Tauroursodeoxycholate prevents estradiol 17ß-d-glucuronide-induced cholestasis and endocytosis of canalicular transporters by switching off pro-cholestatic signaling pathways.

AIMS: Estradiol 17ß-d-glucuronide (E217G) induces cholestasis by triggering endocytosis and further intracellular retention of the canalicular transporters Bsep and Mrp2, in a cPKC- and PI3K-dependent manner, respectively. Pregnancy-induced cholestasis has been associated with E217G cholestatic effect, and is routinely treated with ursodeoxycholic acid (UDCA). Since protective mechanisms of UDCA in E217G-induced cholestasis are still unknown, we ascertained here whether its main metabolite, tauroursodeoxycholate (TUDC), can prevent endocytosis of canalicular transporters by counteracting cPKC and PI3K/Akt activation. MAIN METHODS: Activation of cPKC and PI3K/Akt was evaluated in isolated rat hepatocytes by immunoblotting (assessment of membrane-bound and phosphorylated forms, respectively). Bsep/Mrp2 function was quantified in isolated rat hepatocyte couplets (IRHCs) by assessing the apical accumulation of their fluorescent substrates, CLF and GS-MF, respectively. We also studied, in isolated, perfused rat livers (IPRLs), the status of Bsep and Mrp2 transport function, assessed by the biliary excretion of TC and DNP-SG, respectively, and Bsep/Mrp2 localization by immunofluorescence. KEY FINDINGS: E217G activated both cPKC- and PI3K/Akt-dependent signaling, and pretreatment with TUDC significantly attenuated these activations. In IRHCs, TUDC prevented the E217G-induced decrease in apical accumulation of CLF and GS-MF, and inhibitors of protein phosphatases failed to counteract this protection. In IPRLs, E217G induced an acute decrease in bile flow and in the biliary excretion of TC and DNP-SG, and this was prevented by TUDC. Immunofluorescence studies revealed that TUDC prevented E217G-induced Bsep/Mrp2 endocytosis. SIGNIFICANCE: TUDC restores function and localization of Bsep/Mrp2 impaired by E217G, by preventing both cPKC and PI3K/Akt activation in a protein-phosphatase-independent manner.

NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis.

The endogenous metabolite of estradiol, estradiol 17ß-D-glucuronide (E17G), is considered the main responsible of the intrahepatic cholestasis of pregnancy. E17G alters the activity of canalicular transporters through a signaling pathway-dependent cellular internalization, phenomenon that was attributed to oxidative stress in different cholestatic conditions. However, there are no reports involving oxidative stress in E17G-induced cholestasis, representing this the aim of our work. Using polarized hepatocyte cultures, we showed that antioxidant compounds prevented E17G-induced Mrp2 activity alteration, being this alteration equally prevented by the NADPH oxidase (NOX) inhibitor apocynin. The model antioxidant N-acetyl-cysteine prevented, in isolated and perfused rat livers, E17G-induced impairment of bile flow and Mrp2 activity, thus confirming the participation of reactive oxygen species (ROS) in this cholestasis. In primary cultured hepatocytes, pretreatment with specific inhibitors of ERK1/2 and p38MAPK impeded E17G-induced ROS production; contrarily, NOX inhibition did not affect ERK1/2 and p38MAPK phosphorylation. Both, knockdown of p47phox by siRNA and preincubation with apocynin in sandwich-cultured rat hepatocytes significantly prevented E17G-induced internalization of Mrp2, suggesting a crucial role for NOX in this phenomenon. Concluding, E17G-induced cholestasis is partially mediated by NOX-generated ROS through internalization of canalicular transporters like Mrp2, being ERK1/2 and p38MAPK necessary for NOX activation.

Factors associated with cholestasis in newborns with gastroschisis.

