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Exploring the pathogenesis of and developing therapies for cholestatic liver diseases such as primary sclerosing cholangitis (PSC) remains challenging, partly due to a paucity of in vitro models that capture the complex environments contributing to disease progression and partly due to difficulty in obtaining cholangiocytes. Here we report the development of a human vascularized bile duct-on-a-chip (VBDOC) that uses cholangiocyte organoids derived from normal bile duct tissue and human vascular endothelial cells to model bile ducts and blood vessels structurally and functionally in three dimensions. Cholangiocytes in the duct polarized, formed mature tight junctions and had permeability properties comparable to those measured in ex vivo systems. The flow of blood and bile was modeled by perfusion of the cell-lined channels, and cholangiocytes and endothelial cells displayed differential responses to flow. We also showed that the device can be constructed with biliary organoids from cells isolated from both bile duct tissue and the bile of PSC patients. Cholangiocytes in the duct became more inflammatory under the stimulation of IL-17A, which induced peripheral blood mononuclear cells and differentiated Th17 cells to transmigrate across the vascular channel. In sum, this human VBDOC recapitulated the vascular-biliary interface structurally and functionally and represents a novel multicellular platform to study inflammatory and fibrotic cholestatic liver diseases., (Creative Commons Attribution license.)

Bile formation is a fundamental physiological process that is vital to the survival of all vertebrates. However, little was known about the mechanisms of this secretion until after World War II. Initial studies involved classic physiologic studies in animal models and humans, which progressed to include studies in isolated cells and membrane vesicles. The advent of molecular biology then led to the identification of specific transport systems that are the determinants of this secretion. Progress in this field was reviewed in the American Physiologic Society's series on "Comprehensive Physiology" in 2013. Herein, we provide an in-depth update of progress since that time., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2021. Published by Elsevier B.V.)

Translational studies in human cholestatic diseases have for years been hindered by various challenges, including the rarity of the disorders, the difficulty in obtaining biliary tissue from across the spectrum of the disease stage, and the difficulty culturing and maintaining primary cholangiocytes. Organoid technology is increasingly being viewed as a technological breakthrough in translational medicine as it allows the culture and biobanking of self-organizing cells from various sources that facilitate the study of pathophysiology and therapeutics, including from individual patients in a personalized approach. This review describes current research using biliary organoids for the study of human cholestatic diseases and the emerging applications of organoids to regenerative medicine directed at the biliary tree. Challenges and possible solutions to the current hurdles in this emerging field, particularly the need for standardization of terminology and clarity on source materials and techniques, are also discussed., Competing Interests: J.L.B. received a research grant in 2020 from Gilead Sciences for an investigator-initiated study on patient reported outcomes in primary sclerosing cholangitis, unrelated to the work in this manuscript., (Thieme. All rights reserved.)

Background & Aims: The nuclear factor of activated T-cells (NFAT) plays an important role in immune responses by regulating the expression of inflammatory genes. However, it is not known whether NFAT plays any role in the bile acid (BA)-induced hepatic inflammatory response. Thus, we aimed to examine the functional role of NFATc3 in cholestatic liver injury in mice and humans., Methods: Gene and protein expression and cellular localization were assessed in primary hepatocyte cultures (mouse and human) and cholestatic liver tissues (murine models and patients with primary biliary cholangitis [PBC] or primary sclerosing cholangitis [PSC]) by quantitative PCR, western blot and immunohistochemistry. Specific NFAT inhibitors were used in vivo and in vitro. Gene reporter assays and ChIP-PCR were used to determine promoter activity., Results: NFAT isoforms c1 and c3 were expressed in human and mouse hepatocytes. When treated with cholestatic levels of BAs, nuclear translocation of NFATc3 was increased in both human and mouse hepatocytes and was associated with elevated mRNA levels of IL-8, CXCL2, and CXCL10 in these cells. Blocking NFAT activation with pathway-specific inhibitors or knocking down Nfatc3 expression significantly decreased BA-driven induction of these cytokines in mouse hepatocytes. Nuclear expression of NFATc3/Nfatc3 protein was increased in cholestatic livers, both in mouse models (bile duct ligation or Abcb4 -/- mice) and in patients with PBC and PSC in association with elevated tissue levels of Cxcl2 (mice) or IL-8 (humans). Gene reporter assays and ChIP-PCR demonstrated that the NFAT response element in the IL-8 promoter played a key role in BA-induced human IL-8 expression. Finally, blocking NFAT activation in vivo in Abcb4 -/- mice reduced cholestatic liver injury., Conclusions: NFAT plays an important role in BA-stimulated hepatic cytokine expression in cholestasis. Blocking hepatic NFAT activation may reduce cholestatic liver injury in humans., Lay Summary: Bile acid induces liver injury by stimulating the expression of inflammatory genes in hepatocytes through activation of the transcription factor NFAT. Blocking this activation in vitro (in hepatocyte cultures) and in vivo (in cholestatic mice) decreased the expression of inflammatory genes and reduced liver injury., Competing Interests: Conflict of interest The authors declare no conflicts of interest that pertain to this work. Please refer to the accompanying ICMJE disclosure forms for further details., (Copyright © 2020 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

Primary sclerosing cholangitis (PSC) is a heterogeneous and progressive fibroinflammatory cholangiopathy with no known etiology or effective treatment. Studies of PSC are limited due to difficulty in accessing the cholangiocyte, the small percentage of these cells in the liver, instability of in vitro culture systems, and reliance on samples from end-stage disease. Here, we demonstrate that stem cells can be isolated from the bile of PSC patients undergoing endoscopic retrograde cholangiopancreatography earlier in their clinical course and maintained long term in vitro as three-dimensional (3D) organoids that express a biliary genetic phenotype. Additionally, bile-derived organoids (BDOs) can be biobanked and samples obtained longitudinally over the course of the disease. These BDOs express known cholangiocyte markers including gamma glutamyl transferase, cytokeratin 19, epithelial cellular adhesion molecule, cystic fibrosis transmembrane conductance regulator, and anion exchanger 2. RNA sequence analysis identified 39 genes whose expression differed in organoids from PSC patients compared to non-PSC controls, including human leukocyte antigen DM alpha chain and chemokine (C-C motif) ligand 20 (CCL20), immune-related genes previously described in genome-wide association studies of PSC. Incubation of these BDOs with interleukin 17A or tumor necrosis factor alpha led to an immune-reactive phenotype with a significant increase in secretion of proinflammatory mediators, including CCL20, a T-cell chemoattractant. Conclusion: This study demonstrates that bile can be used as a source of biliary-like cells that can be maintained long term in vitro as 3D organoids; these BDOs retain features of cholangiopathies, including the ability to react to inflammatory stimuli by secreting chemokines and propagating an immune-reactive phenotype reflective of the pathogenesis of these diseases; thus, BDOs represent a platform for the study of the pathogenesis and therapy of cholangiopathies, particularly PSC., (© 2018 by the American Association for the Study of Liver Diseases.)

Cholangiopathies, including primary sclerosing cholangitis, are a group of heterogeneous diseases characterized by inflammation and fibrosis of the intrahepatic and extrahepatic bile duct epithelium. Studies, especially of primary sclerosing cholangitis, have been hampered by the difficulty in accessing the cholangiocyte, instability of in vitro culture systems, and reliance on (limited) samples from end-stage disease. Here we describe a novel method of culturing biliary cells from bile of primary sclerosing cholangitis patients undergoing endoscopic retrograde cholangiopancreatography for clinical indications. These 3D organoid cultures demonstrate a biliary phenotype, can be maintained in vitro, and biobanked for future analyses. Given the need for diagnostic and therapeutic endoscopic retrograde cholangiopancreatography throughout the disease in many primary sclerosing cholangitis patients, this method can provide longitudinal studies in individual patients, allowing for a correlation of gene expression with disease status. These organoids can react to inflammatory stimuli, resulting in the secretion of chemo/cytokines indicative of the reactive immune phenotype characteristic of primary sclerosing cholangitis. Therefore, bile-derived organoids provide a model to study the pathogenesis and pharmacotherapeutic treatment of cholangiopathies.

