Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes by PKCα-associated signalling under cholesterol stimulation.

BACKGROUND AND AIMS: The secretion of bile salts transported by the bile salt export pump (BSEP) is the primary driving force for the generation of bile flow; thus, it is closely related to the formation of cholesterol stones. Caveolin-1 (Cav-1), an essential player in cell signalling and endocytosis, is known to co-localize with cholesterol-rich membrane domains. This study illustrates the role of Cav-1 and BSEP in cholesterol stone formation. METHODS: Adult male C57BL/6 mice were used as an animal model. HepG2 cells were cultured under different cholesterol concentrations and BSEP, Cav-1, p-PKCα and Hax-1 expression levels were determined via Western blotting. Expression levels of BSEP and Cav-1 mRNA were detected using real-time PCR. Immunofluorescence and immunoprecipitation assays were performed to study BSEP and Hax-1 distribution. Finally, an ATPase activity assay was performed to detect BSEP transport activity under different cholesterol concentrations in cells. RESULTS: Under low-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels significantly increased, PKCα phosphorylation significantly decreased, BSEP binding capacity to Hax-1 weakened, and BSEP function increased. Under high-concentration stimulation with cholesterol, Cav-1 and BSEP protein and mRNA expression levels decreased, PKCα phosphorylation increased, BSEP binding capacity to Hax-1 rose, and BSEP function decreased. CONCLUSION: Cav-1 regulates the bile salt export pump on the canalicular membrane of hepatocytes via PKCα-associated signalling under cholesterol stimulation.

Oleanolic acid induces hepatic injury by disrupting hepatocyte tight junction and dysregulation of farnesoid X receptor-mediated bile acid efflux transporters.

Oleanolic acid (OA) is a naturally occurring pentacyclic triterpene compound that has been reported to cause cholestatic liver injury. However, the regulation and pathogenic role of bile acids in OA-induced development of cholestatic liver injury remains largely unclear. Farnesoid X receptor (FXR) is a metabolic nuclear receptor that plays an important role in bile acid homeostasis in the liver by regulating efflux transporters bile salt export pump (BSEP) and multidrug resistance-associated protein 2 (MRP2). The aim of this study was to investigate the effect of OA on hepatocyte tight junction function and determine the role of FXR, BSEP, and MRP2 in the mechanism of impairment of transport of bile acids induced by OA. Both in vivo and in vitro models were used to characterize the OA-induced liver injury. The liquid chromatography-tandem mass spectrometry (LC-MS) was employed to characterize the efflux function of the transporters, and the results showed that OA caused a blockage of bile acids efflux. OA treatment resulted in decreased expression levels of the tight junction proteins zonula occludens-1 and occludin. Immunofluorescence results showed that OA treatment significantly reduced the number of bile ducts and the immunofluorescence intensity. Pretreatment with agonists of FXR and MRP2, respectively, in animal experiments attenuated OA-induced liver injury, while pretreatment with inhibitors of BSEP and MRP2 further aggravated OA-induced liver injury. These results suggest that OA inhibits FXR-mediated BSEP and MRP2, leading to impaired bile acid efflux and disruption of tight junctions between liver cells, resulting in liver damage.

Molecular and Clinical Links between Drug-Induced Cholestasis and Familial Intrahepatic Cholestasis.