OBJECTIVE: To describe the incidence and to analyze risk factors associated with cholestasis in neonates with gastroschisis. METHODS: This is a retrospective cohort study in a tertiary single center analyzing 181 newborns with gastroschisis between 2009 and 2020. The following risk factors associated with cholestasis were analyzed: gestational age, birth weight, type of gastroschisis, silo closure or immediate closure, days of parenteral nutrition, type of lipid emulsion, days of fasting, days to reach a full diet, days with central venous catheter, presence of infections, and outcomes. RESULTS: Among the 176 patients evaluated, 41 (23.3%) evolved with cholestasis. In the univariate analysis, low birth weight (p=0.023), prematurity (p<0.001), lipid emulsion with medium-chain triglycerides and long-chain triglycerides (p=0.001) and death (p<0.001) were associated with cholestasis. In the multivariate analysis, patients who received lipid emulsion with fish oil instead of medium chain triglycerides/long chain triglycerides (MCT/LCT) emulsion had a lower risk of cholestasis. CONCLUSIONS: Our study shows that lipid emulsion with fish oil is associated with a lower risk of cholestasis in neonates with gastroschisis. However, this is a retrospective study and a prospective study should be performed to confirm the results.

Drug-Induced Vanishing Bile Duct Syndrome: From Pathogenesis to Diagnosis and Therapeutics.

The most concerned issue in the context of drug/herb-induced chronic cholestasis is vanishing bile duct syndrome. The progressive destruction of intrahepatic bile ducts leading to ductopenia is usually not dose dependent, and has a delayed onset that should be suspected when abnormal serum cholestasis enzyme levels persist despite drug withdrawal. Immune-mediated cholangiocyte injury, direct cholangiocyte damage by drugs or their metabolites once in bile, and sustained exposure to toxic bile salts when biliary epithelium protective defenses are impaired are the main mechanisms of cholangiolar damage. Current therapeutic alternatives are scarce and have not shown consistent beneficial effects so far. This review will summarize the current literature on the main diagnostic tools of ductopenia and its histological features, and the differential diagnostic with other ductopenic diseases. In addition, pathomechanisms will be addressed, as well as the connection between them and the supportive and curative strategies for ductopenia management.

Serious liver injury induced by Nimesulide: an international collaborative study.

Nimesulide is a non-steroidal anti-inflammatory drug still marketed in many countries. We aim to analyze the clinical phenotype, outcome, and histological features of nimesulide-induced liver injury (nimesulide-DILI). We analyzed 57 cases recruited from the Spanish and Latin American DILI registries. Causality was assessed by the RUCAM scale. Mean age of the whole case series was 59 years (86% women) with a median time to onset of 40 days. A total of 46 patients (81%) were jaundiced. Nimesulide-DILI pattern was hepatocellular in 38 (67%), mixed in 12 (21%), and cholestatic in 7 (12%) cases. Transaminases were elevated with a mean of nearly 20-fold the upper limit of normality (ULN), while alkaline phosphatase showed a twofold mean elevation above ULN. Total bilirubin showed a mean elevation of 13-fold the ULN. Liver histology was obtained in 14 cases (25%), most of them with a hepatocellular pattern. Median time to recovery was 60 days. Overall, 12 patients (21%) developed acute liver failure (ALF), five (8.8%) died, three underwent liver transplantation (5.3%), and the remaining four resolved. Latency was ≤ 15 days in 12 patients (21%) and one patient developed ALF within 7 days from treatment initiation. Increased total bilirubin and aspartate transaminase levels were independently associated with the development of ALF. In summary, nimesulide-DILI affects mainly women and presents typically with a hepatocellular pattern. It is associated with ALF and death in a high proportion of patients. Shorter (≤ 15 days) duration of therapy does not prevent serious nimesulide hepatotoxicity, making its risk/benefit ratio clearly unfavorable.

Anticholestatic mechanisms of ursodeoxycholic acid in lipopolysaccharide-induced cholestasis.