Mechanisms of bile acid-induced (BA-induced) liver injury in cholestasis are controversial, limiting development of new therapies. We examined how BAs initiate liver injury using isolated liver cells from humans and mice and in-vivo mouse models. At pathophysiologic concentrations, BAs induced proinflammatory cytokine expression in mouse and human hepatocytes, but not in nonparenchymal cells or cholangiocytes. These hepatocyte-specific cytokines stimulated neutrophil chemotaxis. Inflammatory injury was mitigated in Ccl2 -/- mice treated with BA or after bile duct ligation, where less hepatic infiltration of neutrophils was detected. Neutrophils in periportal areas of livers from cholestatic patients also correlated with elevations in their serum aminotransferases. This liver-specific inflammatory response required BA entry into hepatocytes via basolateral transporter Ntcp. Pathophysiologic levels of BAs induced markers of ER stress and mitochondrial damage in mouse hepatocytes. Chemokine induction by BAs was reduced in hepatocytes from Tlr9 -/- mice, while liver injury was diminished both in conventional and hepatocyte-specific Tlr9 -/- mice, confirming a role for Tlr9 in BA-induced liver injury. These findings reveal potentially novel mechanisms whereby BAs elicit a hepatocyte-specific cytokine-induced inflammatory liver injury that involves innate immunity and point to likely novel pathways for treating cholestatic liver disease., Competing Interests: Conflict of interest: The authors have declared that no conflict of interest exists.

The multidrug resistance-associated protein 2 (Mrp2) is an ATP-binding cassette transporter that transports a wide variety of organic anions across the apical membrane of epithelial cells. The expression of Mrp2 on the plasma membrane is regulated by protein-protein interactions. Cystic fibrosis transmembrane conductance regulator (CFTR)-associated ligand (CAL) interacts with transmembrane proteins via its PDZ domain and reduces their cell surface expression by increasing lysosomal degradation and intracellular retention. Our results showed that CAL is localized at the trans-Golgi network of rat hepatocytes. The expression of CAL is increased, and Mrp2 expression is decreased, in the liver of mice deficient in sodium/hydrogen exchanger regulatory factor-1. To determine whether CAL interacts with Mrp2 and is involved in the posttranscriptional regulation of Mrp2, we used glutathione S-transferase (GST) fusion proteins with or without the COOH-terminal PDZ binding motif of Mrp2 as the bait in GST pull-down assays. We demonstrated that Mrp2 binds to CAL via its COOH-terminal PDZ-binding motif in GST pull-down assays, an interaction verified by coimmunoprecipitation of these two proteins in cotransfected COS-7 cells. In COS-7 and LLC-PK1 cells transfected with Mrp2 alone, only a mature, high-molecular-mass band of Mrp2 was detected. However, when cells were cotransfected with Mrp2 and CAL, Mrp2 was expressed as both mature and immature forms. Biotinylation and streptavidin pull-down assays confirmed that CAL dramatically reduces the expression level of total and cell surface Mrp2 in Huh-7 cells. Our findings suggest that CAL interacts with Mrp2 and is a negative regulator of Mrp2 expression., (Copyright © 2017 the American Physiological Society.)

Sirtuin1 (Sirt1; mammalian homolog of Saccharomyces cerevisiae enzyme Sir2) is a transcriptional and transactivational regulator of murine farnesoid X receptor (Fxr), which is the primary bile acid (BA) sensor, and critical regulator of BA metabolism in physiological and pathophysiological conditions. Previous studies have suggested compromised Sirt1 expression in rodent models of cholestatic liver injury. We hypothesized that Sirt1 could be potentially targeted to alleviate cholestatic liver injury. In cultured primary human hepatocytes, SIRT1 messenger RNA was down-regulated after GCA treatment, potentially through induction of microRNA (miR)-34a, whereas tauroursodeoxycholic acid induced SIRT1 expression without affecting miR-34a expression. Sirt1 expression was also significantly down-regulated in three mouse models of liver injury (bile duct ligation, 1% cholic acid [CA] fed, and the Mdr2 -/- mouse). Mice fed CA diet also demonstrated hepatic FXR hyperacetylation and induction of the Janus kinase/p53 pathway. Mice fed a CA diet and concurrently administered the Sirt1 activator, SRT1720 (50 mg/kg/day, orally), demonstrated 40% and 45% decrease in plasma alanine aminotransferase and BA levels, respectively. SRT1720 increased hepatic BA hydrophilicity by increasing tri- and tetrahydroxylated and decreasing the dihydroxylated BA fraction. SRT1720 administration also inhibited hepatic BA synthesis, potentially through ileal fibroblast growth factor 15- and Fxr-mediated inhibition of cytochrome p450 (Cyp) 7a1 and Cyp27a1, along with increased hepatic BA hydroxylation in association with Cyp2b10 induction. SRT1720 administration significantly induced renal multidrug resistance-associated protein 2 and 4, peroxisome proliferator-activated receptor gamma coactivator 1-α, and constitutive androstance receptor expression along with ∼2-fold increase in urinary BA concentrations., Conclusion: SRT1720 administration alleviates cholestatic liver injury in mice by increasing hydrophilicity of hepatic BA composition and decreasing plasma BA concentration through increased BA excretion into urine. Thus, use of small-molecule activators of Sirt1 presents a novel therapeutic target for cholestatic liver injury. (Hepatology 2016;64:2151-2164)., (© 2016 by the American Association for the Study of Liver Diseases.)

The heteromeric membrane protein Organic Solute Transporter alpha/beta is the major bile acid efflux transporter in the intestine. Physical association of its alpha and beta subunits is essential for their polarized basolateral membrane localization and function in the transport of bile acids and other organic solutes. We identified a highly conserved acidic dileucine motif (-EL20L21EE) at the extracellular amino-tail of organic solute transporter beta from multiple species. To characterize the role of this protein interacting domain in the association of the human beta and alpha subunits and in membrane localization of the transporter, Leu20 and Leu21 on the amino-tail of human organic solute transporter beta were replaced with alanines by site-directed mutagenesis. Co-immunoprecipitation study in HEK293 cells demonstrated that substitution of the leucine residues with alanines prevented the interaction of the human beta mutant with the alpha subunit. Membrane biotinylation demonstrated that the LL/AA mutant eliminated membrane expression of both subunits. Computational-based modelling of human organic solute transporter beta suggested that the LL/AA mutation substantially alters both the structure and lipophilicity of the surface, thereby not only affecting the interaction with the alpha subunit but also possibly impacting the capacity of the beta subunit to traffick through the cell and interact with the membrane. In summary, our findings indicate that the dileucine motif in the extracellular N-terminal region of human organic solute transporter beta subunit plays a critical role in the association with the alpha subunit and in its polarized plasma membrane localization.

Unlabelled: The intercellular adhesion molecule 1 (ICAM-1) is induced in mouse liver after bile duct ligation (BDL) and plays a key role in neutrophil-mediated liver injury in BDL mice. ICAM-1 has been shown to interact with cytoskeletal ezrin-radixin-moesin (ERM) proteins that also interact with the PDZ protein, Na(+) /H(+) exchanger regulatory factor 1 (NHERF-1/EBP50). In NHERF-1(-/-) mice, ERM proteins are significantly reduced in brush-border membranes from kidney and small intestine. ERM knockdown reduces ICAM-1 expression in response to tumor necrosis factor alpha. Here we show that NHERF-1 assembles ERM proteins, ICAM-1 and F-actin into a macromolecule complex that is increased in mouse liver after BDL. Compared to wild-type (WT) mice, both sham-operated and BDL NHERF-1(-/-) mice have lower levels of activated ERM and ICAM-1 protein in the liver accompanied by significantly reduced hepatic neutrophil accumulation, serum alanine aminotransferase, and attenuated liver injury after BDL. However, total bile acid concentrations in serum and liver of sham and BDL NHERF-1(-/-) mice were not significantly different from WT controls, although hepatic tetrahydroxylated bile acids and Cyp3a11 messenger RNA levels were higher in NHERF-1(-/-) BDL mice., Conclusion: NHERF-1 participates in the inflammatory response that is associated with BDL-induced liver injury. Deletion of NHERF-1 in mice leads to disruption of the formation of ICAM-1/ERM/NHERF-1 complex and reduction of hepatic ERM proteins and ICAM-1, molecules that are up-regulated and are essential for neutrophil-mediated liver injury in cholestasis. Further study of the role of NHERF-1 in the inflammatory response in cholestasis and other forms of liver injury should lead to discovery of new therapeutic targets in hepatic inflammatory diseases., (© 2015 by the American Association for the Study of Liver Diseases.)

