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Medeot AC, Boaglio AC, Salas G, Maidagan PM, Miszczuk GS, Barosso IR, Sánchez Pozzi EJ, Crocenzi FA, Roma MG. Tauroursodeoxycholate prevents estradiol 17β-d-glucuronide-induced cholestasis and endocytosis of canalicular transporters by switching off pro-cholestatic signaling pathways. Life Sci 2024; 352:122839. [PMID: 38876186 DOI: 10.1016/j.lfs.2024.122839] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/26/2024] [Revised: 06/08/2024] [Accepted: 06/11/2024] [Indexed: 06/16/2024]
Abstract
AIMS Estradiol 17β-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.
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Affiliation(s)
- Anabela C Medeot
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina
| | - Andrea C Boaglio
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina
| | - Gimena Salas
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina
| | - Paula M Maidagan
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina
| | - Gisel S Miszczuk
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina
| | - Ismael R Barosso
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina
| | - Enrique J Sánchez Pozzi
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina
| | - Fernando A Crocenzi
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina
| | - Marcelo G Roma
- Institute of Experimental Physiology (IFISE-CONICET), National University of Rosario, 2000 Rosario, Argentina.
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2
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Salas G, Litta AA, Medeot AC, Schuck VS, Andermatten RB, Miszczuk GS, Ciriaci N, Razori MV, Barosso IR, Sánchez Pozzi EJ, Roma MG, Basiglio CL, Crocenzi FA. NADPH oxidase-generated reactive oxygen species are involved in estradiol 17ß-d-glucuronide-induced cholestasis. Biochimie 2024; 223:41-53. [PMID: 38608750 DOI: 10.1016/j.biochi.2024.04.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2024] [Revised: 04/04/2024] [Accepted: 04/08/2024] [Indexed: 04/14/2024]
Abstract
The endogenous metabolite of estradiol, estradiol 17β-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.
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Affiliation(s)
- Gimena Salas
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Alen A Litta
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Anabela C Medeot
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Virginia S Schuck
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Romina B Andermatten
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Gisel S Miszczuk
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Nadia Ciriaci
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Ma Valeria Razori
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Ismael R Barosso
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Enrique J Sánchez Pozzi
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Marcelo G Roma
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Cecilia L Basiglio
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina
| | - Fernando A Crocenzi
- Instituto de Fisiología Experimental (IFISE), Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Departamento de Ciencias Fisiológicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Argentina.
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Gao XX, Lin S, Jiang PY, Ye MY, Chen W, Hu CX, Chen YH. Gestational cholestasis induced intrauterine growth restriction through triggering IRE1α-mediated apoptosis of placental trophoblast cells. FASEB J 2022; 36:e22388. [PMID: 35639049 DOI: 10.1096/fj.202101844rr] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/03/2021] [Revised: 05/19/2022] [Accepted: 05/23/2022] [Indexed: 11/11/2022]
Abstract
Epidemiological and animal experimental studies suggest an association between gestational cholestasis and intrauterine growth restriction (IUGR). Here, we explored the mechanism through which gestational cholestasis induced IUGR. To establish gestational cholestasis model, pregnant mice were subcutaneously injected with 17α-Ethynylestradiol (E2) on gestational day 13 (GD13)-GD17. Some pregnant mice were intraperitoneally injected with 4μ8C on GD13-GD17. The results found that the apoptosis of trophoblast cells was elevated in placentas of mice with gestational cholestasis and in deoxycholic acid (DCA)-treated human trophoblast cell lines and primary mouse trophoblast cells. Correspondingly, the levels of placental cleaved caspase-3 and Bax were increased, while placental Bcl2 level was decreased in mice with gestational cholestasis and in DCA-treated trophoblast cells. Further analysis found that placental IRE1α pathway was activated in mice with gestational cholestasis and in DCA-treated trophoblast cells. Interestingly, 4μ8C, an IRE1α RNase inhibitor, significantly inhibited caspase-3 activity and apoptosis of trophoblast cells in vivo and in vitro. Importantly, 4μ8C rescued gestational cholestasis-induced placental insufficiency and IUGR. Furthermore, a case-control study demonstrated that placental IRE1α and caspase-3 pathways were activated in cholestasis cases. Our results provide evidence that gestational cholestasis induces placental insufficiency and IUGR may be via triggering IRE1α-mediated apoptosis of placental trophoblast cells.