Lipopolysaccharide (LPS) from Gram (-) bacteria induces inflammatory cholestasis by impairing the expression/localization of transporters involved in bile formation (e.g., Bsep, Mrp2). Therapeutic options for this disease are lacking. Ursodeoxycholic acid (UDCA) is the first choice therapy in cholestasis, but its anticholestatic efficacy in this hepatopathy remains to be evaluated. To asses it, male Wistar rats received UDCA for 5 days (25 mg/Kg/day, i.p.) with or without LPS, administered at 8 a.m. of the last 2 days (4 mg/Kg/day, i.p.), plus half of this dose at 8 p.m. of the last day. Then, plasma alkaline phosphatase (ALP), bile flow, basal and taurocholate-stimulated bile acid output, total glutathione output, and total/plasma membrane liver protein expression of Bsep and Mrp2 by confocal microscopy were assessed. mRNA levels of both transporters were assessed by Real-Time PCR. Plasma pro-inflammatory cytokines (IL-6 and TNF-α) were measured by ELISA. Our results showed that UDCA attenuated LPS-induced ALP plasma release and the impairment in the excretion of the Bsep substrate, taurocholate. This was associated with an improved Bsep expression at both mRNA and protein levels, and by an improved localization of Bsep in plasma membrane. UDCA failed to reduce the increase in plasma pro-inflammatory cytokines induced by LPS and Mrp2 expression/function. In conclusion, UDCA protects the hepatocyte against the damaging effect of bile acids accumulated by the LPS-induced secretory failure. This involved an enhanced synthesis of Bsep and an improved membrane stability of the newly synthesized transporters.

Heme oxygenase-1 induction by hemin prevents oxidative stress-induced acute cholestasis in the rat.

We previously demonstrated in in vitro and ex vivo models that physiological concentrations of unconjugated bilirubin (BR) prevent oxidative stress (OS)-induced hepatocanalicular dysfunction and cholestasis. Here, we aimed to ascertain, in the whole rat, whether a similar cholestatic OS injury can be counteracted by heme oxygenase-1 (HO-1) induction that consequently elevates endogenous BR levels. This was achieved through the administration of hemin, an inducer of HO-1, the rate-limiting step in BR generation. We found that BR peaked between 6 and 8 h after hemin administration. During this time period, HO-1 induction fully prevented the pro-oxidant tert-butylhydroperoxide (tBuOOH)-induced drop in bile flow, and in the biliary excretion of bile salts and glutathione, the two main driving forces of bile flow; this was associated with preservation of the membrane localization of their respective canalicular transporters, bile salt export pump (Bsep) and multidrug resistance-associated protein 2 (Mrp2), which are otherwise endocytosed by OS. HO-1 induction counteracted the oxidation of intracellular proteins and membrane lipids induced by tBuOOH, and fully prevented the increase in the oxidized-to-total glutathione (GSHt) ratio, a sensitive parameter of hepatocellular OS. Compensatory elevations of the activity of the antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) were also prevented. We conclude that in vivo HO-1 induction protects the liver from acute oxidative injury, thus preventing consequent cholestasis. This reveals an important role for the induction of HO-1 and the consequently elevated levels of BR in preserving biliary secretory function under OS conditions, thus representing a novel therapeutic tool to limit the cholestatic injury that bears an oxidative background.

Hepatotoxicidad por esteroides anabólicos androgénicos, reporte de 2 casos en Uruguay / Hepatotoxicity induced by anabolic-androgenic steroids, 2 cases report in Uruguay / Hepatotoxicidade por anabolizantes esteróides androgênicos, relato de 2 casos no Uruguai

El consumo ilícito de esteroides anabólicos androgénicos con fines estéticos ha aumentado en los últimos años y, aunque raro, es causa de hepatotoxicidad. Los casos con daño hepatocelular son más frecuentes, pero los colestásicos son más graves y pueden asociarse a falla renal. Salvo por la suspensión del fármaco, la hepatotoxicidad por anabólicos no tiene tratamiento específico. Se describe y discuten las historias clínicas de dos hombres jóvenes, deportistas aficionados que consultaron por ictericia y presentaron colestasis e insuficiencia renal. El reporte de casos, en patologías poco frecuentes, resulta fundamental para difundir y ampliar la información que ayude al clínico a considerar con firmeza este diagnóstico, incluso ante la falta de reconocimiento inicial del consumo por parte del paciente.

Illicit consumption of anabolic-androgenic steroids for aesthetic purposes has increased in recent years. Hepatocellular damage is more frequent, but cholestasis is more dangerous and may be associated with renal failure. The clinical records of two young men, amateur athletes who consulted for jaundice in the last year and denied its consumption at the beginning, are described. Except for the drug interruption, hepatotoxicity by anabolics has no specific treatment. Usually presented as cholestatic liver disease and renal failure, case reports are fundamental to characterize its clinical-evolutionary presentation. This may also allow clinicians to firmly consider diagnosis even when the patient denies consumption.

