterized by impaired bile flow, reduction of bile acids (BAs) in the intestine, and retention of BAs in the liver. The farnesoid X receptor (FXR) is the transcriptional regulator of BA homeostasis. Activation of FXR by BAs reduces circulating BA levels in a feedback mechanism, repressing hepatic cholesterol 7α-hydroxylase (Cyp7a1), the rate-limiting enzyme for the conversion of cholesterol to BAs. This mechanism involves the hepatic nuclear receptor small heterodimer partner and the intestinal fibroblast growth factor (FGF) 19 and 15. We investigated the role of activation of intestine-specific FXR in reducing hepatic levels of BAs and protecting the liver from cholestasis in mice. METHODS We generated transgenic mice that express a constitutively active FXR in the intestine. Using FXR gain- and loss-of-function models, we studied the roles of intestinal FXR in mice with intrahepatic and extrahepatic cholestasis. RESULTS Selective activation of intestinal FXR induced FGF15 and repressed hepatic Cyp7a1, reducing the pool size of BAs and changing the BA pool composition. Activation of intestinal FXR protected mice from obstructive extrahepatic cholestasis after bile duct ligation or administration of α-naphthylisothiocyanate. In Mdr2(-/-) mice, transgenic expression of activated FXR in the intestine protected against liver damage, whereas absence of FXR promoted progression of liver disease. CONCLUSIONS Activation of FXR transcription in the intestine protects the liver from cholestasis in mice by inducing FGF15 expression and reducing the hepatic pool of BA; this approach might be developed to reverse cholestasis in patients.

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.)

Farnesoid X Receptor Critically Determines the Fibrotic Response in Mice but Is Expressed to a Low Extent in Human Hepatic Stellate Cells and Periductal Myofibroblasts

The nuclear bile acid receptor, farnesoid X receptor (FXR), may play a pivotal role in liver fibrosis. We tested the impact of genetic FXR ablation in four different mouse models. Hepatic fibrosis was induced in wild-type and FXR knock-out mice (FXR(-/-)) by CCl(4) intoxication, 3,5-diethoxycarbonyl-1,4-dihydrocollidine feeding, common bile duct ligation, or Schistosoma mansoni (S.m.)-infection. In addition, we determined nuclear receptor expression levels (FXR, pregnane X receptor (PXR), vitamin D receptor, constitutive androstane receptor (CAR), small heterodimer partner (SHP)) in mouse hepatic stellate cells (HSCs), portal myofibroblasts (MFBs), and human HSCs. Cell type-specific FXR protein expression was determined by immunohistochemistry in five mouse models and prototypic human fibrotic liver diseases. Expression of nuclear receptors was much lower in mouse and human HSCs/MFBs compared with total liver expression with the exception of vitamin D receptor. FXR protein was undetectable in mouse and human HSCs and MFBs. FXR loss had no effect in CCl(4)-intoxicated and S.m.-infected mice, but significantly decreased liver fibrosis of the biliary type (common bile duct ligation, 3,5-diethoxycarbonyl-1,4-dihydrocollidine). These data suggest that FXR loss significantly reduces fibrosis of the biliary type, but has no impact on non-cholestatic liver fibrosis. Since there is no FXR expression in HSCs and MFBs in liver fibrosis, our data indicate that these cells may not represent direct therapeutic targets for FXR ligands.

Nuclear Receptors as New Perspective for the Management of Liver Diseases

Nuclear receptors (NRs) are ligand-activated transcription factors that act as sensors for a broad range of natural and synthetic ligands and regulate several key hepatic functions including bile acid homeostasis, bile secretion, lipid and glucose metabolism, as well as drug deposition. Moreover, NRs control hepatic inflammation, regeneration, fibrosis, and tumor formation. Therefore, NRs are key for understanding the pathogenesis and pathophysiology of a wide range of hepatic disorders. Finally, targeting NRs and their alterations offers exciting new perspectives for the treatment of liver diseases.

Differential effects of norUDCA and UDCA in obstructive cholestasis in mice

Background & Aims The quest for effective drugs to treat cholangiopathies led to the development of norUDCA previously shown to have potent choleretic effects and to heal cholangiopathy in Abcb4 knockout (Abcb4−/−) mice. Its mother compound UDCA had detrimental effects in common bile duct ligated (CBDL) mice, presumably related to its choleretic effects. norUDCA choleretic effects may therefore raise safety concerns when used in cholangiopathies with biliary obstruction. We therefore aimed at comparing the effects of UDCA and norUDCA in clear-cut obstructive cholestasis. Methods 0.5% UDCA- or norUDCA-fed wild type and Abcb4−/− mice were subjected to CBDL or selective bile duct ligation (SBDL) and compared to controls with regard to liver injury. Bile flow, bile composition, and biliary manometry were compared in UDCA-fed, norUDCA-fed and control mice. Toxicity of UDCA and norUDCA was compared in vitro. Results Compared to UDCA, liver injury in CBDL mice was significantly lower in almost all norUDCA groups. In SBDL mice, only UDCA induced bile infarcts in the ligated lobes, whereas norUDCA even ameliorated liver injury. In vitro, UDCA induced cellular ATP depletion and was significantly more toxic than norUDCA in HepG2 cells, mouse bile duct epithelial cells, and primary human hepatocytes. Conclusions Compared to norUDCA, UDCA is significantly more toxic in CBDL mice. norUDCA, in contrast to UDCA, significantly ameliorates liver injury in SBDL mice. Our findings uncover profound differences in metabolism and therapeutic mechanisms of both bile acids with important clinical consequences.

