RESUMEN
The organic anion uptake and efflux transporters [organic anion-transporting polypeptide (OATP)1B1, OATP1B3 and multidrug resistance-associated protein (MRP)2 and MRP3] that mediate the transport of the hepatobiliary-specific contrast agent gadoxetate (Gd-EOB-DTPA) are direct or indirect targets of the farnesoid X receptor (FXR), a key regulator of bile acid and lipid homeostasis. In benign liver tumors, FXR expression and activation is not yet characterized. We investigated the expression and activation of FXR and its targets in hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH) and their correlation with Gd-EOB-DTPA-enhanced magnetic resonance imaging (MRI). Gd-EOB-DTPA MRI patterns were assessed by an expert radiologist. The intensity of the lesions on the hepatobiliary phase was correlated to mRNA expression levels of OATP1B1, OATP1B3, MRP2, MRP3, FXR, and small heterodimer partner (SHP) in fresh surgical specimens of patients with FNH or HCA subtypes. Normal and tumor sample pairs of 43 HCA and 14 FNH were included. All FNH (14/14) were hyperintense. Of the 34 HCA with available Gd-EOB-DTPA-enhanced MRI, 6 were hyperintense and 28 HCA were hypointense. OATP1B3 was downregulated in the hypointense tumors compared with normal surrounding liver tissue (2.77±3.59 vs. 12.9±15.6, P < 0.001). A significant positive correlation between FXR expression and activation and OATP1B3 expression level was found in the HCA cohort. SHP showed a trend toward downregulation in hypointense HCA. In conclusion, this study suggests that the MRI relative signal in HCA may reflect expression level and/or activity of SHP and FXR. Moreover, our data confirms the pivotal role of OATP1B3 in Gd-EOB-DTPA uptake in HCA. SIGNIFICANCE STATEMENT: FXR represents a valuable target for the treatment of liver disease and metabolic syndrome. Currently, two molecules, ursodeoxycholate and obeticholate, are approved for the treatment of primary biliary cirrhosis and cholestasis, with several compounds in clinical trials for the treatment of metabolic dysfunction-associated fatty liver disease. Because FXR expression and activation is associated with gadoxetate accumulation in HCA, an atypical gadoxetate-enhanced MRI pattern might arise in patients under FXR-targeted therapy, thereby complicating the differential diagnosis.
Asunto(s)
Adenoma de Células Hepáticas , Hiperplasia Nodular Focal , Neoplasias Hepáticas , Transportadores de Anión Orgánico , Humanos , Transportadores de Anión Orgánico/metabolismo , Neoplasias Hepáticas/diagnóstico por imagen , Neoplasias Hepáticas/metabolismo , Medios de Contraste/metabolismo , Hiperplasia Nodular Focal/diagnóstico , Hiperplasia Nodular Focal/metabolismo , Hiperplasia Nodular Focal/patología , Imagen por Resonancia Magnética/métodos , Proteína 2 Asociada a Resistencia a Múltiples Medicamentos , Aniones/metabolismo , Estudios RetrospectivosRESUMEN
ABCB4 is expressed in hepatocytes and translocates phosphatidylcholine into bile canaliculi. The mechanism of specific lipid recruitment from the canalicular membrane, which is essential to mitigate the cytotoxicity of bile salts, is poorly understood. We present cryogenic electron microscopy structures of human ABCB4 in three distinct functional conformations. An apo-inward structure reveals how phospholipid can be recruited from the inner leaflet of the membrane without flipping its orientation. An occluded structure reveals a single phospholipid molecule in a central cavity. Its choline moiety is stabilized by cation-π interactions with an essential tryptophan residue, rationalizing the specificity of ABCB4 for phosphatidylcholine. In an inhibitor-bound structure, a posaconazole molecule blocks phospholipids from reaching the central cavity. Using a proteoliposome-based translocation assay with fluorescently labeled phosphatidylcholine analogs, we recapitulated the substrate specificity of ABCB4 in vitro and confirmed the role of the key tryptophan residue. Our results provide a structural basis for understanding an essential translocation step in the generation of bile and its sensitivity to azole drugs.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Fosfatidilcolinas/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/química , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Transporte Biológico , Técnicas de Visualización de Superficie Celular , Microscopía por Crioelectrón , Epítopos , Regulación de la Expresión Génica , Variación Genética , Células HEK293 , Humanos , Fragmentos Fab de Inmunoglobulinas , Modelos Moleculares , Conformación ProteicaRESUMEN
The liver fulfills many vital functions for the body, among them bile formation and detoxification. Bile salts are organic anions, are the major constituents of bile and are at high concentrations cytotoxic. Detoxification exposes the liver to many harmful compounds. This function is therefore potentially damaging to the liver. Impaired bile formation may lead to hepatic accumulation of bile salts and subsequently to liver disease. Diagnosis of liver diseases involves the measurement of so-called liver function parameters. This overview aims to characterize and summarize the role of organic anion transporters in bile formation at the protein level under normal physiologic conditions and in liver function tests used for diagnosing liver diseases in pathophysiologic situations.