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Affiliation(s)
- Xing-Xing Gao
- School of Basic Medical Sciences, Anhui Medical University, Hefei, China
| | - Shuai Lin
- School of Basic Medical Sciences, Anhui Medical University, Hefei, China
| | - Pei-Ying Jiang
- School of Basic Medical Sciences, Anhui Medical University, Hefei, China
| | - Meng-Ying Ye
- School of Basic Medical Sciences, Anhui Medical University, Hefei, China
| | - Wei Chen
- School of Basic Medical Sciences, Anhui Medical University, Hefei, China
| | - Chuan-Xiang Hu
- School of Basic Medical Sciences, Anhui Medical University, Hefei, China
| | - Yuan-Hua Chen
- School of Basic Medical Sciences, Anhui Medical University, Hefei, China.,Key Laboratory of Environmental Toxicology of Anhui Higher Education Institutes, Anhui Medical University, Hefei, China
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Zu Y, Yang J, Zhang C, Liu D. The Pathological Mechanisms of Estrogen-Induced Cholestasis: Current Perspectives. Front Pharmacol 2021; 12:761255. [PMID: 34819862 PMCID: PMC8606790 DOI: 10.3389/fphar.2021.761255] [Citation(s) in RCA: 29] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2021] [Accepted: 10/26/2021] [Indexed: 12/31/2022] Open
Abstract
Estrogens are steroid hormones with a wide range of biological activities. The excess of estrogens can lead to decreased bile flow, toxic bile acid (BA) accumulation, subsequently causing intrahepatic cholestasis. Estrogen-induced cholestasis (EIC) may have increased incidence during pregnancy, and within women taking oral contraception and postmenopausal hormone replacement therapy, and result in liver injury, preterm birth, meconium-stained amniotic fluid, and intrauterine fetal death in pregnant women. The main pathogenic mechanisms of EIC may include deregulation of BA synthetic or metabolic enzymes, and BA transporters. In addition, impaired cell membrane fluidity, inflammatory responses and change of hepatocyte tight junctions are also involved in the pathogenesis of EIC. In this article, we review the role of estrogens in intrahepatic cholestasis, and outlined the mechanisms of EIC, providing a greater understanding of this disease.
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Affiliation(s)
- Yue Zu
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Jinyu Yang
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Chengliang Zhang
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Dong Liu
- Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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5
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Basiglio CL, Crocenzi FA, Sánchez Pozzi EJ, Roma MG. Oxidative Stress and Localization Status of Hepatocellular Transporters: Impact on Bile Secretion and Role of Signaling Pathways. Antioxid Redox Signal 2021; 35:808-831. [PMID: 34293961 DOI: 10.1089/ars.2021.0021] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
Significance: Most hepatopathies are primarily or secondarily cholestatic in nature. Oxidative stress (OS) is a frequent trait among them, and impairs the machinery to generate bile by triggering endocytic internalization of hepatocellular transporters, thus causing cholestasis. This is critical, since it leads to accelerated transporter degradation, which could explain the common post-transcriptional downregulation of transporter expression in human cholestatic diseases. Recent Advances: The mechanisms involved in OS-induced hepatocellular transporter internalization are being revealed. Filamentous actin (F-actin) cytoskeleton disorganization and/or detachment of crosslinking actin proteins that afford transporter stability have been characterized as causal factors. Activation of redox-sensitive signaling pathways leading to changes in phosphorylation status of these structures is involved, including Ca2+-mediated activation of "classical" and "novel" protein kinase C (PKC) isoforms or redox-signaling cascades downstream of NADPH oxidase. Critical Issues: Despite the well-known occurrence of hepatocellular transporter internalization in human hepatopathies, the cholestatic implications of this phenomenon have been overlooked. Accordingly, no specific treatment has been established in the clinical practice for its prevention/reversion. Future Directions: We need to improve our knowledge on the pro-oxidant triggering factors and the multiple signaling pathways that mediate this oxidative injury in each cholestatic hepatopathy, so as to envisage tailor-made therapeutic strategies for each case. Meanwhile, administration of antioxidants or heme oxygenase-1 induction to elevate the hepatocellular levels of the endogenous scavenger bilirubin are promising alternatives that need to be re-evaluated and implemented. They may complement current treatments in cholestasis aimed to enhance transcriptional carrier expression, by providing membrane stability to the newly synthesized carriers. Antioxid. Redox Signal. 35, 808-831.