The bile salt export pump (BSEP, ABCB11) is the primary transporter of bile acids from the hepatocyte to the biliary system. This rate-limiting step in bile formation is essential to the formation of bile salt dependent bile flow, the enterohepatic circulation of bile acids, and the digestion of dietary fats. Mutations in BSEP are associated with cholestatic diseases such as progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), drug-induced cholestasis, and intrahepatic cholestasis of pregnancy. Development of clinical therapies for these conditions necessitates a clear understanding of the cell biology of biosynthesis, trafficking, and transcriptional and translational regulation of BSEP. This chapter will focus on the molecular and cell biological aspects of this critical hepatic membrane transporter., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Deficiency of ABCB4 is associated with several forms of cholestasis in humans. Abcb4(-/-) mice also develop cholestasis, but it remains uncertain what role other canalicular transporters play in the development of this disease. We examined the expression of these transporters in Abcb4(-/-) mice compared with their wild-type littermate controls at ages of 10 days and 3, 6, and 12 wk. Elevated plasma bile acid levels were already detected at 10 days and at all ages thereafter in Abcb4(-/-) mice. The expression of Bsep, Mrp2, Atp8b1, Abcg5, and Abcg8 liver proteins did not change at 10 days, but Bsep, Mrp2, and Atp8b1 were reduced, whereas Abcg5 and Abcg8 expression were increased in Abcb4(-/-) mice at all later ages. Lower bile acid concentrations were also detected in the bile of 6-wk-old Abcb4(-/-) mice. Immunofluorescence labeling revealed distorted canalicular architecture in the liver tissue by 12 wk in Abcb4(-/-) mice. Whereas Bsep and Mrp2 remained associated with the apical membrane, Atp8b1 was now localized in discrete punctuate structures adjacent to the canalicular membrane in these mice. Expression of Bsep mRNA was increased in the livers of 10-day-old Abcb4(-/-) mice, whereas Ost-α was decreased. By 12 wk, Bsep, Mrp2, and Abcg5 mRNA were all increased, whereas Ost-α and Ntcp were reduced. These findings indicate that canalicular transporters that determine the formation of bile are altered early in the development of cholestasis in Abcb4(-/-) mice and may contribute to the pathogenesis of cholestasis in this disorder.

Chronic cholestasis results in liver injury and eventually liver failure. Although ursodeoxycholic acid (UDCA) showed limited benefits in primary biliary cirrhosis, there is an urgent need to develop alternative therapy for chronic cholestatic disorders. Previous studies from our laboratory demonstrated that all-trans-retinoic acid (atRA) is a potent suppressor of CYP7A1, the rate-limiting enzyme in bile acid synthesis. atRA also repressed the expression of tumor growth factor-β and collagen 1A1 in activated primary human stellate cells and LX2 cells. When administered together with UDCA to bile duct-ligated rats, this combined therapy significantly reduced the bile acid pool size and improved liver conditions. To further examine whether atRA alone or in combination with UDCA has greater beneficial effects than UDCA treatment alone, we assessed this treatment in two additional chronic cholestatic rodent models: α-naphthylisothiocyanate (ANIT)-treated rats and the Mdr2(-/-) (Abcb4(-/-)) knockout mouse. atRA alone significantly reduced bile duct proliferation, inflammation, and hydroxyproline levels in ANIT-treated rats, whereas the combination of atRA and UDCA significantly reduced plasma bile salt level compared with UDCA treatment. atRA alone or in combination with UDCA significantly reduced plasma levels of alkaline phosphatase and bile salts in 12-week-old Mdr2(-/-) mice. Reduced bile duct proliferation and inflammation were also observed in the livers of these mice. Together, atRA alone or in combination with UDCA significantly reduced the severity of liver injury in these two animal models, further supporting the combination treatment of atRA and UDCA as a potential new therapy for patients with chronic cholestatic liver disease who have not responded fully to UDCA.

Unlabelled: Multidrug resistance transporter 3/ATP-binding cassette protein subfamily B4 (MDR3/ABCB4) is a critical determinant of biliary phosphatidylcholine (PC) secretion. Clinically, mutations and partial deficiencies in MDR3 result in cholestatic liver injury. Thus, MDR3 is a potential therapeutic target for cholestatic liver disease. Fenofibrate is a peroxisome proliferator-activated receptor (PPAR) α ligand that has antiinflammatory actions and regulates bile acid detoxification. Here we examined the mechanism by which fenofibrate regulates MDR3 gene expression. Fenofibrate significantly up-regulated MDR3 messenger RNA (mRNA) and protein expression in primary cultured human hepatocytes, and stimulated MDR3 promoter activity in HepG2 cells. In silico analysis of 5'-upstream region of human MDR3 gene revealed a number of PPARα response elements (PPRE). Electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP) assays demonstrated specific binding of PPARα to the human MDR3 promoter. Targeted mutagenesis of three novel PPREs reduced inducibility of the MDR3 promoter by fenofibrate. In collagen sandwich cultured rat hepatocytes, treatment with fenofibrate increased secretion of fluorescent PC into bile canaliculi., Conclusion: Fenofibrate transactivates MDR3 gene transcription by way of the binding of PPARα to three novel and functionally critical PPREs in the MDR3 promoter. Fenofibrate treatment further stimulates biliary phosphatidylcholine secretion in rat hepatocytes, thereby providing a functional correlate. We have established a molecular mechanism that may contribute to the beneficial use of fenofibrate therapy in human cholestatic liver disease., (© 2014 by the American Association for the Study of Liver Diseases.)

The organic solute transporter OSTα-OSTβ is a key transporter for the efflux of bile acids across the basolateral membrane of ileocytes and the subsequent return of bile acids to the liver. Ostα(-/-) mice exhibit reduced bile acid pools and impaired lipid absorption. In this study, wild-type and Ostα(-/-) mice were characterized at 5 and 12 mo of age. Ostα(-/-) mice were resistant to age-related weight gain, body fat accumulation, and liver and muscle lipid accumulation, and male Ostα(-/-) mice lived slightly longer than wild-type mice. Caloric intake and activity levels were similar for Ostα(-/-) and wild-type male mice. Fecal lipid excretion was increased in Ostα(-/-) mice, indicating that a defect in lipid absorption contributes to decreased fat accumulation. Analysis of genes involved in intestinal lipid absorption revealed changes consistent with decreased dietary lipid absorption in Ostα(-/-) animals. Hepatic expression of cholesterol synthetic genes was upregulated in Ostα(-/-) mice, showing that increased cholesterol synthesis partially compensated for reduced dietary cholesterol absorption. Glucose tolerance was improved in male Ostα(-/-) mice, and insulin sensitivity was improved in male and female Ostα(-/-) mice. Akt phosphorylation was measured in liver and muscle tissue from mice after acute administration of insulin. Insulin responses were significantly larger in male and female Ostα(-/-) than wild-type mice. These findings indicate that loss of OSTα-OSTβ protects against age-related weight gain and insulin resistance.

Background & Aims: Oltipraz (4-methyl-5(pyrazinyl-2)-1-2-dithiole-3-thione), a promising cancer preventive agent, has an antioxidative activity and ability to enhance glutathione biosynthesis, phase II detoxification enzymes and multidrug resistance-associated protein-mediated efflux transporters. Oltipraz can protect against hepatotoxicity caused by carbon tetrachloride, acetaminophen and alpha-naphthylisothiocyanate. Whether oltipraz has hepato-protective effects on obstructive cholestasis is unknown., Methods: We administered oltipraz to mice for 5 days prior to bile duct ligation (BDL) for 3 days. Liver histology, liver function markers, bile flow rates and hepatic expression of profibrogenic genes were evaluated., Results: Mice pretreated with oltipraz prior to BDL demonstrated higher levels of serum aminotransferases and more severe liver damage than in control mice. Higher bile flow and glutathione secretion rates were observed in unoperated mice treated with oltipraz than in control mice, suggesting that liver necrosis in oltipraz-treated BDL mice may be related partially to increased bile-acid independent flow and biliary pressure. Oltipraz treatment in BDL mice enhanced α-smooth muscle actin expression, consistent with activation of hepatic stellate cells and portal fibroblasts. Matrix metalloproteinases (Mmp) 9 and 13 and tissue inhibitors of metalloproteinases (Timp) 1 and 2 levels were increased in the oltipraz-treated BDL group, suggesting that the secondary phase of liver injury induced by oltipraz might be due to excessive Mmp and Timp secretions, which induce remodeling of the extracellular matrix., Conclusions: Oltipraz treatment exacerbates the severity of liver injury following BDL and should be avoided as therapy for extrahepatic cholestatic disorders due to bile duct obstruction., (Copyright © 2013 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved.)