RESUMEN
Bile salts, such as cholate, glycocholate, taurocholate, and glycochenodeoxycholate, are taken up from the portal blood into hepatocytes via transporters, such as the Na+-taurocholate-cotransporting polypeptide (NTCP) and organic anion-transporting polypeptides (OATPs). These bile salts are later secreted into bile across the canalicular membrane, which is facilitated by the bile salt export pump (BSEP). Apart from bile salt transport, some of these proteins (e.g., OATPs) are also key transporters for drug uptake into hepatocytes. In vivo studies of transporter function in patients by using tracer compounds have emerged as an important diagnostic tool to complement classic liver parameter measurements by determining dynamic liver function both for diagnosis and monitoring progression or improvement of liver diseases. Such approaches include use of radioactively labeled bile salts (e.g., for positron emission tomography) and fluorescent bile salt derivatives or dyes (e.g., indocyanine green). To expand the list of liver function markers, we synthesized fluorescent derivatives of cholic and chenodeoxycholic acid by conjugating small organic dyes to the bile acid side chain. These novel fluorescent probes were able to block substrate transport in a concentration-dependent manner of NTCP, OATP1B1, OATP1B3, OATP2B1, BSEP, and intestinal apical sodium-dependent bile salt transporter (ASBT). Whereas the fluorescent bile acid derivatives themselves were transported across the membrane by OATP1B1, OATP1B3, and OATP2B1, they were not transport substrates for NTCP, ASBT, BSEP, and multidrug resistance-related protein 2. Accordingly, these novel fluorescent bile acid probes can potentially be used as imaging agents to monitor the function of OATPs. SIGNIFICANCE STATEMENT: Synthetic modification of common bile acids by attachment of small organic fluorescent dyes to the bile acid side chain resulted in bright, fluorescent probes that interact with hepatic and intestinal organic anion [organic anion-transporting polypeptide (OATP) 1B1, OATP1B3, OATP2B1], bile salt uptake (Na+-taurocholate-cotransporting polypeptide, apical sodium-dependent bile salt transporter), and bile salt efflux (bile salt export pump, multidrug resistance-related protein 2) transporters. Although the fluorescent bile salt derivatives are taken up into cells via the OATPs, the efflux transporters do not transport any of them but one.
Asunto(s)
Transportadores de Anión Orgánico , Subfamilia B de Transportador de Casetes de Unión a ATP , Ácidos y Sales Biliares , Hepatocitos , Miembro 4 de la Subfamilia B de Casete de Unión a ATPRESUMEN
Herbal supplements are generally considered safe; however, drug disposition is influenced by the interactions of herbal supplements and food constituents with transport and metabolic processes. Although the interference of herbal supplements with drug metabolism has been studied extensively, knowledge of how they interact with the drug transport processes is less advanced. Therefore, we describe here specific examples of experimental and human interaction studies of herbal supplement components with drug transporters addressing, for example, organic anion transporting polypeptides or P-glycoprotein, as such interactions may lead to severe side effects and altered drug efficacy. Hence, it is clearly necessary to increase the awareness of the clinical relevance of the interference of herbal supplements with the drug transport processes.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Hepatocitos/metabolismo , Mucosa Intestinal/metabolismo , Transportadores de Anión Orgánico/metabolismo , Preparaciones de Plantas/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Interacciones Farmacológicas/fisiología , Hepatocitos/efectos de los fármacos , Humanos , Mucosa Intestinal/efectos de los fármacos , Preparaciones Farmacéuticas/metabolismo , Preparaciones de Plantas/farmacología , Preparaciones de Plantas/uso terapéutico , Resultado del TratamientoRESUMEN