O uso ilícito de esteróides androgênicos anabólicos para fins estéticos tem aumentado nos últimos anos e, apesar de raro, é causa de hepatotoxicidade. Casos com dano hepatocelular são mais freqüentes, mas colestesia é mais grave e pode estar associada à insuficiência renal. Com exceção da suspensão do medicamento, a hepatotoxicidade anabólica não possui tratamento específico. As histórias clínicas de dois homens jovens, atletas amadores que consultaram para icterícia e apresentaram colestase e insuficiência renal, são descritos e discutidos. O relato de casos, em patologias pouco freqüentes, é fundamental para disseminar e ampliar as informações que auxiliam o clínico a considerar com firmeza esse diagnóstico antes mesmo do não reconhecimento inicial do consumo pelo paciente.

Mechanisms of canalicular transporter endocytosis in the cholestatic rat liver.

Impaired canalicular secretion due to increased endocytosis and intracellular retention of canalicular transporters such as BSEP and MRP2 is a main, common pathomechanism of cholestasis. Nevertheless, the mechanisms governing this process are unknown. We characterized this process in estradiol 17 ß-d-glucuronide (E17G)-induced cholestasis, an experimental model which partially mimics pregnancy-induced cholestasis. Inhibitors of clathrin-mediated endocytosis (CME) such as monodansylcadaverine (MDC) or K+ depletion, but not the caveolin-mediated endocytosis inhibitors filipin and genistein, prevented E17G-induced endocytosis of BSEP and MRP2, and the associated impairment of activity of these transporters in isolated rat hepatocyte couplets (IRHC). Immunofluorescence and confocal microscopy studies showed that, in E17G-treated IRHC, there was a significant increase in the colocalization of MRP2 with clathrin, AP2, and Rab5, three essential members of the CME machinery. Knockdown of AP2 by siRNA in sandwich-cultured rat hepatocytes completely prevented E17G-induced endocytosis of BSEP and MRP2. MDC significantly prevented this endocytosis, and the impairment of bile flow and biliary secretion of BSEP and MRP2 substrates, in isolated and perfused livers. BSEP and MRP2, which were mostly present in raft (caveolin-enriched) microdomains in control rats, were largely found in non-raft (clathrin-enriched) microdomains in livers from E17G-treated animals, from where they can be readily recruited for CME. In conclusion, our findings show that CME is the mechanism responsible for the internalization of the canalicular transporters BSEP and MRP2 in E17G-induced cholestasis. The shift of these transporters from raft to non-raft microdomains could be a prerequisite for the transporters to be endocytosed under cholestatic conditions.

Activation of insulin-like growth factor 1 receptor participates downstream of GPR30 in estradiol-17ß-D-glucuronide-induced cholestasis in rats.

Estradiol-17ß-D-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets. In vitro, IGF-1R inhibition by tyrphostin AG1024 (TYR, 100 nM), or its knock-down with siRNA, strongly prevented E17G-induced impairment of Abcc2 and Abcb11 function and localization. The protection by TYR was not additive to that produced by wortmannin (PI3K inhibitor, 100 nM), and both protections share the same dependency on microtubule integrity, suggesting that IGF-1R shared the signaling pathway of PI3K/Akt. Further analysis of the activation of Akt and IGF-1R induced by E17G indicated a sequence of activation GPR30-IGF-1R-PI3K/Akt. In IPRL, an intraportal injection of E17G triggered endocytosis of Abcc2 and Abcb11, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of Abcc2 and Abcb11 substrates. TYR did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of transporters into the canalicular membrane. In conclusion, the activation of IGF-1R is a key factor in the alteration of canalicular transporter function and localization induced by E17G, and its activation follows that of GPR30 and precedes that of PI3K/Akt.