Idiosyncratic Drug-Induced Liver Injury (iDILI) represents an actual health challenge, accounting for more than 40% of hepatitis cases in adults over 50 years and more than 50% of acute fulminant hepatic failure cases. In addition, approximately 30% of iDILI are cholestatic (drug-induced cholestasis (DIC)). The liver's metabolism and clearance of lipophilic drugs depend on their emission into the bile. Therefore, many medications cause cholestasis through their interaction with hepatic transporters. The main canalicular efflux transport proteins include: 1. the bile salt export pump (BSEP) protein (ABCB11); 2. the multidrug resistance protein-2 (MRP2, ABCC2) regulating the bile salts' independent flow by excretion of glutathione; 3. the multidrug resistance-1 protein (MDR1, ABCB1) that transports organic cations; 4. the multidrug resistance-3 protein (MDR3, ABCB4). Two of the most known proteins involved in bile acids' (BAs) metabolism and transport are BSEP and MDR3. BSEP inhibition by drugs leads to reduced BAs' secretion and their retention within hepatocytes, exiting in cholestasis, while mutations in the ABCB4 gene expose the biliary epithelium to the injurious detergent actions of BAs, thus increasing susceptibility to DIC. Herein, we review the leading molecular pathways behind the DIC, the links with the other clinical forms of familial intrahepatic cholestasis, and, finally, the main cholestasis-inducing drugs.

Disordered farnesoid X receptor signaling is associated with liver carcinogenesis in Abcb11-deficient mice.

ABCB11 encodes the bile salt export pump (BSEP), a key regulator in maintaining bile acid (BA) homeostasis. Although inherited ABCB11 mutations have previously been linked to primary liver cancer, whether ABCB11 deficiency leads to liver cancer remains unknown. Here, we analyzed ABCB11 mRNA expression levels in liver tumor specimens [29 with hepatocellular carcinoma (HCC), one with intrahepatic cholangiocarcinoma (ICC), and one with mixed HCC/ICC] with adjacent normal specimens and published human datasets. Liver tissues obtained from Abcb11-deficient (Abcb11-/- ) mice and wild-type mice at different ages were compared by histologic, RNA-sequencing, and BA analyses. ABCB11 was significantly downregulated in human liver tumors compared with normal controls. Abcb11-/- mice demonstrated progressive intrahepatic cholestasis and liver fibrosis, and spontaneously developed HCC and ICC over 12 months of age. Abcb11 deficiency increased BAs in the liver and serum in mice, most of which are farnesoid X receptor (FXR) antagonists/non-agonists. Accordingly, the hepatic expression and transcriptional activity of FXR were downregulated in Abcb11-/- mouse livers. Administration of the FXR agonist obeticholic acid reduced liver injury and tumor incidence in Abcb11-/- mice. In conclusion, ABCB11 is aberrantly downregulated and plays a vital role in liver carcinogenesis. The cholestatic liver injury and liver tumors developed in Abcb11-/- mice are associated with increased FXR antagonist BAs and thereby decreased activation of FXR. FXR activation might be a therapeutic strategy in ABCB11 deficiency diseases. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Farnesoid X receptor contributes to oleanolic acid-induced cholestatic liver injury in mice.

Farnesoid X receptor (FXR) is a nuclear receptor involved in the metabolism of bile acid. However, the molecular signaling of FXR in bile acid homeostasis in cholestatic drug-induced liver injury remains unclear. Oleanolic acid (OA), a natural triterpenoid, has been reported to produce evident cholestatic liver injury in mice after a long-term use. The present study aimed to investigate the role of FXR in OA-induced cholestatic liver injury in mice using C57BL/6J (WT) mice and FXR knockout (FXR-/- ) mice. The results showed that a significant alleviation in OA-induced cholestatic liver injury was observed in FXR-/- mice as evidenced by decreases in serum alanine aminotransferase, aspartate aminotransferase, and alkaline phosphatase as well as reduced hepatocyte necrosis. UPLC-MS analysis of bile acids revealed that the contents of bile acids decreased significantly in liver and serum, while increased in the bile in FXR-/- mice compared with in WT mice. In addition, the mRNA expressions of hepatic transporter Bsep, bile acid synthesis enzymes Bacs and Baat, and bile acids detoxifying enzymes Cyp3a11, Cyp2b10, Ephx1, Ugt1a1, and Ugt2b5 were increased in liver tissues of FXR-/- mice treated with OA. Furthermore, the expression of membrane protein BSEP was significantly higher in livers of FXR-/- mice compared with WT mice treated with OA. These results demonstrate that knockout of FXR may alleviate OA-induced cholestatic liver injury in mice by decreasing accumulation of bile acids both in the liver and serum, increasing the export of bile acids via the bile, and by upregulation of bile acids detoxification enzymes.