Mitochondrial damage is considered a hallmark of drug-induced liver injury (DILI). However, despite the common molecular etiology, the evolution of the injury is usually unpredictable, with some cases that are mild and reversible upon discontinuation of the treatment and others characterized by irreversible acute liver failure. This suggests that additional mechanisms of damage play a role in determining the progression of the initial insult. To uncover novel pathways potentially involved in DILI, we investigated in vitro the metabolic perturbations associated with nefazodone, an antidepressant associated with acute liver failure. Several pathways associated with ATP production, including gluconeogenesis, anaerobic glycolysis, and oxidative phosphorylation, were altered in human hepatocellular carcinoma-derived (Huh7) cells after 2-hour exposure to a 50 µM extracellular concentration of nefazodone. In the presence or absence of glucose, ATP production of Huh7 cells was glycolysis- and oxidative phosphorylation-dependent, respectively. In glucose-containing medium, nefazodone-induced ATP depletion from Huh7 cells was biphasic. Huh7 cells in glucose-free medium were more sensitive to nefazodone than those in glucose-containing medium, losing the biphasic inhibition. Nefazodone-induced ATP depletion in primary cultured mouse hepatocytes, mainly dependent on oxidative phosphorylation, was monophasic. At lower extracellular concentrations, nefazodone inhibited the oxygen consumption of Huh7 cells, whereas at higher extracellular concentrations, it also inhibited the extracellular acidification. ATP content was rescued by increasing the extracellular concentration of glucose. In conclusion, nefazodone has a dual inhibitory effect on mitochondrial-dependent and mitochondrial-independent ATP production. SIGNIFICANCE STATEMENT: Mitochondrial damage is a hallmark of drug-induced liver injury, yet other collateral alterations might contribute to the severity and evolution of the injury. Our in vitro study supports previous results arguing that a deficit in hepatic glucose metabolism, concomitant to the mitochondrial injury, might be cardinal in the prognosis of the initial insult to the liver. From a drug development standpoint, coupling anaerobic glycolysis and mitochondrial function assessment might increase the drug-induced liver injury preclinical screening performance.
Asunto(s)
Antidepresivos/efectos adversos , Hígado/efectos de los fármacos , Hígado/metabolismo , Metabolómica , Piperazinas/efectos adversos , Triazoles/efectos adversos , Adenosina Trifosfato/metabolismo , Anaerobiosis/efectos de los fármacos , Animales , Transporte Biológico/efectos de los fármacos , Línea Celular , Glucosa/metabolismo , Glucólisis/efectos de los fármacos , Humanos , RatonesRESUMEN
P-Glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) in the canalicular membrane of hepatocytes mediate the biliary excretion of drugs and drug metabolites. To measure hepatic ABCB1 and ABCG2 activity, we performed positron emission tomography (PET) scans with the ABCB1/ABCG2 substrate [11C]tariquidar in healthy volunteers and wild-type, Abcb1a/b(-/-), Abcg2(-/-), and Abcb1a/b(-/-)Abcg2(-/-) mice without and with coadministration of unlabeled tariquidar. PET data were analyzed with a three-compartment pharmacokinetic model. [11C]Tariquidar underwent hepatobiliary excretion in both humans and mice, and tariquidar coadministration caused a significant reduction in the rate constant for the transfer of radioactivity from the liver into bile (by -74% in humans and by -62% in wild-type mice), suggesting inhibition of canalicular efflux transporter activity. Radio-thin-layer chromatography analysis revealed that the majority of radioactivity (>87%) in the mouse liver and bile was composed of unmetabolized [11C]tariquidar. PET data in transporter knockout mice revealed that both ABCB1 and ABCG2 mediated biliary excretion of [11C]tariquidar. In vitro experiments indicated that tariquidar is not a substrate of major hepatic basolateral uptake transporters (SLCO1B1, SLCO1B3, SLCO2B1, SLC22A1, and SLC22A3). Our data suggest that [11C]tariquidar can be used to measure hepatic canalicular ABCB1/ABCG2 transport activity without a confounding effect of uptake transporters.