Hepatotoxicidad colestásica asociada al uso de quimioterapia en esquema con lomustina: reporte de un caso y revisión de la literatura / Colestasic hepatotoxicity associated with the use of chemotherapy in scheme with lomustine: case report and literature review

Drug-induced liver injury (DILI) is a rare entity associated with high morbidity and mortality. It includes a broad spectrum of clinical patterns, from acute hepatitis to cirrhosis. Among the common associated drugs are antimicrobial like anti-TBC, antineoplastic, CNS agents and non-steroidal anti-inflammatory drugs. Establishing causality between DILI and a certain drug is a challenge. Some scoring systems have been evaluated, considering RUCAM score as the gold standard. We present the case of a 35-year-old woman with a history of a high-grade glioma treated with surgery and chemotherapy with lomustine, procarbazine and vincristine. She evolved with altered liver tests, predominantly cholestatic pattern, but asymptomatic. Etiologic study negative and abdominal imaging were normal. The liver biopsy was compatible with 40% ductopenia, without inflammatory elements. We consider DILI associated with the use of lomustine, with RUCAM score suggesting. After discontinuing chemotherapy and using ursodeoxycholic acid for the treatment of cholestasis there was an improvement in liver tests. There is limited evidence in the literature regarding hepatotoxicity associated with lomustine, mainly in experimental animal models. Cases of cholestatic hepatotoxicity have been described with the use of other similar nitrosureas. In relation to procarbazine and vincristine, DILI is reported mainly reversible and predominantly with hepatocellular pattern, not consistent with our case. We find it interesting to communicate with review of the literature about it.

El daño hepático inducido por drogas (DILI) es una entidad poco frecuente, con alta morbimortalidad asociada. Incluye un amplio espectro de patrones clínicos, desde hepatitis aguda a cirrosis. Dentro de los fármacos frecuentemente asociados se encuentran antibióticos como anti-TBC, agentes antineoplásicos, de acción en el SNC y anti-inflamatorios no esteroidales. Establecer una causalidad entre DILI y una determinada droga constituye un desafío. Para ello, se han evaluado diversos sistemas de puntuación, considerándose gold estándar el RUCAM score. Se presenta el caso de una mujer de 35 años de edad con antecedentes de glioma de alto grado operado y en quimioterapia con lomustina, procarbazina y vincristina. En su evolución presenta alteración de pruebas hepáticas de predominio colestásico de manera asintomática, con estudio etiológico causal negativo e imagenológico normal. La biopsia hepática fue compatible con ductopenia de 40% sin elementos inflamatorios. Se plantea DILI asociado al uso de lomustina con un score de RUCAM sugerente, decidiéndose interrumpir sus ciclos de quimioterapia e inicia tratamiento con ácido ursodesoxicólico, presentando mejoría progresiva de pruebas hepáticas. Existe evidencia limitada en la literatura en relación a hepatotoxicidad asociada a este fármaco, principalmente en modelos experimentales, y con el uso de otras nitrosureas similares se han descrito casos de hepatotoxicidad de predominio colestásico. En relación con procarbazina y vincristina existen reportes de DILI principalmente reversible y con patrón de predominio hepatocelular, lo que no es concordante con nuestro caso, por lo cual nos parece de interés comunicarlo con revisión de la literatura al respecto.

Adenovirus-mediated human aquaporin-1 expression in hepatocytes improves lipopolysaccharide-induced cholestasis.

Lipopolysaccharides (LPS) are known to cause cholestasis in sepsis. There is evidence that a defective expression of canalicular aquaporin water channels contributes to bile secretory failure in LPS-induced cholestasis. Thus, we studied whether the hepatic adenovirus-mediated transfer of human aquaporin-1 gene (haqp1) can improve the cholestasis induced by LPS. Adenoviral vector encoding hAQP1 (AdhAQP1) or control vector was administered to rats by retrograde intrabiliary infusion. Hepatocyte canalicular hAQP1 expression was assessed by liver immunostaining and immunoblotting in purified plasma membranes. LPS reduced bile flow and biliary bile acid excretion by 30% and 45%, respectively. AdhAQP1-treatment normalized both bile flow and biliary bile acid excretion in LPS-induced cholestasis. Moreover, markedly elevated serum bile acid levels in cholestatic rats, were also normalized with the AdhAQP1 hepatic transduction. Bile flow and serum or biliary bile acids in normal rats were not significantly altered by AdhAQP1. AdhAQP1 delivery unaffected the downregulated protein expression of canalicular bile salt export pump (BSEP/ABCB11) in cholestasis, but improved its transport activity restoring reduced canalicular cholesterol content. Our data suggest that the adenovirus-mediated hepatocyte hAQP1 expression improves LPS-induced cholestasis in rats by stimulating the BSEP/ABCB11-mediated biliary bile acid excretion; a finding that might contribute to the understanding and treatment of sepsis-associated cholestatic diseases. © 2017 IUBMB Life, 69(12):978-984, 2017.