Metabolic profiling of emodin drug-induced liver injury and silybin treatment in rats using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry: A metabolomic and mechanistic approach.

Silybin, an active component in the plant Silybum marianum (L.) Gaertn., is commonly used to protect against liver disease. We investigated silybin's protective potential in rat liver against emodin-induced liver injury 4 weeks. It was found that aspartate aminotransferase and direct bilirubin serum biomarkers for liver toxicity significantly increased, and liver histopathology revealed cholestasis and necrosis in rats administered emodin alone, whereas aspartate aminotransferase and total bile acid levels in rats administered emodin and silybin simultaneously were changed compared to rats administered emodin alone. Liver mRNA and protein levels of Cyp7a1-which plays roles in cholesterol metabolism and bile acid synthesis-and Abcb11 (Bsep)-which facilitates bile salt secretion in hepatocyte canaliculi-were significantly altered with emodin, whereas cotreatment with silybin attenuated emodin's adverse effect. Metabolomic analysis using ultra-performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry determined eight potential metabolite biomarkers in serum, urine, and liver tissue. Network analysis was conducted to conceptualize the interplay of genes, metabolites, and metabolic pathways for cholesterol metabolism and bile acid synthesis for liver injury. Overall, rats administered only emodin were shown to be a sound model to investigate fat-associated drug-induced hepatoxicity or liver injury and cotreatment of emodin with silybin prevents fatty liver injury. This metabolomic study revealed that emodin-induced fatty liver injury disrupted bile acid synthesis, vitamin B6 , and glycerophospholipid metabolism pathways and that silybin ameliorates liver injury on these compromised pathways.

In Vitro Rescue of the Bile Acid Transport Function of ABCB11 Variants by CFTR Potentiators.

ABCB11 is responsible for biliary bile acid secretion at the canalicular membrane of hepatocytes. Variations in the ABCB11 gene cause a spectrum of rare liver diseases. The most severe form is progressive familial intrahepatic cholestasis type 2 (PFIC2). Current medical treatments have limited efficacy. Here, we report the in vitro study of Abcb11 missense variants identified in PFIC2 patients and their functional rescue using cystic fibrosis transmembrane conductance regulator potentiators. Three ABCB11 disease-causing variations identified in PFIC2 patients (i.e., A257V, T463I and G562D) were reproduced in a plasmid encoding an Abcb11-green fluorescent protein. After transfection, the expression and localization of the variants were studied in HepG2 cells. Taurocholate transport activity and the effect of potentiators were studied in Madin-Darby canine kidney (MDCK) clones coexpressing Abcb11 and the sodium taurocholate cotransporting polypeptide (Ntcp/Slc10A1). As predicted using three-dimensional structure analysis, the three variants were expressed at the canalicular membrane but showed a defective function. Ivacaftor, GLP1837, SBC040 and SBC219 potentiators increased the bile acid transport of A257V and T463I and to a lesser extent, of G562D Abcb11 missense variants. In addition, a synergic effect was observed when ivacaftor was combined with SBC040 or SBC219. Such potentiators could represent new pharmacological approaches for improving the condition of patients with ABCB11 deficiency due to missense variations affecting the function of the transporter.

Heterozygous mutations of ATP8B1, ABCB11 and ABCB4 cause mild forms of Progressive Familial Intrahepatic Cholestasis in a pediatric cohort.