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Affiliation(s)
- Cecilia L Basiglio
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, U.N.R., Rosario, Argentina
| | - Fernando A Crocenzi
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, U.N.R., Rosario, Argentina
| | - Enrique J Sánchez Pozzi
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, U.N.R., Rosario, Argentina
| | - Marcelo G Roma
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas, CONICET, U.N.R., Rosario, Argentina
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6
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Kralj T, Brouwer KLR, Creek DJ. Analytical and Omics-Based Advances in the Study of Drug-Induced Liver Injury. Toxicol Sci 2021; 183:1-13. [PMID: 34086958 PMCID: PMC8502468 DOI: 10.1093/toxsci/kfab069] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/12/2022] Open
Abstract
Drug-induced liver injury (DILI) is a significant clinical issue, affecting 1-1.5 million patients annually, and remains a major challenge during drug development-toxicity and safety concerns are the second-highest reason for drug candidate failure. The future prevalence of DILI can be minimized by developing a greater understanding of the biological mechanisms behind DILI. Both qualitative and quantitative analytical techniques are vital to characterizing and investigating DILI. In vitro assays are capable of characterizing specific aspects of a drug's hepatotoxic nature and multiplexed assays are capable of characterizing and scoring a drug's association with DILI. However, an even deeper insight into the perturbations to biological pathways involved in the mechanisms of DILI can be gained through the use of omics-based analytical techniques: genomics, transcriptomics, proteomics, and metabolomics. These omics analytical techniques can offer qualitative and quantitative insight into genetic susceptibilities to DILI, the impact of drug treatment on gene expression, and the effect on protein and metabolite abundance. This review will discuss the analytical techniques that can be applied to characterize and investigate the biological mechanisms of DILI and potential predictive biomarkers.
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Affiliation(s)
- Thomas Kralj
- Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
| | - Kim L R Brouwer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7569, USA
| | - Darren J Creek
- Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, VIC, Australia
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7
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Wei G, Cao J, Huang P, An P, Badlani D, Vaid KA, Zhao S, Wang DQH, Zhuo J, Yin L, Frassetto A, Markel A, Presnyak V, Gandham S, Hua S, Lukacs C, Finn PF, Giangrande PH, Martini PGV, Popov YV. Synthetic human ABCB4 mRNA therapy rescues severe liver disease phenotype in a BALB/c.Abcb4 -/- mouse model of PFIC3. J Hepatol 2021; 74:1416-1428. [PMID: 33340584 PMCID: PMC8188846 DOI: 10.1016/j.jhep.2020.12.010] [Citation(s) in RCA: 46] [Impact Index Per Article: 11.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/25/2020] [Revised: 12/08/2020] [Accepted: 12/10/2020] [Indexed: 12/14/2022]
Abstract
BACKGROUND & AIMS Progressive familial intrahepatic cholestasis type 3 (PFIC3) is a rare lethal autosomal recessive liver disorder caused by loss-of-function variations of the ABCB4 gene, encoding a phosphatidylcholine transporter (ABCB4/MDR3). Currently, no effective treatment exists for PFIC3 outside of liver transplantation. METHODS We have produced and screened chemically and genetically modified mRNA variants encoding human ABCB4 (hABCB4 mRNA) encapsulated in lipid nanoparticles (LNPs). We examined their pharmacological effects in a cell-based model and in a new in vivo mouse model resembling human PFIC3 as a result of homozygous disruption of the Abcb4 gene in fibrosis-susceptible BALB/c.Abcb4-/- mice. RESULTS We show that treatment with liver-targeted hABCB4 mRNA resulted in de novo expression of functional hABCB4 protein and restored phospholipid transport in cultured cells and in PFIC3 mouse livers. Importantly, repeated injections of the hABCB4 mRNA effectively rescued the severe disease phenotype in young Abcb4-/- mice, with rapid and dramatic normalisation of all clinically relevant parameters such as inflammation, ductular reaction, and liver fibrosis. Synthetic mRNA therapy also promoted favourable hepatocyte-driven liver regeneration to restore normal homeostasis, including liver weight, body weight, liver enzymes, and portal vein blood pressure. CONCLUSIONS Our data provide strong preclinical proof-of-concept for hABCB4 mRNA therapy as a potential treatment option for patients with PFIC3. LAY SUMMARY This report describes the development of an innovative mRNA therapy as a potential treatment for PFIC3, a devastating rare paediatric liver disease with no treatment options except liver transplantation. We show that administration of our mRNA construct completely rescues severe liver disease in a genetic model of PFIC3 in mice.