Mitogen-activated protein kinases are involved in hepatocanalicular dysfunction and cholestasis induced by oxidative stress.

In previous studies, we showed that the pro-oxidant model agent tert-butyl hydroperoxide (tBuOOH) induces alterations in hepatocanalicular secretory function by activating Ca2+-dependent protein kinase C isoforms (cPKC), via F-actin disorganization followed by endocytic internalization of canalicular transporters relevant to bile formation (Mrp2, Bsep). Since mitogen-activated protein kinases (MAPKs) may be downstream effectors of cPKC, we investigated here the involvement of the MAPKs of the ERK1/2, JNK1/2, and p38MAPK types in these deleterious effects. tBuOOH (100 µM, 15 min) increased the proportion of the active, phosphorylated forms of ERK1/2, JNK1/2, and p38MAPK, and panspecific PKC inhibition with bisindolylmaleimide-1 (100 nM) or selective cPKC inhibition with Gö6976 (1 µM) prevented the latter two events. In isolated rat hepatocyte couplets, tBuOOH (100 µM, 15 min) decreased the canalicular vacuolar accumulation of the fluorescent Bsep and Mrp2 substrates, cholylglycylamido fluorescein, and glutathione-methylfluorescein, respectively, and selective inhibitors of ERK1/2 (PD098059), JNK1/2 (SP600125), and p38MAPK (SB203580) partially prevented these alterations. In in situ perfused rat livers, these three MAPK inhibitors prevented tBuOOH (75 µM)-induced impairment of bile flow and the decrease in the biliary output of the Bsep and Mrp2 substrates, taurocholate, and dinitrophenyl-S-glutathione, respectively. The changes in Bsep/Mrp2 and F-actin localization induced by tBuOOH, as assessed by (immuno)fluorescence staining followed by analysis of confocal images, were prevented total or partially by the MAPK inhibitors. We concluded that MAPKs of the ERK1/2, JNK1/2, and p38MAPK types are all involved in cholestasis induced by oxidative stress, by promoting F-actin rearrangement and further endocytic internalization of canalicular transporters critical for bile formation.

Hepatic gene transfer of human aquaporin-1 improves bile salt secretory failure in rats with estrogen-induced cholestasis.

UNLABELLED: The adenoviral gene transfer of human aquaporin-1 (hAQP1) water channels to the liver of 17α-ethinylestradiol-induced cholestatic rats improves bile flow, in part by enhancing canalicular hAQP1-mediated osmotic water secretion. To gain insight into the mechanisms of 17α-ethinylestradiol cholestasis improvement, we studied the biliary output of bile salts (BS) and the functional expression of the canalicular BS export pump (BSEP; ABCB11). Adenovector encoding hAQP1 (AdhAQP1) or control vector was administered by retrograde intrabiliary infusion. AdhAQP1-transduced cholestatic rats increased the biliary output of major endogenous BS (50%-80%, P < 0.05) as well as that of taurocholate administered in choleretic or trace radiolabel amounts (around 60%, P < 0.05). Moreover, liver transduction with AdhAQP1 normalized serum BS levels, otherwise markedly elevated in cholestatic animals. AdhAQP1 treatment was unable to improve BSEP protein expression in cholestasis; however, its transport activity, assessed by adenosine triphosphate-dependent taurocholate transport in canalicular membrane vesicles, was induced by 90% (P < 0.05). AdhAQP1 administration in noncholestatic rats induced no significant changes in either biliary BS output or BSEP activity. Canalicular BSEP, mostly present in raft (high cholesterol) microdomains in control rats, was largely found in nonraft (low cholesterol) microdomains in cholestasis. Considering that BSEP activity directly depends on canalicular membrane cholesterol content, decreased BSEP presence in rafts may contribute to BSEP activity decline in 17α-ethinylestradiol cholestasis. In AdhAQP1-transduced cholestatic rats, BSEP showed a canalicular microdomain distribution similar to that of control rats, which provides an explanation for the improved BSEP activity. CONCLUSION: Hepatocyte canalicular expression of hAQP1 through adenoviral gene transfer promotes biliary BS output by modulating BSEP activity in estrogen-induced cholestasis, a novel finding that might help us to better understand and treat cholestatic disorders. (Hepatology 2016;64:535-548).