INTRODUCTION: Heterozygous defects in genes implicated in Progressive Familial Intrahepatic Cholestasis have been described in milder forms of cholestatic diseases. Our aim is to describe clinical, laboratory and imaging characteristics as well as treatment and outcome of a cohort of pediatric patients with heterozygous mutations in ATP8B1, ABCB11 or ABCB4. PATIENTS AND METHODS: We present a retrospective descriptive study including pediatric patients with at least one heterozygosis defect in ATP8B1, ABCB11 or ABCB4 diagnosed after a cholestatic episode. Clinical, diagnostic and outcome data were collected including gene analysis (panel of PFIC NextGeneDx®). RESULTS: 7 patients showed a heterozygous mutation: 3 patients in ABCB4, 1 in ABCB11, 2 in ABCB4 and ABCB11 and 1 in ATP8B1. The median onset age was 5.5 years with a median time of follow-up of 6 years. The initial presentation was pruritus followed by asymptomatic hypertransaminasemia and persistent cholestasis. Two patients had family history of gallbladder stones and mild hepatitis. All showed elevated transaminases and bile acids, high gamma glutamyl-transferase (GGT) in 3 and conjugated bilirubin in 2 patients. Liver biopsy showed inflammatory infiltrate or mild fibrosis with normal immunohistochemistry. All patients were treated with ursodeoxycholic acid, two patients requiring the addition of resincholestyramine. During follow-up, 3 patients suffered limited relapses of pruritus. No disease progression was observed. CONCLUSION: Heterozygous mutations in genes coding proteins of the hepatocellular transport system can cause cholestatic diseases with great phenotypic variability. The presence of repeated episodes of hypertransaminasemia or cholestasis after a trigger should force us to rule out the presence of these heterozygous mutations in genes involved in CIFP.

Bile Acid Toxicity and Protein Kinases.

If the bile acids reach to pathological concentrations due to cholestasis, accumulation of hydrophobic bile acids within the hepatocyte may result in cell death. Thus, hydrophobic bile acids induce apoptosis in hepatocytes, while hydrophilic bile acids increase intracellular adenosine 3',5'-monophosphate (cAMP) levels and activate mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K) pathways to protect hepatocytes from apoptosis.Two apoptotic pathways have been described in bile acids-induced death. Both are controlled by multiple protein kinase signaling pathways. In mitochondria-controlled pathway, caspase-8 is activated with death domain-independent manner, whereas, Fas-dependent classical pathway involves ligand-independent oligomerization of Fas.Hydrophobic bile acids dose-dependently upregulate the inflammatory response by further stimulating production of inflammatory cytokines. Death receptor-mediated apoptosis is regulated at the cell surface by the receptor expression, at the death-inducing signaling complex (DISC) by expression of procaspase-8, the death receptors Fas-associated death domain (FADD), and cellular FADD-like interleukin 1-beta (IL-1ß)-converting enzyme (FLICE) inhibitory protein (cFLIP). Bile acids prevent cFLIP recruitment to the DISC and thereby enhance initiator caspase activation and lead to cholestatic apoptosis. At mitochondria, the expression of B-cell leukemia/lymphoma-2 (Bcl-2) family proteins contribute to apoptosis by regulating mitochondrial cytochrome c release via Bcl-2, Bcl-2 homology 3 (BH3) interacting domain death agonist (Bid), or Bcl-2 associated protein x (Bax). Fas receptor CD95 activation by hydrophobic bile acids is initiated by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent reactive oxygen species (ROS) signaling. However, activation of necroptosis by ligands of death receptors requires the kinase activity of receptor interacting protein1 (RIP1), which mediates the activation of RIP3 and mixed lineage kinase domain-like protein (MLKL). In this chapter, mainly the effect of protein kinases signal transduction on the mechanisms of hydrophobic bile acids-induced inflammation, apoptosis, necroptosis and necrosis are discussed.

The Bile Salt Export Pump: Molecular Structure, Study Models and Small-Molecule Drugs for the Treatment of Inherited BSEP Deficiencies.