Asunto(s)
Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/metabolismo , Hígado/diagnóstico por imagen , Proteínas de Neoplasias/metabolismo , Tomografía de Emisión de Positrones , Quinolinas/farmacocinética , Radiofármacos/farmacocinética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Transportador de Casetes de Unión a ATP, Subfamilia G, Miembro 2/genética , Adulto , Animales , Bilis/metabolismo , Isótopos de Carbono/química , Vesícula Biliar/diagnóstico por imagen , Humanos , Hígado/metabolismo , Masculino , Ratones , Ratones Noqueados , Quinolinas/química , Distribución TisularRESUMEN
The bile salt export pump (BSEP) is the primary canalicular transporter responsible for the secretion of bile acids from hepatocytes into bile canaliculi, and inhibition of this transporter has been associated with drug-induced liver injury (DILI). A common variant (rs2287622; p.V444A) in the gene encoding BSEP has been associated with an increased risk of cholestatic DILI. Although p.444V BSEP (reference) and p.444A BSEP (variant) do not differ in their transport kinetics of taurocholic acid (TCA), transport of the more abundant glycocholic acid (GCA) has not been investigated. Importantly, differences in the susceptibility of p.444V and p.444A BSEP to inhibition by drugs causing cholestatic DILI have not been investigated. To address these issues, the transport kinetics of GCA were evaluated by incubating membrane vesicles expressing either p.444V or p.444A BSEP with GCA over a range of concentrations (1, 10, 25, 50, and 100 µM). The abilities of commonly used cholestatic medications to inhibit the transport of TCA and GCA by the reference and variant proteins were compared. Resulting data indicated that GCA transport kinetics for reference and variant BSEP followed Michaelis-Menten kinetics and were not statistically different [ Vmax values of 1132 ± 246 and 959 ± 256 pmol min-1 (mg of protein)-1, respectively, and Km values of 32.7 ± 18.2 and 45.7 ± 25.5 µM, respectively]. There were no statistically significant differences between the reference and variant BSEP in the inhibition of TCA or GCA transport by the cholestatic drugs tested. In conclusion, differential inhibition of TCA or GCA transport cannot account for an association between the variant BSEP and the risk for cholestatic DILI due to the drugs tested.
Asunto(s)
Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/genética , Ácidos y Sales Biliares/metabolismo , Colagogos y Coleréticos/uso terapéutico , Colestasis/tratamiento farmacológico , Polimorfismo de Nucleótido Simple , Miembro 11 de la Subfamilia B de Transportador de Casetes de Unión al ATP/antagonistas & inhibidores , Animales , Baculoviridae , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Colagogos y Coleréticos/farmacología , Dipiridamol/farmacología , Eritromicina/farmacología , Ácido Glicocólico/antagonistas & inhibidores , Ácido Glicocólico/metabolismo , Cetoconazol/farmacología , Cinética , Proteínas de Transporte de Membrana/metabolismo , Células Sf9 , Transducción de Señal/efectos de los fármacos , Spodoptera/virología , Ácido Taurocólico/antagonistas & inhibidores , Ácido Taurocólico/metabolismo , Vesículas Transportadoras/metabolismoRESUMEN
We report an unusual case of intermittent episodes of cholestasis in a young patient. The cholestatic attacks were preceded in each case by an infection and subsequent antibiotic therapies. After ruling out many possible causes of cholestatic hepatitis, the differential diagnoses were a benign recurrent intrahepatic cholestasis or a drug-induced liver injury. We discuss here the diagnostic approach and interpretation of the genetic analysis.
Asunto(s)
Colestasis Intrahepática/diagnóstico , Colestasis Intrahepática/genética , Ictericia Obstructiva/etiología , Adolescente , Enfermedad Hepática Inducida por Sustancias y Drogas , Diagnóstico Diferencial , Predisposición Genética a la Enfermedad , Pruebas Genéticas , Humanos , Masculino , RecurrenciaRESUMEN
BACKGROUND & AIMS: The organic anion-transporting polypeptide 1B1 (OATP1B1) is an anion exchanger expressed at the hepatocyte sinusoidal membrane, which mediates the uptake of several endogenous metabolites and drugs. OATP1B1 expression level and activity are major sources of inter-patient variability of pharmacokinetics and pharmacodynamics of several drugs. Besides the genotype, factors that contribute to the inter-individual variability in OATP1B1 expression level are practically unknown. The aim of this work was to uncover novel epigenetic mechanisms of OATP1B1 regulation. METHODS: A functional screening strategy to assess the effect of microRNAs on the uptake of estrone-3-sulphate, an OATP1B1 substrate, into human hepatocellular carcinoma (Huh-7) cells was used. microRNA-206 (miR-206) expression in human liver tissues was measured by real-time RT-PCR. OATP1B1 expression in Huh-7 and in human liver tissues was assessed by real-time RT-PCR, Western blotting and immunostaining. The mRNA-miRNA interaction was assessed by reporter assay. RESULTS: miR-206 mimic repressed mRNA and protein expression of OATP1B1 in Huh-7 cells. The intracellular accumulation of estrone-3-sulphate was reduced by 30% in cells overexpressing miR-206. The repressive effect of miR-206 on the activity of the firefly luciferase gene 2 under the control of the OATP1B1 3' untranslated region was lost upon deletion of the predicted miR-206 binding site. Hepatic miR-206 level negatively correlated with OATP1B1 mRNA and protein levels extracted from normal human liver tissues. CONCLUSIONS: miR-206 exerts a suppressive effect on OATP1B1 expression by an epigenetic mechanism. Individuals with high hepatic levels of miR-206 appear to display lower level of OATP1B1.