Sertraline-Associated Cholestasis and Ductopenia Consistent with Vanishing Bile Duct Syndrome.

An adolescent with depression treated with sertraline developed cholestasis and bile duct paucity, which resolved with medication discontinuation. Vanishing bile duct syndrome is an acquired destruction of interlobular bile ducts. This type of drug-induced liver injury has been associated with other medications and requires practitioners' awareness of potential hepatotoxicity.

EGFR participates downstream of ERα in estradiol-17ß-D-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets.

Estradiol-17ß-D-glucuronide (E17G) induces acute endocytic internalization of canalicular transporters, including multidrug resistance-associated protein 2 (Abcc2) in rat, generating cholestasis. Several proteins organized in at least two different signaling pathways are involved in E17G cholestasis: one pathway involves estrogen receptor alpha (ERα), Ca(2+)-dependent protein kinase C and p38-mitogen activated protein kinase, and the other pathway involves GPR30, PKA, phosphoinositide 3-kinase/AKT and extracellular signal-related kinase 1/2. EGF receptor (EGFR) can potentially participate in both pathways since it interacts with GPR30 and ERα. Hence, the aim of this study was to analyze the potential role of this receptor and its downstream effectors, members of the Src family kinases in E17G-induced cholestasis. In vitro, EGFR inhibition by Tyrphostin (Tyr), Cl-387785 or its knockdown with siRNA strongly prevented E17G-induced impairment of Abcc2 function and localization. Activation of EGFR was necessary but not sufficient to impair the canalicular transporter function, whereas the simultaneous activation of EGFR and GPR30 could impair Abcc2 transport. The protection of Tyr was not additive to that produced by the ERα inhibitor ICI neither with that produced by Src kinase inhibitors, suggesting that EGFR shared the signaling pathway of ERα and Src. Further analysis of ERα, EGFR and Src activations induced by E17G, demonstrated that ERα activation precedes that of EGFR and EGFR activation precedes that of Src. In conclusion, activation of EGFR is a key factor in the alteration of canalicular transporter function and localization induced by E17G and it occurs before that of Src and after that of ERα.

Cholestatic hepatitis C after chemotherapy containing rituximab in diffuse large B cell lymphoma.

Rituximab is a chimeric anti-CD20 monoclonal antibody that is a widely used for the treatment of B cells non-Hodgkin lymphoma. The use of chemotherapy regimens containing rituximab in HCV-positive patients with non-Hodgkin lymphoma has been associated with liver dysfunction, but no cases of cholestatic hepatitis C were described. To our knowledge, this is the first case of cholestatic hepatitis C in an HCV-positive patient with diffuse large B-cell lymphoma describes in the literature. We discuss the pathogenetic mechanisms underlying this severe form of hepatitis and describe its evolution after antiviral treatment.

Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis.