The bile salt export pump (BSEP/ABCB11) is responsible for the transport of bile salts from hepatocytes into bile canaliculi. Malfunction of this transporter results in progressive familial intrahepatic cholestasis type 2 (PFIC2), benign recurrent intrahepatic cholestasis type 2 (BRIC2) and intrahepatic cholestasis of pregnancy (ICP). Over the past few years, several small molecular weight compounds have been identified, which hold the potential to treat these genetic diseases (chaperones and potentiators). As the treatment response is mutation-specific, genetic analysis of the patients and their families is required. Furthermore, some of the mutations are refractory to therapy, with the only remaining treatment option being liver transplantation. In this review, we will focus on the molecular structure of ABCB11, reported mutations involved in cholestasis and current treatment options for inherited BSEP deficiencies.

Genotype correlates with the natural history of severe bile salt export pump deficiency.

BACKGROUND & AIMS: Mutations in ABCB11 can cause deficiency of the bile salt export pump (BSEP), leading to cholestasis and end-stage liver disease. Owing to the rarity of the disease, the associations between genotype and natural history, or outcomes following surgical biliary diversion (SBD), remain elusive. We aimed to determine these associations by assembling the largest genetically defined cohort of patients with severe BSEP deficiency to date. METHODS: This multicentre, retrospective cohort study included 264 patients with homozygous or compound heterozygous pathological ABCB11 mutations. Patients were categorized according to genotypic severity (BSEP1, BSEP2, BSEP3). The predicted residual BSEP transport function decreased with each category. RESULTS: Genotype severity was strongly associated with native liver survival (NLS, BSEP1 median 20.4 years; BSEP2, 7.0 years; BSEP3, 3.5 years; p <0.001). At 15 years of age, the proportion of patients with hepatocellular carcinoma was 4% in BSEP1, 7% in BSEP2 and 34% in BSEP3 (p = 0.001). SBD was associated with significantly increased NLS (hazard ratio 0.50; 95% CI 0.27-0.94: p = 0.03) in BSEP1 and BSEP2. A serum bile acid concentration below 102 µmol/L or a decrease of at least 75%, each shortly after SBD, reliably predicted NLS of ≥15 years following SBD (each p <0.001). CONCLUSIONS: The genotype of severe BSEP deficiency strongly predicts long-term NLS, the risk of developing hepatocellular carcinoma, and the chance that SBD will increase NLS. Serum bile acid parameters shortly after SBD can predict long-term NLS. LAY SUMMARY: This study presents data from the largest genetically defined cohort of patients with severe bile salt export pump deficiency to date. The genotype of patients with severe bile salt export pump deficiency is associated with clinical outcomes and the success of therapeutic interventions. Therefore, genotypic data should be used to guide personalized clinical care throughout childhood and adulthood in patients with this disease.

Tanshinone IIA prevents rifampicin-induced liver injury by regulating BSEP/NTCP expression via epigenetic activation of NRF2.

BACKGROUND & AIMS: Rifampicin (RFP)-induced cholestatic liver injury is characterized by impaired hepatic bile acid (BA) transport. Bile salt efflux pump (BSEP) and Na+/taurocholate cotransporter (NTCP) are the major BA transporters. However, little is known about the mechanisms underlying these transporters. METHODS: The role of tanshinone IIA (TAN IIA) in preventing RFP-induced liver injury was evaluated in vitro and in vivo, based on the regulatory mechanism of nuclear factor erythroid 2-related factor 2 (NRF2)-BSEP/NTCP signalling. The epigenetic induction of NRF2 by TAN IIA was investigated as well as the influence on BSEP and NTCP transcriptional activation and NRF2 DNA-binding ability. RESULTS: TAN IIA strongly induced BSEP and NTCP expression in hepatocytes. NRF2 knockdown abrogated the induction. We found two NRF2 binding sites on the human BSEP promoter, called musculoaponeurotic fibrosarcoma recognition elements (MAREs), and one MARE on the NTCP promoter. Human BSEP and NTCP promoter luciferase reporter gene plasmids were stimulated by NRF2. Mutations of the predicted MAREs abolished NRF2 transcriptional activation. TAN IIA induced the expression of ten-eleven translocation 2 (TET2) to mediate the demethylation of NRF2, which promoted NRF2 DNA-binding on the BSEP and NTCP promoters and their transcriptional activation. Finally, in vivo, Nrf2 played an important role in RFP-induced liver injury (more serious liver injury in Nrf2-/- mice), and TAN IIA prevented it. CONCLUSIONS: These results indicate that NRF2 regulates the target transporters BSEP and NTCP, depending on the DNA demethylation by TET2. Pharmacological activation of NRF2 by TAN IIA may be beneficial for RFP-induced liver injury.