The sea lamprey (Petromyzon marinus) is a genetically programmed animal model for biliary atresia, as it loses its bile ducts and gallbladder during metamorphosis. However, in contrast to patients with biliary atresia or other forms of cholestasis who develop progressive disease, the postmetamorphosis lampreys grow normally to adult size. To understand how the adult lamprey thrives without the ability to secrete bile, we examined bile salt homeostasis in larval and adult lampreys. Adult livers were severely cholestatic, with levels of bile salts >1 mM, but no evidence of necrosis, fibrosis, or inflammation. Interestingly, both larvae and adults had normal plasma levels (∼10 μM) of bile salts. In larvae, petromyzonol sulfate (PZS) was the predominant bile salt, whereas the major bile salts in adult liver were sulfated C27 bile alcohols. Cytotoxicity assays revealed that PZS was highly toxic. Pharmacokinetic studies in free-swimming adults revealed that ∼35% of intravenously injected bromosulfophthalein (BSP) was eliminated over a 72-hour period. Collection of urine and feces demonstrated that both endogenous and exogenous organic anions, including biliverdin, bile salts, and BSP, were predominantly excreted by way of the kidney, with minor amounts also detected in feces. Gene expression analysis detected marked up-regulation of orthologs of known organic anion and bile salt transporters in the kidney, with lesser effects in the intestine and gills in adults compared to larvae. These findings indicate that adult lampreys tolerate cholestasis by altering hepatic bile salt composition, while maintaining normal plasma bile salt levels predominantly through renal excretion of bile products. Therefore, we conclude that strategies to accelerate renal excretion of bile salt and other toxins should be beneficial for patients with cholestasis. (HEPATOLOGY 2013;57:2418-2426)., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Unlabelled: The liver-specific bile salt export pump (BSEP) is crucial for bile acid-dependent bile flow at the apical membrane. BSEP, a member of the family of structurally related adenosine triphosphate (ATP)-binding cassette (ABC) proteins, is composed of 12 transmembrane segments (TMS) and two large cytoplasmic nucleotide-binding domains (NBDs). The regulation of trafficking of BSEP to and from the cell surface is not well understood, but is believed to play an important role in cholestatic liver diseases such as primary familial intrahepatic cholestasis type 2 (PFIC2). To address this issue, BSEP endocytosis was studied by immunofluorescence and a cell surface enzyme-linked immunosorbent assay (ELISA) endocytosis reporter system using a chimera of the interleukin-2 receptor α (previously referred to as Tac) and the C-terminal tail of BSEP (TacCterm). An autonomous endocytosis motif in the carboxyl cytoplasmic terminus of BSEP was identified. We define this endocytic motif by site-directed mutagenesis as a canonical tyrosine-based motif (1310) YYKLV(1314) (YxxØ). When expressed in HEK293T cells, TacCterm is constitutively internalized via a dynamin- and clathrin-dependent pathway. Mutation of the Y(1310) Y(1311) amino acids in TacCterm and in full-length human BSEP blocks the internalization. Subsequent sequence analysis reveals this motif to be highly conserved between the closely related ABCB subfamily members that mediate ATP-dependent transport of broad substrate specificity., Conclusion: Our results indicate that constitutive internalization of BSEP is clathrin-mediated and dependent on the tyrosine-based endocytic motif at the C-terminal end of BSEP., (Copyright © 2012 American Association for the Study of Liver Diseases.)

The transcription factor nuclear factor-E2-related factor 2 (Nrf2) is a key regulator for induction of hepatic detoxification and antioxidant mechanisms, as well as for certain hepatobiliary transporters. To examine the role of Nrf2 in bile acid homeostasis and cholestasis, we assessed the determinants of bile secretion and bile acid synthesis and transport before and after bile duct ligation (BDL) in Nrf2(-/-) mice. Our findings indicate reduced rates of biliary bile acid and GSH excretion, higher levels of intrahepatic bile acids, and decreased expression of regulators of bile acid synthesis, Cyp7a1 and Cyp8b1, in Nrf2(-/-) compared with wild-type control mice. The mRNA expression of the bile acid transporters bile salt export pump (Bsep) and organic solute transporter (Ostα) were increased in the face of impaired expression of the multidrug resistance-associated proteins Mrp3 and Mrp4. Deletion of Nrf2 also decreased ileal apical sodium-dependent bile acid transporter (Asbt) expression, leading to reduced bile acid reabsorption and increased loss of bile acid in feces. Finally, when cholestasis is induced by BDL, liver injury was not different from that in wild-type BDL mice. These Nrf2(-/-) mice also had increased pregnane X receptor (Pxr) and Cyp3a11 mRNA expression in association with enhanced hepatic bile acid hydroxylation. In conclusion, this study finds that Nrf2 plays a major role in the regulation of bile acid homeostasis in the liver and intestine. Deletion of Nrf2 results in a cholestatic phenotype but does not augment liver injury following BDL.

Bile acid homeostasis is tightly maintained through interactions between the liver, intestine, and kidney. During cholestasis, the liver is incapable of properly clearing bile acids from the circulation, and alternative excretory pathways are utilized. In obstructive cholestasis, urinary elimination is often increased, and this pathway is further enhanced after bile duct ligation in mice that are genetically deficient in the heteromeric, basolateral organic solute transporter alpha-beta (Ostα-Ostβ). In this study, we examined renal and intestinal function in Ostα-deficient and wild-type mice in a model of bile acid overload. After 1% cholic acid feeding, Ostα-deficient mice had significantly lower serum ALT levels compared with wild-type controls, indicating partial protection from liver injury. Urinary clearance of bile acids, but not clearance of [(3)H]inulin, was significantly higher in cholic acid-fed Ostα-deficient mice compared with wild-type mice but was not sufficient to account for the protection. Fecal excretion of bile acids over the 5 days of cholic acid feeding was responsible for almost all of the bile acid loss in Ostα-deficient mice, suggesting that intestinal losses of bile acids accounted for the protection from liver injury. Thus fecal loss of bile acids after bile acid overload reduced the need for the kidney to filter and excrete the excess bile acids. In conclusion, Ostα-deficient mice efficiently eliminate excess bile acids via the feces. Inhibition of intestinal bile acid absorption might be an effective therapeutic target in early stages of cholestasis when bile acids are still excreted into bile.

Breast cancer resistance protein (Bcrp) is a member of the ATP-binding cassette membrane transporter family, which is expressed apically in liver, kidney, and intestine epithelium. Recent reports suggest that in addition to xenobiotics, porphyrins, and food toxins, Bcrp can also transport bile acids and, therefore, may participate in the adaptive response to cholestasis. Bile duct ligation (BDL), an experimental model of obstructive cholestasis, was performed in male wild-type (WT) and Bcrp knockout (KO) mice. An initial time course of 3, 7, and 14 days of BDL in WT mice revealed that Bcrp expression was significantly reduced in liver but increased in ileum by 7 days. Subsequent experiments using 7-day BDL in WT and Bcrp KO mice demonstrated that there was no difference in liver necrosis, serum glutamic pyruvate aminotransferase, bilirubin, or bile acid levels in serum, hepatic tissue, bile, urine, or feces between the two groups. Protein expression levels for liver organic solute transporter (Ost) α and multidrug resistance protein 1 and kidney multidrug resistance-associated protein (Mrp) 2, Mrp3, and Mrp4 were significantly greater in the sham Bcrp KO versus sham WT mice. The expression of Mrp2 and Mrp4 in KO kidneys was further increased after BDL. In contrast, the adaptive response of transporters to BDL in the liver was similar in KO and WT BDL mice, including Ostα and Ostβ expression, which increased in liver and kidney but decreased in the ileum. These findings suggest that Bcrp does not have a significant role in the adaptive response to cholestasis in the liver but may be more important for solute export in the kidney and intestine.

Multidrug resistance protein 4 (MRP4; ABCC4) is an ATP binding cassette transporter that facilitates the excretion of bile salt conjugates and other conjugated steroids in hepatocytes and renal proximal tubule epithelium. MRP4/Mrp4 undergoes adaptive upregulation in response to oxidative and cholestatic liver injury in human and animal models of cholestasis. However, the molecular mechanism of this regulation remains to be determined. The aryl hydrocarbon receptor (AhR) and NF-E2-related factor 2 (Nrf2) play important roles in protecting cells from oxidative stress. Here we examine the role of these two nuclear factors in the regulation of the expression of human MRP4. HepG2 cells and human hepatocytes were treated with the AhR and Nrf2 activators, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 3-methylcholanthrene (3-MC), or oltipraz and other nuclear receptor agonists. TCDD, 3-MC, and oltipraz significantly increased MRP4 expression at mRNA and protein levels. Computer program analysis revealed three Xenobiotic response element (XRE) and one Maf response element sites within the first 500 bp of the MRP4 proximal promoter. Luciferase reporter assay detected strong promoter activity (53-fold higher than vector control) in this region. TCDD and 3-MC also induced promoter activity in the reporter assays. Mutation of any of these XRE sites significantly decreased MRP4 promoter activity in reporter assays, although XRE2 demonstrated the strongest effects on both basal and TCDD-inducible activity. EMSA and chromatin immunoprecipitation assays further confirmed that both AhR and Nrf2 bind to the proximal promoter of MRP4. Our findings indicate that AhR and Nrf2 play important roles in regulating MRP4 expression and suggest that agents that activate their activity may be of therapeutic benefit for cholestasis.