Asunto(s)
Estrona/análogos & derivados , Transportador 1 de Anión Orgánico Específico del Hígado/metabolismo , Hígado/metabolismo , MicroARNs/metabolismo , Animales , Células CHO , Línea Celular Tumoral , Simulación por Computador , Cricetulus , Estrona/metabolismo , Femenino , Humanos , Transportador 1 de Anión Orgánico Específico del Hígado/genética , Masculino , Persona de Mediana EdadRESUMEN
Positron emission tomography (PET) using the cationic compound [18F]fluoromethylcholine (FCH) enhances the sensitivity for noninvasive classification of hepatic tumors due to peculiar patterns of accumulation. The underlying transporters are not known. We aim to identify the carriers mediating uptake of FCH in liver and to correlate their expression pattern with PET intrahepatic signal distribution to clarify the role of membrane transporters in FCH accumulation. FCH transport was characterized in cells overexpressing organic cation transporters (OCTs). OCT mRNA levels were determined in different types of hepatic lesions and correlated with FCH PET signal intensity. Additionally, OCT1 and OCT3 protein was analyzed in a subset of patients by Western blotting. HEK293 cells overexpressing OCT1, OCT2, or OCT3 showed higher intracellular levels of FCH in comparison with wild-type cells. mRNA levels of OCT1 paralleled protein levels and were significantly downregulated in hepatocellular carcinoma (HCC), hepatocellular adenoma (HCA), and, to a lesser extent, in focal nodular hyperplasia compared with matched nontumor tissues. In three patients with HCA, the FCH PET signal intensity was reduced relative to normal liver. This correlated with the simultaneous downregulation of OCT1 and OCT3 mRNA. In another patient with HCA, lesion and surrounding tissue did not show a difference in signal, coinciding with downregulation of OCT1 and upregulation of OCT3. Therefore, OCT1 is very likely a key transporter for the accumulation of FCH in the liver. The data support the hypothesis that the varying expression levels of OCT1 and OCT3 in focal liver lesions determine FCH PET signal intensity.
Asunto(s)
Adenoma de Células Hepáticas/diagnóstico por imagen , Carcinoma Hepatocelular/diagnóstico por imagen , Neoplasias Hepáticas/diagnóstico por imagen , Proteínas de Transporte de Catión Orgánico/metabolismo , Transportador 1 de Catión Orgánico/metabolismo , Tomografía de Emisión de Positrones/métodos , Adenoma de Células Hepáticas/metabolismo , Carcinoma Hepatocelular/metabolismo , Colina/análogos & derivados , Colina/farmacocinética , Diagnóstico Diferencial , Femenino , Radioisótopos de Flúor , Células HEK293 , Humanos , Hígado/diagnóstico por imagen , Hígado/metabolismo , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana Edad , Proteínas de Transporte de Catión Orgánico/genética , Transportador 1 de Catión Orgánico/genética , Distribución TisularRESUMEN
PURPOSE: Vincristine is known to interfere with OATP-mediated uptake of other compounds, hinting that vincristine itself could be a substrate of OATP transporters. The present study therefore aimed to investigate the role of OATP transporters in the hepatocellular disposition of vincristine. METHODS: Vincristine uptake was studied in suspended rat and human hepatocytes as well as OATP-transfected Chinese hamster ovary (CHO) cells in the absence and presence of OATP transporter inhibitors. Membrane vesicles containing MDR1 or MRP1/2/3 were used to directly assess the role of these efflux transporters in vincristine disposition. RESULTS: Uptake in suspended rat hepatocytes was temperature-dependent and could be inhibited by a range of OATP inhibitors. Furthermore, the MRP-inhibitor benzbromarone, but none of the tested MDR1 inhibitors, reduced vincristine efflux in rat and human suspended hepatocytes. OATP1B1-, OATP1B3- and OATP2B1- transfected CHO cells showed significantly increased vincristine uptake as compared to wild-type cells. Moreover, uptake in OATP-transfected CHO cells was reduced by OATP inhibitors. However, uptake studies in suspended human hepatocytes showed that only 10% of the total vincristine uptake process could be attributed to OATP-mediated transport. Studies with transporter-expressing membrane vesicles confirmed vincristine as an MDR1 substrate, while MRP1/2/3-mediated transport of vincristine could not be observed with this model system. CONCLUSIONS: Our findings show the involvement of OATP transporters in the disposition of vincristine in rat and human hepatocytes. However, in both species, hepatic uptake is overshadowed by a benzbromarone-sensitive efflux mechanism, possibly MRP3.