At present, it has not been systematically evaluated whether the functional alterations induced by cholestatic compounds in canalicular transporters involved in bile formation can be reproduced in sandwich-cultured rat hepatocytes (SCRHs). Here, we focused on two clinically relevant cholestatic agents, such as estradiol 17ß-D-glucuronide (E17G) and taurolithocholate (TLC), also testing the ability of dibutyryl cyclic AMP (DBcAMP) to prevent their effects. SCRHs were incubated with E17G (200 µM) or TLC (2.5 µM) for 30 min, with or without pre-incubation with DBcAMP (10 µM) for 15 min. Then, the increase in glutathione methyl fluorescein (GS-MF)-associated fluorescence inside the canaliculi was monitored by quantitative time-lapse imaging, and Mrp2 transport activity was calculated by measuring the slope of the time-course fluorescence curves during the initial linear phase, which was considered to be the Mrp2-mediated initial transport rate (ITR). E17G and TLC impaired canalicular bile formation, as evidenced by a decrease in both the bile canaliculus volume and the bile canaliculus width, estimated from 3D and 2D confocal images, respectively. These compounds decreased ITR and induced retrieval of Mrp2, a main pathomechanism involved in their cholestatic effects. Finally, DBcAMP prevented these effects, and its well-known choleretic effect was evident from the increase in the canalicular volume/width values; this choleretic effect is associated in part with its capability to increase Mrp2 activity, evidenced here by the increase in ITR of GS-MF. Our study supports the use of SCRHs as an in vitro model useful to quantify canalicular transport function under conditions of cholestasis and choleresis.

Adenoviral transfer of human aquaporin-1 gene to rat liver improves bile flow in estrogen-induced cholestasis.

Estrogens can cause liver cholestatic disease. As downregulation of hepatocyte canalicular aquaporin-8 (AQP8) water channels has been involved in estrogen-induced bile secretory failure, we tested whether the archetypal water channel AQP1 improves 17α-ethinylestradiol (EE)-induced cholestasis. EE administration to rats reduced bile flow by 50%. A recombinant adenoviral (Ad) vector encoding human AQP1 (hAQP1), AdhAQP1, or a control vector was administered by retrograde bile ductal infusion. Hepatocyte canalicular hAQP1 expression was confirmed by liver immunostaining and immunoblotting in purified membrane fractions. Accordingly, canalicular osmotic water permeability was markedly increased. Bile flow, either basal or bile salt-stimulated was significantly augmented by over 50%. The choleretic efficiency of endogenous bile salts (that is, volume of bile per µmol of excreted bile salt) was significantly increased by 45% without changes in the biliary bile salt composition. Our data suggest that the adenoviral transfer of hAQP1 gene to the livers of EE-induced cholestatic rats improves bile flow by enhancing the AQP-mediated bile salt-induced canalicular water secretion. This novel finding might have potential therapeutic implications for cholestatic diseases.

G-protein-coupled receptor 30/adenylyl cyclase/protein kinase A pathway is involved in estradiol 17ß-D-glucuronide-induced cholestasis.

UNLABELLED: Estradiol-17ß-D-glucuronide (E17G) activates different signaling pathways (e.g., Ca(2+) -dependent protein kinase C, phosphoinositide 3-kinase/protein kinase B, mitogen-activated protein kinases [MAPKs] p38 and extracellular signal-related kinase 1/2, and estrogen receptor alpha) that lead to acute cholestasis in rat liver with retrieval of the canalicular transporters, bile salt export pump (Abcb11) and multidrug resistance-associated protein 2 (Abcc2). E17G shares with nonconjugated estradiol the capacity to activate these pathways. G-protein-coupled receptor 30 (GPR30) is a receptor implicated in nongenomic effects of estradiol, and the aim of this study was to analyze the potential role of this receptor and its downstream effectors in E17G-induced cholestasis. In vitro, GPR30 inhibition by G15 or its knockdown with small interfering RNA strongly prevented E17G-induced impairment of canalicular transporter function and localization. E17G increased cyclic adenosine monophosphate (cAMP) levels, and this increase was blocked by G15, linking GPR30 to adenylyl cyclase (AC). Moreover, AC inhibition totally prevented E17G insult. E17G also increased protein kinase A (PKA) activity, which was blocked by G15 and AC inhibitors, connecting the links of the pathway, GPR30-AC-PKA. PKA inhibition prevented E17G-induced cholestasis, whereas exchange protein activated directly by cyclic nucleotide/MAPK kinase, another cAMP downstream effector, was not implicated in cAMP cholestatic action. In the perfused rat liver model, inhibition of the GPR30-AC-PKA pathway totally prevented E17G-induced alteration in Abcb11 and Abcc2 function and localization. CONCLUSION: Activation of GPR30-AC-PKA is a key factor in the alteration of canalicular transporter function and localization induced by E17G. Interaction of E17G with GPR30 may be the first event in the cascade of signaling activation.