Multidrug resistance-associated protein 2 (Mrp2, Abcc2) is an ATP-binding cassette transporter localized at the canalicular membrane of hepatocytes that plays an important role in bile formation and detoxification. Prior in vitro studies suggest that Mrp2 can bind to Na(+)/H(+) exchanger regulatory factor 1 (NHERF-1), a PDZ protein that cross-links membrane proteins to actin filaments. However the role of NHERF-1 in the expression and functional regulation of Mrp2 remains largely unknown. Here we examine the interaction of Mrp2 and NHERF-1 and its physiological significance in HEK293 cells and NHERF-1 knock-out mice. Mrp2 co-precipitated with NHERF-1 in co-transfected HEK293 cells, an interaction that required the PDZ-binding motif of Mrp2. In NHERF-1(-/-) mouse liver, Mrp2 mRNA was unchanged but Mrp2 protein was reduced in whole cell lysates and membrane-enriched fractions to approximately 50% (p < 1 x 10(-6)) and approximately 70% (p < 0.05), respectively, compared with wild-type mice, suggesting that the down-regulation of Mrp2 expression was caused by post-transcriptional events. Mrp2 remained localized at the apical/canalicular membrane of NHERF-1(-/-) mouse hepatocytes, although its immunofluorescent labeling was noticeably weaker. Bile flow in NHERF-1(-/-) mice was reduced to approximately 70% (p < 0.001) in association with a 50% reduction in glutathione excretion (p < 0.05) and a 60% reduction in glutathione-methylfluorescein (GS-MF) excretion in isolated mouse hepatocyte (p < 0.01). Bile acid and bilirubin excretion remained unchanged compared with wild-type mice. These findings strongly suggest that NHERF-1 binds to Mrp2, and plays a critical role in the canalicular expression of Mrp2 and its function as a determinant of glutathione-dependent, bile acid-independent bile flow.

Organic solute transporter alpha-beta (OSTalpha-OSTbeta) is a unique heteromeric transporter localized to the basolateral membrane of epithelial cells involved in sterol transport. It is believed to be the primary bile acid efflux transporter in the intestine of mammals and is therefore essential to bile acid homeostasis and the enterohepatic circulation. First described in the evolutionarily primitive small skate, LEUCORAJA ERINACEA, this facilitated transporter requires expression of both subunits for its function. It can transport a variety of bile acids, as well as estrone 3-sulfate, dehydroepiandrosterone 3-sulfate, digoxin, and prostaglandin E (2). Expression of both subunits is variable between species and tissues; in humans high expression is noted in the liver, small intestine, kidney, testis, and adrenal gland. OSTalpha-OSTbeta is directly regulated by the bile acid sensing nuclear receptor, farnesoid X receptor (FXR). Furthermore, it is part of the complex regulatory pathway that controls bile acid synthesis and homeostasis. Hepatic OSTalpha-OSTbeta is upregulated in cholestasis in both humans and rodents, where it appears to play a protective role. Additional studies are necessary to determine its role in liver injury, bile acid malabsorption, and lipid and glucose metabolism, as well as a potential protective role for kidney OSTalpha-OSTbeta in cholestasis.

The primary transporter responsible for bile salt secretion is the bile salt export pump (BSEP, ABCB11), a member of the ATP-binding cassette (ABC) superfamily, which is located at the bile canalicular apical domain of hepatocytes. In humans, BSEP deficiency results in several different genetic forms of cholestasis, which include progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), as well as other acquired forms of cholestasis such as drug-induced cholestasis (DIC) and intrahepatic cholestasis of pregnancy (ICP). Because bile salts play a pivotal role in a wide range of physiologic and pathophysiologic processes, regulation of BSEP expression has been a subject of intense research. The authors briefly describe the molecular characteristics of BSEP and then summarize what is known about its role in the pathogenesis of genetic and acquired cholestatic disorders, emphasizing experimental observations from animal models and cell culture in vitro systems.

Unlabelled: Organic solute transporter alpha-beta (Ostalpha-Ostbeta) is a heteromeric bile acid and sterol transporter that facilitates the enterohepatic and renal-hepatic circulation of bile acids. Hepatic expression of this basolateral membrane protein is increased in cholestasis, presumably to facilitate removal of toxic bile acids from the liver. In this study, we show that the cholestatic phenotype induced by common bile duct ligation (BDL) is reduced in mice genetically deficient in Ostalpha. Although Ostalpha(-/-) mice have a smaller bile acid pool size, which could explain lower serum and hepatic levels of bile acids after BDL, gallbladder bilirubin and urinary bile acid concentrations were significantly greater in Ostalpha(-/-) BDL mice, suggesting additional alternative adaptive responses. Livers of Ostalpha(-/-) mice had higher messenger RNA levels of constitutive androstane receptor (Car) than wild-type BDL mice and increased expression of Phase I enzymes (Cyp7a1, Cyp2b10, Cyp3a11), Phase II enzymes (Sult2a1, Ugt1a1), and Phase III transporters (Mrp2, Mrp3). Following BDL, the bile acid pool size increased in Ostalpha(-/-) mice and protein levels for the hepatic basolateral membrane export transporters, multidrug resistance-associated protein 3 (Mrp3) and Mrp4, and for the apical bilirubin transporter, Mrp2, were all increased. In the kidney of Ostalpha(-/-) mice after BDL, the apical bile acid uptake transporter Asbt is further reduced, whereas the apical export transporters Mrp2 and Mrp4 are increased, resulting in a significant increase in urinary bile acid excretion., Conclusion: These findings indicate that loss of Ostalpha provides protection from liver injury in obstructive cholestasis through adaptive responses in both the kidney and liver that enhance clearance of bile acids into urine and through detoxification pathways most likely mediated by the nuclear receptor Car.

Unlabelled: The bile salt export pump (BSEP) is the major determinant of bile salt-dependent bile secretion, and its deficiency leads to cholestatic liver injury. BSEP/Bsep gene expression is regulated by the nuclear farnesoid X receptor. However, BSEP expression, though reduced, is retained in the livers of Fxr(-/-) mice, indicating that additional transcriptional factors may regulate its expression. Nuclear factor erythroid 2-related factor 2 (Nrf2) plays a major role in response to oxidative stress by binding to antioxidant-responsive elements that regulate many hepatic phase I and II enzymes as well as hepatic efflux transporters. Computer software analysis of human BSEP reveals two musculo-aponeurotic fibrosacroma (Maf) recognition elements (MAREs) from the sequence in the proximal promoter region where Nrf2 may bind. In this study, we assessed whether Nrf2 plays a role in human BSEP expression and if this might be mediated by MAREs. Oltipraz, a potent activator of Nrf2, increased BSEP messenger RNA expression by approximately seven-fold in HepG2 cells and protein by approximately 70% in human hepatocytes. Small interfering RNAs lowered NRF2 expression in HepG2 cells and prevented the up-regulation of BSEP by oltipraz. Human BSEP promoter activity was stimulated by Nrf2 in a dose-dependent manner in luciferase reporter assays. Mutations of the predicted MARE1, but not MARE2, abolished this Nrf2 transcriptional activation. Chromatin immunoprecipitation assays also demonstrated that Nrf2 specifically bound to MARE1, but not MARE2 regions in the BSEP promoter in HepG2 cells. Electrophoretic mobility shift assays further demonstrated direct binding of MARE1 in the BSEP promoter., Conclusion: Nrf2 is a positive transcriptional regulator of human BSEP expression. Pharmacological activation of Nrf2 may be beneficial for cholestatic liver injury.

Background & Aims: Progressive familial intrahepatic cholestasis 1 (PFIC1) results from mutations in ATP8B1, a putative aminophospholipid flippase. Conflicting hypotheses have been proposed for the pathogenesis of PFIC1. The aim of this study was to determine whether ATP8B1 deficiency produces cholestasis by altering the activity of the farnesoid X receptor (FXR) or by impairing the structure of the canalicular membrane., Methods: ATP8B1/Atp8b1 was knocked down in human and rat hepatocytes and Caco2 cells using adenoviral and oligonucleotide small interfering RNAs., Results: ATP8B1 messenger RNA and protein expression was greatly reduced in human and rat cells. In contrast, FXR expression and several FXR-dependent membrane transporters (bile salt export pump [BSEP], multidrug resistance-associated protein [MRP] 2) were unchanged at messenger RNA or protein levels in ATP8B1-deficient cells, whereas Mrp3 and Mrp4 were up-regulated in rat hepatocytes. FXR activity remained intact in these cells, as evidenced by 6alpha-ethyl chenodeoxycholic acid-mediated induction of small heterodimer partner, BSEP, and multidrug-resistant protein (MDR) 3/Mdr2. Fluorescent substrate excretion assays indicate that Bsep function was significantly reduced in Atp8b1-deficient rat hepatocytes, although Bsep remained localized to the canalicular membrane. Exposure to the hydrophobic bile acid CDCA resulted in focal areas of canalicular membrane disruption by electron microscopy and luminal accumulation of NBD-phosphatidylserine, consistent with the function of Atp8b1 as an aminophospholipid flippase., Conclusions: ATP8B1 deficiency predisposes to cholestasis by favoring bile acid-induced injury in the canalicular membrane but does not directly affect FXR expression, which may occur in PFIC1 as a secondary phenomenon associated with cholestasis.