Asunto(s)
Antineoplásicos/farmacología , Benzbromarona/farmacología , Transportadores de Anión Orgánico/metabolismo , Vincristina/farmacología , Animales , Antineoplásicos/metabolismo , Transporte Biológico , Células CHO , Cricetinae , Cricetulus , Hepatocitos/metabolismo , Hígado/metabolismo , Masculino , Transportadores de Anión Orgánico/antagonistas & inhibidores , Transportadores de Anión Orgánico/genética , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Ratas Wistar , Vincristina/metabolismoRESUMEN
Drug transporters are now recognized as major actors in pharmacokinetics, involved notably in drug-drug interactions and drug adverse effects. Factors that govern their activity, localization and expression are therefore important to consider. In the present review, the implications of protein kinases C (PKCs) in transporter regulations are summarized and discussed. Both solute carrier (SLC) and ATP-binding cassette (ABC) drug transporters can be regulated by PKCs-related signaling pathways. PKCs thus target activity, membrane localization and/or expression level of major influx and efflux drug transporters, in various normal and pathological types of cells and tissues, often in a PKC isoform-specific manner. PKCs are notably implicated in membrane insertion of bile acid transporters in liver and, in this way, are thought to contribute to cholestatic or choleretic effects of endogenous compounds or drugs. The exact clinical relevance of PKCs-related regulation of drug transporters in terms of drug resistance, pharmacokinetics, drug-drug interactions and drug toxicity remains however to be precisely determined. This issue is likely important to consider in the context of the development of new drugs targeting PKCs-mediated signaling pathways, for treating notably cancers, diabetes or psychiatric disorders.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Proteína Quinasa C/metabolismo , Transducción de Señal , Proteínas Transportadoras de Solutos/metabolismo , Transportadoras de Casetes de Unión a ATP/genética , Transporte Biológico , Regulación de la Expresión Génica , Humanos , Isoenzimas/metabolismo , Preparaciones Farmacéuticas/metabolismo , Fosforilación , Proteínas Transportadoras de Solutos/genéticaRESUMEN
Drug-induced liver injury is an important clinical entity resulting in a considerable number of hospitalizations. While drug-induced cholestasis due to the inhibition of the bile salt export pump (BSEP) is well investigated, only limited information on the interaction of drugs with multidrug resistance protein 3 (MDR3) exists and its role in the pathogenesis of drug-induced cholestasis is poorly understood. Therefore, we aimed to study the interaction of drugs with MDR3 and the effect of drugs on canalicular lipid secretion in a newly established polarized cell line system that serves as a model of canalicular lipid secretion. LLC-PK1 cells were stably transfected with human Na(+)-taurocholate cotransporting polypeptide, BSEP, MDR3, and ABCG5/G8 and grown in the Transwell system. Apical phospholipid secretion and taurocholate transport were assayed to investigate the effect of selected drugs on MDR3-mediated phospholipid secretion as well as inhibition of BSEP. The established cell line displayed vectorial bile salt transport and specific phosphatidylcholine secretion into the apical compartment. The antifungal azoles, posaconazole, itraconazole, and ketoconazole, significantly inhibited MDR3-mediated phosphatidylcholine secretion. In contrast, amoxicillin clavulanate and troglitazone did not interfere with MDR3 activity. Drugs interfering with MDR3 activity did not display a parallel inhibition of BSEP. Our in vitro model for MDR3-mediated phospholipid secretion facilitates parallel screening for MDR3 and BSEP inhibitors. Our data demonstrate that the cholestatic potential of certain drugs may be aggravated by simultaneous inhibition of BSEP and MDR3.