Metformin Disrupts Bile Acid Efflux by Repressing Bile Salt Export Pump Expression.

PURPOSE: The bile salt export pump (BSEP), a key player in hepatic bile acid clearance, has been the center of research on drug-induced cholestasis. However, such studies focus primarily on the direct inhibition of BSEP, often overlooking the potential impact of transcriptional repression. This work aims to explore the disruption of bile acid efflux caused by drug-induced BSEP repression. METHODS: BSEP activity was analyzed in human primary hepatocytes (HPH) using a traditional biliary-clearance experiment and a modified efflux assay, which includes a 72-h pretreatment prior to efflux measurement. Relative mRNA and protein expressions were examined by RT-PCR and Western blotting, respectively. RESULTS: Metformin concentration-dependently repressed BSEP expression in HPH. Although metformin did not directly inhibit BSEP activity, longer metformin exposure reduced BSEP transport function in HPH by down-regulating BSEP expression. BSEP repression by metformin was found to be AMP-activated protein kinase-independent. Additional screening of 10 reported cholestatic non-BSEP inhibitors revealed that the anti-cancer drug tamoxifen also markedly repressed BSEP expression and reduced BSEP activity in HPH. CONCLUSIONS: Repression of BSEP alone is sufficient to disrupt hepatic bile acid efflux. Metformin and tamoxifen appear to be prototypes of a class of BSEP repressors that may cause drug-induced cholestasis through gene repression instead of direct BSEP inhibition.

Proposal of a liver histology-based scoring system for bile salt export pump deficiency.

AIM: Bile salt export pump (BSEP) deficiency manifests a form of progressive intrahepatic cholestasis. This study aimed to establish a scoring system of liver histology for the uncommon genetic condition. METHODS: After a roundtable discussion and histology review, a scoring system for BSEP deficiency was established. Eleven tissue samples were independently evaluated by three pathologists based on the proposed standard for an interobserver agreement analysis. In four cases with serial tissue samples available, correlation between changes in histology scores and clinical outcome was examined. RESULTS: Of 14 initially listed histopathological findings, 12 were selected for scoring and grouped into the following four categories: cholestasis, parenchymal changes, portal tract changes and fibrosis. Each category consisted of two to four microscopic findings that were further divided into three to six scores; therefore, each category had a maximum score of 8-11. Interobserver agreement was highest for pericellular fibrosis (κ = 0.849) and lowest for hepatocellular cholestasis (κ = 0.241) with the mean and median κ values of the 12 parameters being 0.561 and 0.602, respectively. For two patients whose clinical features worsened, score changes between two time points were interpreted as deteriorated. In two patients, who showed a good clinical response to preprandial treatment with sodium 4-phenylbutyrate, histological changes were evaluated as improved or unchanged. CONCLUSIONS: The proposed histology-based scoring system for BSEP deficiency with moderate interobserver agreement may be useful not only for monitoring microscopic changes in clinical practice but also for a surrogate endpoint in clinical trials.

In vitro assessment of farnesoid X receptor antagonism to predict drug-induced liver injury risk.