The bile salt export pump (Bsep) represents the major bile salt transport system at the canalicular membrane of hepatocytes. When examined in model cell lines, genetic mutations in the BSEP gene impair its targeting and transport function, contributing to the pathogenesis of progressive familial intrahepatic cholestasis type II (PFIC II). PFIC II mutations are known to lead to a deficiency of BSEP in human hepatocytes, suggesting that PFIC II mutants are unstable and degraded in the cell. To investigate this further, we have characterized the impact of several PFIC II mutations on the processing and stability of rat Bsep. G238V, D482G, G982R, R1153C, and R1286Q all retain Bsep to the endoplasmic reticulum (ER) to different extents. Except for R1153C, the PFIC II mutants are degraded with varying half-lives. G238V and D482G are partially misfolded and can be stabilized by low temperature and glycerol. The proteasome provides the major degradation pathway for the PFIC II mutants, whereas the lysosome also contributes to the degradation of D482G. The PFIC II mutants appear to be more heavily ubiquitinated compared with the wild-type (wt) Bsep, and their ubiquitination is increased by the proteasome inhibitors. Overexpression of several E3 ubiquitin ligases, which are involved in ER-associated degradation (ERAD), lead to the decrease of both mutant and wt Bsep. Gene knockdown studies showed that the ERAD E3s Rma1 and TEB4 contribute to the degradation of G238V, whereas HRD1 contributes to the degradation of a mutant lacking the lumenal glycosylation domain (DeltaGly). Furthermore, we present evidence that G982R weakly associates with various components of the ER quality control system. These data together demonstrate that the PFIC II mutants except R1153C and DeltaGly are degraded by the ERAD pathway.

Background: The organic solute transporter (OSTalpha-OSTbeta) is a heteromeric transporter that is expressed on the basolateral membrane of epithelium in intestine, kidney, liver, testis and adrenal gland and facilitates efflux of bile acids and other steroid solutes. Both subunits are required for plasma membrane localization of the functional transporter but it is unclear how and where the subunits interact and whether glycosylation is required for functional activity. We sought to examine these questions for the human OSTalpha-OSTbeta transporter using the human hepatoma cell line, HepG2, and COS7 cells transfected with constructs of human OSTalpha-FLAG and OSTbeta-Myc., Results: Tunicamycin treatment demonstrated that human OSTalpha is glycosylated. In COS7 cells Western blotting identified the unglycosylated form (approximately 31 kD), the core precursor form (approximately 35 kD), and the mature, complex glycoprotein (approximately 40 kD). Immunofluorescence of both cells indicated that, in the presence of OSTbeta, the alpha subunit could still be expressed on the plasma membrane after tunicamycin treatment. Furthermore, the functional uptake of 3H-estrone sulfate was unchanged in the absence of N-glycosylation. Co-immunoprecipitation indicates that the immature form of OSTalpha interact with OSTbeta. However, immunoprecipitation of OSTbeta using an anti-Myc antibody did not co-precipitate the mature, complex glycosylated form of OSTalpha, suggesting that the primary interaction occurs early in the biosynthetic pathway and may be transient., Conclusion: In conclusion, human OSTalpha is a glycoprotein that requires interaction with OSTbeta to reach the plasma membrane. However, glycosylation of OSTalpha is not necessary for interaction with the beta subunit or for membrane localization or function of the heteromeric transporter.

Human BSEP (ABCB11) mutations are the molecular basis for at least three clinical forms of liver disease, progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2), and intrahepatic cholestasis of pregnancy (ICP). To better understand the pathobiology of these disease phenotypes, we hypothesized that different mutations may cause significant differences in protein defects. Therefore we compared the effect of two PFIC2 mutations (D482G, E297G) with two BRIC2 mutations (A570T and R1050C) and one ICP mutation (N591S) with regard to the subcellular localization, maturation, and function of the rat Bsep protein. Bile salt transport was retained in all but the E297G mutant. Mutant proteins were expressed at reduced levels on the plasma membrane of transfected HEK293 cells compared with wild-type (WT) Bsep in the following order: WT > N591S > R1050C approximately A570T approximately E297G >> D482G. Total cell protein and surface protein expression were reduced to the same extent, suggesting that trafficking of these mutants to the plasma membrane is not impaired. All Bsep mutants accumulate in perinuclear aggresome-like structures in the presence of the proteasome inhibitor MG-132, suggesting that mutations are associated with protein instability and ubiquitin-dependent degradation. Reduced temperature, sodium butyrate, and sodium 4-phenylbutyrate enhanced the expression of the mature and cell surface D482G protein in HEK293 cells. These results suggest that the clinical phenotypes of PFIC2, BRIC2, and ICP may directly correlate with the amount of mature protein that is expressed at the cell surface and that strategies to stabilize cell surface mutant protein may be therapeutic.

Phloracetophenone (2,4,6-trihydroxyacetophenone, THA) is a potent choleretic in the bile fistula rat, although the mechanism is unknown. In the present study, we examined how THA enhances bile secretion. Stepwise infusions of THA (1-4 micromol/min) in the isolated perfused rat liver resulted in an immediate and dose-dependent increase in bile flow (BF), which reached saturation. The increase in BF was not associated with a change in the excretion of bile acids, suggesting that THA stimulated bile acid-independent bile flow. To further define the mechanism, the effect of THA on the excretion of sulfobromophthalein (BSP) and disulfobromophthalein (DBSP), typical multidrug resistance protein-2 (Mrp2) substrates was examined. THA inhibited the biliary excretion of both substrates. Because DBSP is excreted without conjugation to glutathione, in contrast to BSP, the findings suggest that THA might compete with DBSP and BSP metabolites at a common canalicular transport site, presumably Mrp2. THA infusions had no effect on the subcellular localization and distribution of either Mrp2 or the bile salt export pump (Bsep), nor the integrity of the tight junction. In contrast, the choleretic activity of THA was completely absent in the TR(-) rat, an animal model that lacks Mrp2, directly implicating this canalicular export pump as the mechanisms by which THA is excreted in bile. THA also partially reversed the cholestatic effects of estradiol-17beta-D-glucuronide, a process also dependent on Mrp2. In conclusion, the choleretic activity of THA and its possible metabolites is dependent on Mrp2. THA appears to stimulate BF by its osmotic effects and may attenuate the cholestatic effects of hepatotoxins undergoing biotransformation and excretion via similar pathways.

Multidrug resistance-associated protein MRP3/Mrp3 (ABCC3) is upregulated in cholestasis, an adaptive response that may protect the liver from accumulation of toxic compounds, such as bile salts and bilirubin conjugates. However, the mechanism of this upregulation is poorly understood. We and others have previously reported that fetoprotein transcription factor/liver receptor homolog-1 is an activator of MRP3/Mrp3 expression. In searching for additional regulatory elements in the human MRP3 promoter, we have now identified nuclear receptor retinoic X receptor-alpha:retinoic acid receptor-alpha (RXRalpha:RARalpha) as a repressor of MRP3 activation by transcription factor Sp1. A luciferase reporter assay demonstrated that cotransfection of transcription factor Sp1 stimulates the MRP3 promoter activity and that additions of RXRalpha:RARalpha abrogated this activation in a dose-dependent manner. Site mutations and gel shift assays have identified a Sp1 binding GC box motif at -113 to -108 nts upstream from the MRP3 translation start site, where RXRalpha:RARalpha specifically reduced Sp1 binding to this site. Mutation of the GC box also reduced MRP3 promoter activity. The functional role of RXRalpha:RARalpha as a repressor of MRP3 expression was further confirmed by RARalpha small-interfering RNA knockdown in HepG2 cells, which upregulated endogenous MRP3 expression. In summary, our results indicate that activator Sp1 and repressor RXRalpha:RARalpha act in concert to regulate MRP3 expression. Since RXRalpha:RARalpha expression is diminished by cholestatic liver injury, loss of RXRalpha:RARalpha may lead to upregulation of MRP3/Mrp3 expression in these disorders.