Asunto(s)
Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Antifúngicos/efectos adversos , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Colestasis/inducido químicamente , Colestasis/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Azoles/farmacología , Ácidos y Sales Biliares/metabolismo , Transporte Biológico/efectos de los fármacos , Western Blotting , Colagogos y Coleréticos/metabolismo , Humanos , Células LLC-PK1 , Fosfolípidos/metabolismo , Reproducibilidad de los Resultados , Porcinos , Ácido Taurocólico/metabolismoRESUMEN
Type VII collagen is the main component of anchoring fibrils, structures that are integral to basement membrane homeostasis in skin. Mutations in the gene encoding type VII collagen COL7A1 cause recessive dystrophic epidermolysis bullosa (RDEB) an inherited skin blistering condition complicated by frequent aggressive cutaneous squamous cell carcinoma (cSCC). OATP1B3, which is encoded by the gene SLCO1B3, is a member of the OATP (organic anion transporting polypeptide) superfamily responsible for transporting a wide range of endogenous and xenobiotic compounds. OATP1B3 expression is limited to the liver in healthy tissues, but is frequently detected in multiple cancer types and is reported to be associated with differing clinical outcome. The mechanism and functional significance of tumour-specific expression of OATP1B3 has yet to be determined. Here, we identify SLCO1B3 expression in tumour keratinocytes isolated from RDEB and UV-induced cSCC and demonstrate that SLCO1B3 expression and promoter activity are modulated by type VII collagen. We show that reduction of SLCO1B3 expression upon expression of full-length type VII collagen in RDEB cSCC coincides with acquisition of front-to-rear polarity and increased organisation of 3D spheroid cultures. In addition, we show that type VII collagen positively regulates the abundance of markers implicated in cellular polarity, namely ELMO2, PAR3, E-cadherin, B-catenin, ITGA6 and Ln332.
Asunto(s)
Carcinoma de Células Escamosas/metabolismo , Polaridad Celular , Colágeno Tipo VII/fisiología , Epidermólisis Ampollosa Distrófica/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Neoplasias Cutáneas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos CD , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Cadherinas/genética , Cadherinas/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Técnicas de Cocultivo , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Epidermólisis Ampollosa Distrófica/genética , Epidermólisis Ampollosa Distrófica/patología , Regulación Neoplásica de la Expresión Génica , Humanos , Integrina alfa6/genética , Integrina alfa6/metabolismo , Queratinocitos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Trasplante de Neoplasias , Transportadores de Anión Orgánico Sodio-Independiente/genética , Regiones Promotoras Genéticas , Transporte de Proteínas , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Miembro 1B3 de la Familia de los Transportadores de Solutos de Aniones Orgánicos , Transcripción Genética , Células Tumorales Cultivadas , beta Catenina/genética , beta Catenina/metabolismo , KalininaRESUMEN
UNLABELLED: The Na(+) -taurocholate cotransporting polypeptide (NTCP) mediates uptake of conjugated bile acids (BAs) and is localized at the basolateral membrane of hepatocytes. It has recently been recognized as the receptor mediating hepatocyte-specific entry of hepatitis B virus and hepatitis delta virus. Myrcludex B, a peptide inhibitor of hepatitis B virus entry, is assumed to specifically target NTCP. Here, we investigated BA transport and Myrcludex B binding in the first Slc10a1-knockout mouse model (Slc10a1 encodes NTCP). Primary Slc10a1(-/-) hepatocytes showed absence of sodium-dependent taurocholic acid uptake, whereas sodium-independent taurocholic acid uptake was unchanged. In vivo, this was manifested as a decreased serum BA clearance in all knockout mice. In a subset of mice, NTCP deficiency resulted in markedly elevated total serum BA concentrations, mainly composed of conjugated BAs. The hypercholanemic phenotype was rapidly triggered by a diet supplemented with ursodeoxycholic acid. Biliary BA output remained intact, while fecal BA excretion was reduced in hypercholanemic Slc10a1(-/-) mice, explained by increased Asbt and Ostα/ß expression. These mice further showed reduced Asbt expression in the kidney and increased renal BA excretion. Hepatic uptake of conjugated BAs was potentially affected by down-regulation of OATP1A1 and up-regulation of OATP1A4. Furthermore, sodium-dependent taurocholic acid uptake was inhibited by Myrcludex B in wild-type hepatocytes, while Slc10a1(-/-) hepatocytes were insensitive to Myrcludex B. Finally, positron emission tomography showed a complete abrogation of hepatic binding of labeled Myrcludex B in Slc10a1(-/-) mice. CONCLUSION: The Slc10a1-knockout mouse model supports the central role of NTCP in hepatic uptake of conjugated BAs and hepatitis B virus preS1/Myrcludex B binding in vivo; the NTCP-independent hepatic BA uptake machinery maintains a (slower) enterohepatic circulation of BAs, although it is occasionally insufficient to clear BAs from the circulation.