Bile acids trigger cholemic nephropathy in common bile‐duct–ligated mice

Tubular epithelial injury represents an underestimated but important cause of renal dysfunction in patients with cholestasis and advanced liver disease, but the underlying mechanisms are unclear. To address the hypothesis that accumulation and excessive alternative urinary elimination of potentially toxic bile acids (BAs) may contribute to kidney injury in cholestasis, we established a mouse model for detailed in vivo time course as well as treatment studies. Three‐day common bile duct ligation (CBDL) induced renal tubular epithelial injury predominantly at the level of aquaporin 2–positive collecting ducts with tubular epithelial and basement membrane defects. This was followed by progressive interstitial nephritis and tubulointerstitial renal fibrosis in 3‐, 6‐, and 8‐week CBDL mice. Farnesoid X receptor knockout mice (with a hydrophilic BA pool) were completely protected from CBDL‐induced renal fibrosis. Prefeeding of hydrophilic norursodeoxycholic acid inhibited renal tubular epithelial injury in CBDL mice. In addition, we provide evidence for renal tubular injury in cholestatic patients with cholemic nephropathy. Conclusion: We characterized a novel in vivo model for cholemic nephropathy, which offers new perspectives to study the complex pathophysiology of this condition. Our findings suggest that urinary‐excreted toxic BAs represent a pivotal trigger for renal tubular epithelial injury leading to cholemic nephropathy in CBDL mice. (Hepatology 2013; 58:2056–2069)

Inhibition of intestinal bile acid absorption improves cholestatic liver and bile duct injury in a mouse model of sclerosing cholangitis.

BACKGROUND AND AIMS Approximately 95% of bile acids (BAs) excreted into bile are reabsorbed in the gut and circulate back to the liver for further biliary secretion. Therefore, pharmacological inhibition of the ileal apical sodium-dependent BA transporter (ASBT/SLC10A2) may protect against BA-mediated cholestatic liver and bile duct injury. METHODS Eight week old Mdr2(-/-) (Abcb4(-/-)) mice (model of cholestatic liver injury and sclerosing cholangitis) received either a diet supplemented with A4250 (0.01% w/w) - a highly potent and selective ASBT inhibitor - or a chow diet. Liver injury was assessed biochemically and histologically after 4weeks of A4250 treatment. Expression profiles of genes involved in BA homeostasis, inflammation and fibrosis were assessed via RT-PCR from liver and ileum homogenates. Intestinal inflammation was assessed by RNA expression profiling and immunohistochemistry. Bile flow and composition, as well as biliary and fecal BA profiles were analyzed after 1week of ASBT inhibitor feeding. RESULTS A4250 improved sclerosing cholangitis in Mdr2(-/-) mice and significantly reduced serum alanine aminotransferase, alkaline phosphatase and BAs levels, hepatic expression of pro-inflammatory (Tnf-α, Vcam1, Mcp-1) and pro-fibrogenic (Col1a1, Col1a2) genes and bile duct proliferation (mRNA and immunohistochemistry for cytokeratin 19 (CK19)). Furthermore, A4250 significantly reduced bile flow and biliary BA output, which correlated with reduced Bsep transcription, while Ntcp and Cyp7a1 were induced. Importantly A4250 significantly reduced biliary BA secretion but preserved HCO3(-) and biliary phospholipid secretion resulting in an increased HCO3(-)/BA and PL/BA ratio. In addition, A4250 profoundly increased fecal BA excretion without causing diarrhea and altered BA pool composition, resulting in diminished concentrations of primary BAs tauro-β-muricholic acid and taurocholic acid. CONCLUSIONS Pharmacological ASBT inhibition attenuates cholestatic liver and bile duct injury by reducing biliary BA concentrations in mice.

Nuclear Receptors as Drug Targets in Cholestatic Liver Diseases

Cholestatic liver diseases encompass a wide spectrum of disorders with different causes, resulting in impaired bile flow and accumulation of bile acids and other potentially hepatotoxic cholephils. The understanding of the molecular mechanisms of bile formation and cholestasis has recently improved significantly through new insights into nuclear receptor (patho)biology. Nuclear receptors are ligand-activated transcription factors, which act as central players in the regulation of genes responsible for elimination and detoxification of biliary constituents accumulating in cholestasis. They also control other pathophysiologic processes such as inflammation, fibrogenesis, and carcinogenesis involved in the pathogenesis and disease progression of cholestasis liver diseases.