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MESH Headings
- ATP Binding Cassette Transporter, Subfamily B/administration & dosage
- ATP Binding Cassette Transporter, Subfamily B/deficiency
- ATP Binding Cassette Transporter, Subfamily B/genetics
- ATP Binding Cassette Transporter, Subfamily B/metabolism
- Animals
- Cholestasis, Intrahepatic/drug therapy
- Cholestasis, Intrahepatic/genetics
- Cholestasis, Intrahepatic/metabolism
- Disease Models, Animal
- Gene Deletion
- HEK293 Cells
- Homozygote
- Humans
- Liposomes/chemistry
- Liver/metabolism
- Mice
- Mice, Inbred BALB C
- Mice, Knockout
- Nanoparticle Drug Delivery System/chemistry
- Nanoparticles/chemistry
- Phenotype
- RNA, Messenger/administration & dosage
- RNA, Messenger/genetics
- Transfection
- Treatment Outcome
- ATP-Binding Cassette Sub-Family B Member 4
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Affiliation(s)
- Guangyan Wei
- Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; Department of Radiation Oncology, The First Affiliated Hospital, Sun Yat-sen University, Guangzhou, China
| | | | - Pinzhu Huang
- Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Ping An
- Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA; Division of Gastroenterology and Hepatology, Renmin Hospital of Wuhan University, Wuhan, Hubei, China
| | - Disha Badlani
- Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Kahini A Vaid
- Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - Shuangshuang Zhao
- Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
| | - David Q-H Wang
- Department of Medicine and Genetics, Division of Gastroenterology and Liver Diseases, Marion Bessin Liver Research Center, Einstein-Mount Sinai Diabetes Research Center, Albert Einstein College of Medicine, Bronx, NY, USA
| | - Jenny Zhuo
- Rare Diseases, Moderna Inc, Cambridge, MA, USA
| | - Ling Yin
- Rare Diseases, Moderna Inc, Cambridge, MA, USA
| | | | - Arianna Markel
- Rare Diseases, Moderna Inc, Cambridge, MA, USA; Stem Cell Program, Boston Children's Hospital, Boston, MA, USA
| | | | | | - Serenus Hua
- Analytical Development, Moderna Inc, Cambridge, MA, USA
| | | | | | | | | | - Yury V Popov
- Division of Gastroenterology and Hepatology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
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8
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Alamoudi JA, Li W, Gautam N, Olivera M, Meza J, Mukherjee S, Alnouti Y. Bile acid indices as biomarkers for liver diseases I: Diagnostic markers. World J Hepatol 2021; 13:433-455. [PMID: 33959226 PMCID: PMC8080550 DOI: 10.4254/wjh.v13.i4.433] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/05/2021] [Revised: 02/11/2021] [Accepted: 03/22/2021] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND Hepatobiliary diseases result in the accumulation of toxic bile acids (BA) in the liver, blood, and other tissues which may contribute to an unfavorable prognosis. AIM To discover and validate diagnostic biomarkers of cholestatic liver diseases based on the urinary BA profile. METHODS We analyzed urine samples by liquid chromatography-tandem mass spectrometry and compared the urinary BA profile between 300 patients with hepatobiliary diseases vs 103 healthy controls by statistical analysis. The BA profile was characterized using BA indices, which quantifies the composition, metabolism, hydrophilicity, and toxicity of the BA profile. BA indices have much lower inter- and intra-individual variability compared to absolute concentrations of BA. In addition, BA indices demonstrate high area under the receiver operating characteristic curves, and changes of BA indices are associated with the risk of having a liver disease, which demonstrates their use as diagnostic biomarkers for cholestatic liver diseases. RESULTS Total and individual BA concentrations were higher in all patients. The percentage of secondary BA (lithocholic acid and deoxycholic acid) was significantly lower, while the percentage of primary BA (chenodeoxycholic acid, cholic acid, and hyocholic acid) was markedly higher in patients compared to controls. In addition, the percentage of taurine-amidation was higher in patients than controls. The increase in the non-12α-OH BA was more profound than 12α-OH BA (cholic acid and deoxycholic acid) causing a decrease in the 12α-OH/ non-12α-OH ratio in patients. This trend was stronger in patients with more advanced liver diseases as reflected by the model for end-stage liver disease score and the presence of hepatic decompensation. The percentage of sulfation was also higher in patients with more severe forms of liver diseases. CONCLUSION BA indices have much lower inter- and intra-individual variability compared to absolute BA concentrations and changes of BA indices are associated with the risk of developing liver diseases.
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Affiliation(s)
- Jawaher Abdullah Alamoudi
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Wenkuan Li
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Nagsen Gautam
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Marco Olivera
- Department of Internal Medicine, College of Medicine, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Jane Meza
- Department of Biostatistics, College of Public Health, University of Nebraska Medical Center, Omaha, NE 68198, United States
| | - Sandeep Mukherjee
- Department of Internal Medicine, College of Medicine, Creighton University Medical Center, Omaha, NE 68124, United States
| | - Yazen Alnouti
- Department of Pharmaceutical Sciences, College of Pharmacy, University of Nebraska Medical Center, Omaha, NE 68198, United States.