Drug-induced liver injury (DILI) continues to be a major cause of drug attrition and restrictive labeling. Given the importance of farnesoid X receptor (FXR) in bile acid homeostasis, drug-related FXR antagonism may be an important mechanism of DILI. However, a comprehensive assessment of this phenomenon broadly in the context of DILI is lacking. As such, we used an orthogonal approach comprising a FXR target gene assay in primary human hepatocytes and a commercially available FXR reporter assay to investigate the potential FXR antagonistic effects of an extensive test set of 159 compounds with and without association with clinical DILI. Data were omitted from analysis based on the presence of cytotoxicity to minimize false positive assay signals and other complications in data interpretation. Based on the experimental approaches employed and corresponding data, the prevalence of FXR antagonism was relatively low across this broad DILI test set, with 16-24% prevalence based on individual assay results or combined signals in both assays. Moreover, FXR antagonism was not highly predictive for identifying clinically relevant hepatotoxicants retrospectively, where FXR antagonist classification alone had minimal to moderate predictive value as represented by positive and negative likelihood ratios of 2.24-3.84 and 0.72-0.85, respectively. The predictivity did not increase significantly when considering only compounds with high clinical exposure (maximal or efficacious plasma exposures > 1.0 µM). In contrast, modest gains in predictive value of FXR antagonism were observed considering compounds that also inhibit bile salt export pump. In addition, we have identified novel FXR antagonistic effects of well-studied hepatotoxic drugs, including bosentan, tolcapone and ritonavir. In conclusion, this work represents a comprehensive evaluation of FXR antagonism in the context of DILI, including its overall predictivity and challenges associated with detecting this phenomenon in vitro.

Histological demonstration of BSEP/ABCB11 inhibition in transient neonatal cholestasis: a case report.

BACKGROUND: Idiopathic or transient neonatal cholestasis (TNC) represents a group of cholestatic disorders with unidentified origin and remains a diagnosis of exclusion. Dysfunction of hepatobiliary transporters mediating excretion of biliary constituents from hepatocytes may play a central role in the pathogenesis of cholestasis. Despite variants of bile salt (BS) export pump (BSEP/ABCB11) have already been described in TNC, the pathogenic role of BSEP dysfunction in TNC remained so far elusive. CASE PRESENTATION: We report on a newly-identified heterozygous ABCB11 missense variant (c.1345G > A, p.Glu449Lys) which was associated with prolonged cholestasis in a term infant after a complicated neonatal period. Moreover, we show for the first time almost completely abolished BSEP expression on the hepatocellular membrane in TNC. CONCLUSION: This report demonstrates for the first time a close association between the prolonged cholestasis in infancy and impaired BSEP expression on the hepatocyte canalicular membrane in a heterozygous carrier of newly-identified ABCB11 variant.

Effects of Hepatic Ischemia-Reperfusion Injuries and NRF2 on Transcriptional Activities of Bile Transporters in Rats.

BACKGROUND: The effect of hepatic ischemia-reperfusion injury (IRI) on bile transporter (BT) gene expression is unknown. We hypothesized that abnormal expression of BTs during hepatic IRI is dependent on nuclear factor erythroid 2-related factor 2 (NRF2), which contributes to the cholestasis after reperfusion. METHODS: Sham surgery and short (60 min) or long (90 min) periods of warm ischemia time (WIT) with or without reperfusion for 24 h were applied to wild-type Sprague-Dawley rats and Nrf2 knockout rats (n = 5 per group). At each stage of IRI, the serum levels of aminotransferase, total bilirubin, and bile acids were measured. In addition, hepatic tissue was sampled to determine the histologic score of IRI (Suzuki score), measure adenosine triphosphate (ATP), and identify the quantitative real-time polymerase chain reactions of BTs (Oatp1, Ntcp, Mrp2, Bsep, and Mrp3). RESULTS: In short periods of WIT, BT expression increased during the ischemia stage and returned to the baseline after reperfusion. However, in long periods of WIT, BT expression did not increase after ischemia and decreased further after reperfusion. Short WIT did not increase BT expression in Nrf2 knockout animals. The level of BT expression was correlated with the Suzuki score, the serum levels of aminotransferase, bilirubin, and bile acids, and tissue ATP level. Stepwise multiple regression analysis derived equations to predict the Suzuki score (R2 = 76.8, P < 0.001), serum total bilirubin (R2 = 61.2, P < 0.001), and tissue ATP (R2 = 61.1, P < 0.001). CONCLUSIONS: Short WIT induces the transcriptional activities of BT, whereas long WIT depresses them, and the effect was blunted by Nrf2 knockout status. BT expression can be considered a surrogate marker for hepatic IRI.