Background & Aims: Ezrin-radixin-moesin proteins are cross-linkers between the plasma membrane and actin filaments. Radixin, the dominant ezrin-radixin-moesin protein in hepatocytes, has been reported to selectively tether multidrug-resistance-associated protein 2 to the apical canalicular membrane. However, it remains to be determined if this is its primary function., Methods: An adenovirus-mediated short interfering RNA (siRNA) was used to down-regulate radixin expression in collagen sandwich-cultured rat hepatocytes and morphologic and functional changes were characterized quantitatively., Results: In control cultures, an extensive bile canalicular network developed with properly localized apical and basolateral transporters that provided for functional excretion of fluorescent cholephiles into the bile canalicular lumina. siRNA-induced suppression of radixin was associated with a marked reduction in the canalicular membrane structure as observed by differential interference contrast microscopy and F-actin staining, in contrast to control cells exposed to adenovirus encoding scrambled siRNA. Indirect immunofluorescence showed that apical transporters (multidrug-resistance-associated protein 2, bile salt export pump, and multidrug-resistance protein 1) dissociated from their normal location at the apical membrane and were found largely associated with Rab11-containing endosomes. Localization of the basolateral membrane transporter, organic anion transporting polypeptide 2 (Oatp2), was not affected. Consistent with this dislocation of apical transporters, the biliary excretion of glutathione-methylfluorescein and cholylglycylamido-fluorescein was decreased significantly in the radixin-deficient cells, but not in the control siRNA cells., Conclusions: Radixin is essential for maintaining the polarized targeting and/or retaining of canalicular membrane transporters and is a critical determinant of the overall structure and function of the apical membrane of hepatocytes.

The ability of the kidney and small intestine to synthesize and subsequently eliminate dinitrophenyl-S-glutathione (DNP-SG), a substrate for the multidrug resistance-associated proteins (Mrps), was assessed in bile duct-ligated (BDL) rats 1, 7, and 14 days after surgery, using an in vivo perfused jejunum model with simultaneous urine collection. A single i.v. dose of 30 micromol/kg b.wt. of 1-chloro-2,4-dinitrobenzene (CDNB) was administered, and its glutathione conjugate DNP-SG and dinitrophenyl cysteinyl glycine derivative, which is the result of gamma-glutamyl-transferase action on DNP-SG, were determined in urine and intestinal perfusate by high-performance liquid chromatography. Intestinal excretion of these metabolites was unchanged at day 1, and decreased at days 7 and 14 (-39% and -33%, respectively) after surgery with respect to shams. In contrast, renal excretion was increased by 114%, 150%, and 128% at days 1, 7, and 14. Western blot studies revealed decreased levels of apical Mrp2 in liver and jejunum but increased levels in renal cortex from BDL animals, these changes being maximal between days 7 and 14. Assessment of expression of basolateral Mrp3 at day 14 postsurgery indicated preserved levels in renal cortex, duodenum, jejunum, distal ileum, and colon. Analysis of expression of glutathione-S-transferases alpha, mu, and pi, as well as activity toward CDNB, indicates that formation of DNP-SG was impaired in liver, preserved in intestine, and increased in renal cortex. In conclusion, increased renal tubular conversion of CDNB to DNP-SG followed by subsequent Mrp2-mediated secretion into urine partially compensates for altered liver function in experimental obstructive cholestasis.

Organic solute transporter (OSTalpha-OSTbeta) is a novel heteromeric bile acid and sterol transporter expressed at the basolateral membranes of epithelium in the ileum, kidney, and liver. To determine whether OSTalpha-OSTbeta undergoes farnesoid X receptor (FXR)-dependent adaptive regulation following cholestatic liver injury, mRNA and protein expression levels were analyzed in patients with primary biliary cirrhosis (PBC) and following common bile duct ligation (CBDL) in rats and Fxr null and wild-type mice. Hepatic OSTalpha and OSTbeta mRNA increased 3- and 32-fold, respectively, in patients with PBC compared with controls, whereas expression of Ostalpha and Ostbeta also increased in the liver of rats and mice following CBDL. In contrast, expression of Ostalpha and Ostbeta mRNA was generally lower in Fxr null mice, and CBDL failed to enhance expression of Ostalpha and Ostbeta compared with wild-type mice. HepG2 cells treated for 24 h with chenodeoxycholic acid, a selective FXR ligand, had higher levels of OSTalpha and OSTbeta mRNA and protein. Increases in OST protein were visualized by confocal microscopy at the plasma membrane. These results indicate that expression of Ostalpha and Ostbeta are highly regulated in response to cholestasis and that this response is dependent on the FXR bile acid receptor.

Mrp4 is a member of the multidrug resistance-associated gene family that is expressed on the basolateral membrane of hepatocytes and undergoes adaptive upregulation in response to cholestatic injury or bile acid feeding. However, the relative importance of Mrp4 in a protective adaptive response to cholestatic injury is not known. To address this issue, common bile duct ligation (CBDL) was performed in wild-type and Mrp4-/- mice and animals followed for 7 days. Histological analysis and serum aminotransferase levels revealed more severe liver injury in the absence of Mrp4 expression. Western analyses revealed that Mrp4, but not Mrp3, was significantly increased after CBDL in wild-type mice. Serum bile acid levels were significantly lower in Mrp4-/- mice than in wild-type CBDL mice, whereas serum bilirubin levels were the same, suggesting that Mrp4 was required to effectively extrude bile acids from the cholestatic liver. Mrp3 and Ostalpha-Ostbeta were upregulated in Mrp4-/- mice but were unable to compensate for the loss of Mrp4. High-performance liquid chromatography analysis on liver extracts revealed that taurine tetrahydroxy bile acid/beta-muricholic acid ratios were increased twofold in Mrp4-/- mice. In conclusion, hepatic Mrp4 plays a unique and essential protective role in the adaptive response to obstructive cholestatic liver injury.

Aim: To assess the effects of obstructive cholestasis on a wider range of gene expression using microarray technology., Methods: Male C57BL/6J mice underwent common bile duct ligation (BDL) and were matched with pair-fed sham-operated controls. After 7 d, the animals were sacrificed and total RNA was isolated from livers and kidneys. Equal amounts of RNA from each tissue were pooled for each group and hybridized to Affymetrix GeneChip MG-U74Av2 containing a total of 12488 probe sets. Data analysis was performed using GeneSpring 6.0 software. Northern analysis and immunofluorescence were used for validation., Results: In sham-operated and BDL mice, 44 and 50% of 12488 genes were expressed in livers, whereas 49 and 51% were expressed in kidneys, respectively. Seven days after BDL, 265 liver and 112 kidney genes with GeneOntology annotation were up-regulated and 113 liver and 36 kidney genes were down-regulated in comparison with sham-operated controls. Many genes were commonly regulated in both tissues and metabolism-related genes represented the largest functional group., Conclusion: Following BDL, microarray analysis reveals a broad range of gene alterations in both liver and kidney.

The cellular and subcellular localization and mechanism of transport of the heteromeric organic solute transporter (OST) OSTalpha-OSTbeta was examined in human and rodent epithelia. The two subunits of the transporter were expressed together in human small intestine, kidney, and liver, tissues that also express the apical sodium-dependent bile acid uptake transporter ASBT (SLC10A2). Indirect immunofluorescence microscopy localized OSTalpha and OSTbeta to the basolateral membrane of mouse, rat, and human ileal enterocytes, renal proximal tubular cells, and cholangiocytes. Transport in OSTalpha-OSTbeta-expressing Xenopus laevis oocytes was unaffected by depletion of intracellular adenosine triphosphate, or by changes in transmembrane Na(+), K(+), H(+), or Cl(-) concentration gradients. However, the oocytes demonstrated robust substrate efflux and trans-stimulation, indicating that transport occurs by facilitated diffusion. Madin Darby canine kidney cells coexpressing mouse Ostalpha and Ostbeta exhibited enhanced apical to basolateral transport of the major glycine and taurine conjugated bile acid species. In conclusion, the selective localization of OSTalpha and OSTbeta to the basolateral plasma membrane of epithelial cells responsible for bile acid and sterol reabsorption, the substrate selectivity of the transporter, and the facilitated diffusion transport mode collectively indicate that OSTalpha-OSTbeta is a key basolateral transporter for the reabsorption of these important steroid-derived molecules.