Asunto(s)
Virus de la Hepatitis B/metabolismo , Hígado/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismo , Ácido Taurocólico/sangre , Proteínas del Envoltorio Viral/metabolismo , Animales , Bilis/química , Heces/química , Femenino , Lipopéptidos , Masculino , Ratones Noqueados , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Fenotipo , Simportadores/genética , Ácido Taurocólico/orina , Ácido UrsodesoxicólicoRESUMEN
The bile salt export pump (BSEP) is an ABC-transporter expressed at the canalicular membrane of hepatocytes. Its physiological role is to expel bile salts into the canaliculi from where they drain into the bile duct. Inhibition of this transporter may lead to intrahepatic cholestasis. Predictive computational models of BSEP inhibition may allow for fast identification of potentially harmful compounds in large databases. This article presents a predictive in silico model based on physicochemical descriptors that is able to flag compounds as potential BSEP inhibitors. This model was built using a training set of 670 compounds with available BSEP inhibition potencies. It successfully predicted BSEP inhibition for two independent test sets and was in a further step used for a virtual screening experiment. After in vitro testing of selected candidates, a marketed drug, bromocriptin, was identified for the first time as BSEP inhibitor. This demonstrates the usefulness of the model to identify new BSEP inhibitors and therefore potential cholestasis perpetrators.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/antagonistas & inhibidores , Bromocriptina/farmacología , Animales , Células CHO , Línea Celular , Colestasis/prevención & control , Simulación por Computador , Cricetulus , PorcinosRESUMEN
Obesity has been reported as an independent risk factor for chronic kidney disease, leading to glomerulosclerosis and renal insufficiency. To assess the relationship between a reduced nephron number and a particular susceptibility to obesity-induced renal damage, mice underwent uninephrectomy (UNX) followed by either normal chow or high-fat diet (HFD) and were compared with sham-operated control mice. After 20 weeks of dietary intervention, HFD-fed control mice presented characteristic features of progressive nephropathy, including albuminuria, glomerulosclerosis, renal fibrosis and oxidative stress. These changes were more pronounced in HFD-fed mice that had undergone uninephrectomy. Analysis of gene expression in mouse kidney by whole genome microarrays indicated that high fat diet led to more changes in gene expression than uninephrectomy. HFD affected mainly genes involved in lipid metabolism and transport, whereas the combination of UNX and HFD additionally altered the expression of genes belonging to cytoskeleton remodeling, fibrosis and hypoxia pathways. Canonical pathway analyses identified the farnesoid X receptor (FXR) as a potential key mediator for the observed changes in gene expression associated with UNX-HFD. In conclusion, HFD-induced kidney damage is more pronounced following uninephrectomy and is associated with changes in gene expression that implicate FXR as a central regulatory pathway.
Asunto(s)
Biomarcadores/metabolismo , Dieta Alta en Grasa/efectos adversos , Fibrosis/etiología , Perfilación de la Expresión Génica , Enfermedades Renales/etiología , Nefrectomía/efectos adversos , Obesidad/complicaciones , Animales , Fibrosis/metabolismo , Fibrosis/patología , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Obesidad/metabolismo , Obesidad/patología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Organic anion transporting polypeptides (OATPs) are polyspecific organic anion transporters, which are expressed in the blood-brain barrier, the choroid plexus, and other organs. The physiologic function of OATPs in extrahepatic tissues remains ambiguous. In rat retina, members of the OATP family are expressed. We therefore investigated the human retina for the expression of OATP1A2 and OATP2B1 and extended the study to human brain. Furthermore, we searched for peptide neurotransmitters as novel OATP substrates. OATP1A2 displayed a broad expression pattern in human retina as assessed by immunofluorescence localization. It is expressed in photoreceptor bodies and somas of amacrine cells. OATP1B2 expression is restricted to the inner nuclear layer and to the inner plexiform layer. Using paraffin sections from human cortex, cerebellum, and hippocampus, OATP1A2 was localized to neurons and neuronal processes, while OATP2B1 is expressed in endothelial cells of brain capillaries. Substance P and vasoactive intestinal peptide were identified as substrates for OATP1A2 and OATP2B1. Double-labeling immunofluorescence of human retina demonstrated the presence of substance P and of vasoactive intestinal peptides in neurons expressing OATP1A2 and OATP2B1, respectively. The expression of OATP1A2 and OATP2B1 in retinal neurons implies a role of these transporters in the reuptake of peptide neurotransmitters released from retinal neurons. The abundant expression of OATP1A2 in brain neurons points to the possibility that OATP1A2 could be involved in the homeostasis of neurosteroids. The high expression of OATP2B1 in brain capillaries supports an important function of OATPs in substance penetration across the blood-brain barrier.