Analyzing the Mechanisms Behind Macrolide Antibiotic-Induced Liver Injury Using Quantitative Systems Toxicology Modeling.

PURPOSE: Macrolide antibiotics are commonly prescribed treatments for drug-resistant bacterial infections; however, many macrolides have been shown to cause liver enzyme elevations and one macrolide, telithromycin, has been pulled from the market by its provider due to liver toxicity. This work seeks to assess the mechanisms responsible for the toxicity of macrolide antibiotics. METHODS: Five macrolides were assessed in in vitro systems designed to test for bile acid transporter inhibition, mitochondrial dysfunction, and oxidative stress. The macrolides were then represented in DILIsym, a quantitative systems pharmacology (QST) model of drug-induced liver injury, placing the in vitro results in context with each compound's predicted liver exposure and known biochemistry. RESULTS: DILIsym results suggest that solithromycin and clarithromycin toxicity is primarily due to inhibition of the mitochondrial electron transport chain (ETC) while erythromycin toxicity is primarily due to bile acid transporter inhibition. Telithromycin and azithromycin toxicity was not predicted by DILIsym and may be caused by mechanisms not currently incorporated into DILIsym or by unknown metabolite effects. CONCLUSIONS: The mechanisms responsible for toxicity can be significantly different within a class of drugs, despite the structural similarity among the drugs. QST modeling can provide valuable insight into the nature of these mechanistic differences.

Biliary transporter gene mutations in severe intrahepatic cholestasis of pregnancy: Diagnostic and management implications.

BACKGROUND AND AIMS: Clinical syndromes associated with biallelic mutations of bile acid (BA) transporters usually present in childhood. Subtle mutations may underlie intrahepatic cholestasis of pregnancy (ICP) and oral contraceptive steroid (OCS) induced cholestasis. In five women with identified genetic mutations of such transporters, with eight observed pregnancies complicated by ICP, we examined relationships between transporter mutations, clinical phenotypes, and treatment outcomes. METHODS: Gene mutation analysis for BA transporter deficiencies was performed using Next Generation/Sanger sequencing, with analysis for gene deletions/duplications. RESULTS: Intrahepatic cholestasis of pregnancy was early-onset (9-32 weeks gestation) and severe (peak BA 74-370 µmol/L), with premature delivery (28+1 -370 weeks gestation) in 7/8 pregnancies, in utero passage of meconium in 4/8, but overall good perinatal outcomes, with no stillbirths. There was generally no response to ursodeoxycholic acid and variable responses to rifampicin and chelation therapies; naso-biliary drainage appeared effective in 2/2 episodes persisting post-partum in each of the two sisters. Episodic jaundice occurring spontaneously or provoked by non-specific infections, and OCS-induced cholestasis, had previously occurred in 3/5 women. Two cases showed biallelic heterozygosity for several ABCB11 mutations, one was homozygous for an ABCB4 mutation and a fourth case was heterozygous for another ABCB4 mutation. CONCLUSIONS: Early-onset or recurrent ICP, especially with previous spontaneous or OCS-induced episodes of cholestasis and/or familial cholestasis, may be attributable to transporter mutations, including biallelic mutations of one or more transporters. Response to standard therapies for ICP is often incomplete; BA sequestering therapy or naso-biliary drainage may be effective. Optimized management can produce good outcomes despite premature birth and evidence of fetal compromise.