The liver plays a major role in biotransformation and elimination of various therapeutic agents and xenobiotics, many of which are organic cations and substrates of the organic cation transporter 1 (Oct1, Slc22a1). Oct1 is expressed at the basolateral membranes of hepatocytes and proximal renal tubules. Although Oct1 is the major uptake mechanism in hepatocytes for many pharmaceutical compounds, little is known about the effects of liver injury on this process. Our aim was to investigate the effects of obstructive cholestasis on Oct1 expression and function in liver and kidney. The effects of bile duct ligation (BDL) on Oct1 protein, messenger RNA (mRNA) expression, and tissue localization were determined in rat liver and kidney with Western analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunofluorescence. To assess Oct1 function, the model substrate tetraethylammonium ([(14)C]TEA) was administered intravenously to BDL and control rats and distribution of radioactivity was determined. Oct1 protein significantly decreased in cholestatic livers to 42.1 +/- 17.7% (P <.001), 15.5 +/- 4.7% (P <.05), and 8.6 +/- 2.7% (P <.05) of controls after 3, 7, and 14 days, respectively, but not in kidneys. Hepatic Oct1 mRNA decreased to 77.2 +/- 12.7%, 40.7 +/- 8.1% (P <.05), and 50.3 +/- 7.5% (P <.05) 3, 7, and 14 days after BDL, respectively. Tissue immunofluorescence corroborated these data. Hepatic accumulation of [(14)C]TEA in 14-day BDL rats was reduced to 29.6 +/- 10.9% of controls (P <.0005). In conclusion, obstructive cholestasis down-regulates Oct1 and impairs Oct1-mediated uptake in rat liver, suggesting that hepatic uptake of small cationic drugs may be impaired in cholestatic liver injury.

Background/aims: Multidrug resistance-associated protein 4 (Mrp4, ABCC4) transports cyclic nucleotides, anti-retroviral compounds, and sulfated bile acids. Mrp4 expression is increased in farnesyl/bile acid receptor knockout mice. Our aim was to investigate Mrp4 expression and function in rat liver and kidney in obstructive cholestasis., Methods: Male Sprague-Dawley rats were subjected to bile duct ligation (BDL) or sham-surgery. Animals were sacrificed after 3, 7, and 14 days and tissues were harvested for Western blot analysis, real-time reverse transcriptase-mediated polymerase chain reaction (RT-PCR), and immunohistochemistry., Results: Western blot analysis revealed a progressive, more than seven-fold increase (P < 0.05) of Mrp4 expression in cholestatic livers, 14 days after BDL. In contrast, Mrp4 in 14-day BDL kidneys decreased to 26+/-4% of controls (P < 0.005). Immunohistochemistry localized Mrp4 to the basolateral hepatocyte membrane and corroborated its hepatic up-regulation after BDL. Real-time RT-PCR demonstrated no major changes of Mrp4 mRNA levels in liver and kidney after BDL. Cyclic adenosine monophosphate, an MRP4 substrate, was increased in plasma and urine, consistent with these findings., Conclusions: Obstructive cholestasis in rats results in progressive up-regulation of Mrp4 protein in liver but down-regulation in kidney. The absence of corresponding changes in Mrp4 mRNA suggests posttranscriptional mechanisms as predominant regulators of Mrp4 expression in BDL rats.

Taurolithocholic acid (TLCA) is a potent cholestatic agent. Our recent work suggested that TLCA impairs hepatobiliary exocytosis, insertion of transport proteins into apical hepatocyte membranes, and bile flow by protein kinase Cepsilon (PKCepsilon)-dependent mechanisms. Products of phosphatidylinositol 3-kinases (PI3K) stimulate PKCepsilon. We studied the role of PI3K for TLCA-induced cholestasis in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets (IRHC). In IPRL, TLCA (10 micromol/liter) impaired bile flow by 51%, biliary secretion of horseradish peroxidase, a marker of vesicular exocytosis, by 46%, and the Mrp2 substrate, 2,4-dinitrophenyl-S-glutathione, by 95% and stimulated PI3K-dependent protein kinase B, a marker of PI3K activity, by 154% and PKCepsilon membrane binding by 23%. In IRHC, TLCA (2.5 micromol/liter) impaired canalicular secretion of the fluorescent bile acid, cholylglycylamido fluorescein, by 50%. The selective PI3K inhibitor, wortmannin (100 nmol/liter), and the anticholestatic bile acid tauroursodeoxycholic acid (TUDCA, 25 micromol/liter) independently and additively reversed the effects of TLCA on bile flow, exocytosis, organic anion secretion, PI3K-dependent protein kinase B activity, and PKCepsilon membrane binding in IPRL. Wortmannin also reversed impaired bile acid secretion in IRHC. These data strongly suggest that TLCA exerts cholestatic effects by PI3K- and PKCepsilon-dependent mechanisms that are reversed by tauroursodeoxycholic acid in a PI3K-independent way.

Multidrug resistance protein Mrp2 (symbol Abcc2) in liver plays a significant role in the biliary excretion of organic anionic conjugates. Mutations in human MRP2 result in defects in excretion of conjugated bilirubin and other cholephiles known as the Dubin-Johnson syndrome. Previous studies indicate that transporters with Mrp2-like functions are present in ancient vertebrates. We have now characterized an Mrp2 ortholog at the molecular level from the liver of the small skate, Raja erinacea, a marine vertebrate that evolved approximately 200 million years ago. The full-length skate Mrp2 cDNA is 6 kb and encodes for a 1,564-amino acid peptide with 56% identity to human Mrp2. Northern blot analysis demonstrated that skate Mrp2 is abundantly expressed in skate liver, intestine, and kidney. Immunoblots reveal a 180-kDa protein in skate liver. Immunofluorescence studies locate skate Mrp2 to the apical membrane of hepatocytes, renal tubules, and intestine. A PDZ-interacting motif is also found at its COOH terminus. Further sequence analysis indicates that transmembrane domains 1, 9, 11, 16, and 17 are the most highly conserved transmembrane domains between skate Mrp2 and mammalian MRP2/Mrp2s. This analysis indicates that Mrp2 orthologs evolved early in vertebrate evolution and that conserved domains may be important determinants of Mrp2 substrate specificity.

PFIC II is a subtype of progressive familial intrahepatic cholestasis (PFIC) that is associated with mutations in the ABCB11 gene encoding the bile salt export pump (BSEP). However it is not known how these mutations cause this disease. To evaluate these mechanisms, we introduced seven PFIC II-associated missense mutations into rat Bsep and assessed their effects on Bsep membrane localization and transport function in MDCK and Sf9 cells, respectively. Five mutations, G238V, E297G, G982R, R1153C, and R1268Q, prevented the protein from trafficking to the apical membrane, and E297G, G982R, R1153C, and R1268Q also abolished taurocholate transport activity, possibly by causing Bsep to misfold. Mutation C336S affected neither Bsep transport activity nor the apical trafficking of Bsep, suggesting that this mutation alone may not cause this disease. D482G did not affect the apical expression but partially decreased the transport activity of Bsep. Mutant G238V was rapidly degraded in both MDCK and Sf9 cells, and proteasome inhibitor resulted in intracellular accumulation of this and other mutants, suggesting proteasome-mediated degradation plays an important role in expression of these PFIC II mutants. Our studies highlight the heterogeneous nature of PFIC II mutations and illustrate the significance of these mutations in the function and expression of Bsep.

Ursodeoxycholic acid (UDCA) can protect hepatocytes from apoptosis induced by a variety of stimuli including anti-Fas antibody. However, in vivo the Fas receptor is activated by its natural ligand, Fas-L, whereas anti-Fas antibodies are not a physiologic stimulus. We therefore have assessed the anti-apoptotic effects of UDCA and other bile acids in a novel coculture model where apoptosis is induced in murine hepatocytes by membrane-bound Fas-L expressing fibroblasts. Primary hepatocytes were cultured overnight on collagen-coated coverslips with increasing concentrations of different bile acids and overlaid with either NIH 3T3 Fas-L(+) or Fas-L(-) expressing fibroblasts. After 6 hours cells were fixed and apoptosis was evaluated by TUNEL assay and DAPI staining using digital imaging. Fas-L protein expression and Fas trimerization were evaluated by Western blot analysis. FITC-UDCA and Mitotracker Red were used to evaluate UDCA localization with mitochondria. UDCA (up to 100 micromol/L, P <.0001), TUDCA (up to 400 micromol/L, P <.0001), and TCDCA (up to 200 micromol/L, P <.0001), but not TCA (up to 500 micromol/L), significantly protected hepatocytes in Fas-L(+) cocultures. UDCA had no significant effect on hepatocytes in Fas-L(-) cocultures. TUDCA, 50 micromol/L (P <.001) and TCDCA up to 200 micromol/L (P <.0001) also reduced the hepatocytes apoptotic rate in Fas-L(-) cocultures. Bile acids did not affect Fas-L expression in fibroblasts or Fas trimerization. FITC-UDCA colocalized with the mitochondrial probe. In conclusion, UDCA, TUDCA, and TCDCA but not TCA are capable of protecting hepatocytes from Fas-L-induced apoptosis. This protective effect is not associated with reductions in Fas trimerization, but may be related to a direct effect on the mitochondrial membrane.