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9
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Gijbels E, Pieters A, De Muynck K, Vinken M, Devisscher L. Rodent models of cholestatic liver disease: A practical guide for translational research. Liver Int 2021; 41:656-682. [PMID: 33486884 PMCID: PMC8048655 DOI: 10.1111/liv.14800] [Citation(s) in RCA: 40] [Impact Index Per Article: 10.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/07/2020] [Revised: 01/08/2021] [Accepted: 01/15/2021] [Indexed: 12/12/2022]
Abstract
Cholestatic liver disease denotes any situation associated with impaired bile flow concomitant with a noxious bile acid accumulation in the liver and/or systemic circulation. Cholestatic liver disease can be subdivided into different types according to its clinical phenotype, such as biliary atresia, drug-induced cholestasis, gallstone liver disease, intrahepatic cholestasis of pregnancy, primary biliary cholangitis and primary sclerosing cholangitis. Considerable effort has been devoted to elucidating underlying mechanisms of cholestatic liver injuries and explore novel therapeutic and diagnostic strategies using animal models. Animal models employed according to their appropriate applicability domain herein play a crucial role. This review provides an overview of currently available in vivo animal models, fit-for-purpose in modelling different types of cholestatic liver diseases. Moreover, a practical guide and workflow is provided which can be used for translational research purposes, including all advantages and disadvantages of currently available in vivo animal models.
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Affiliation(s)
- Eva Gijbels
- Department of In Vitro Toxicology and Dermato‐CosmetologyVrije Universiteit BrusselBrusselsBelgium,Gut‐Liver Immunopharmacology Unit, Basic and Applied Medical SciencesLiver Research Center GhentFaculty of Medicine and Health SciencesGhent UniversityGhentBelgium
| | - Alanah Pieters
- Department of In Vitro Toxicology and Dermato‐CosmetologyVrije Universiteit BrusselBrusselsBelgium
| | - Kevin De Muynck
- Gut‐Liver Immunopharmacology Unit, Basic and Applied Medical SciencesLiver Research Center GhentFaculty of Medicine and Health SciencesGhent UniversityGhentBelgium,Hepatology Research UnitInternal Medicine and PaediatricsLiver Research Center GhentFaculty of Medicine and Health SciencesGhent UniversityGhentBelgium
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato‐CosmetologyVrije Universiteit BrusselBrusselsBelgium
| | - Lindsey Devisscher
- Gut‐Liver Immunopharmacology Unit, Basic and Applied Medical SciencesLiver Research Center GhentFaculty of Medicine and Health SciencesGhent UniversityGhentBelgium
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10
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Tocchetti GN, Domínguez CJ, Zecchinati F, Arana MR, Rigalli JP, Ruiz ML, Villanueva SSM, Mottino AD. Intraluminal nutrients acutely strengthen rat intestinal MRP2 barrier function by a glucagon-like peptide-2-mediated mechanism. Acta Physiol (Oxf) 2020; 230:e13514. [PMID: 32476256 DOI: 10.1111/apha.13514] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Revised: 05/22/2020] [Accepted: 05/24/2020] [Indexed: 01/02/2023]
Abstract
AIM MRP2 is an intestinal ABC transporter that prevents the absorption of dietary xenobiotics. The aims of this work were: (1) to evaluate whether a short-term regulation of intestinal MRP2 barrier function takes place in vivo after luminal incorporation of nutrients and (2) to explore the underlying mechanism. METHODS MRP2 activity and localization were assessed in an in vivo rat model with preserved irrigation and innervation. Nutrients were administered into distal jejunum. After 30-minutes treatments, MRP2 activity was assessed in proximal jejunum by quantifying the transport of the model substrate 2,4-dinitrophenyl-S-glutathione. MRP2 localization was determined by quantitative confocal microscopy. Participation of extracellular mediators was evaluated using selective inhibitors and by immunoneutralization. Intracellular pathways were explored in differentiated Caco-2 cells. RESULTS Oleic acid, administered intraluminally at dietary levels, acutely stimulated MRP2 insertion into brush border membrane. This was associated with increased efflux activity and, consequently, enhanced barrier function. Immunoneutralization of the gut hormone glucagon-like peptide-2 (GLP-2) prevented oleic acid effect on MRP2, demonstrating the participation of this trophic factor as a main mediator. Further experiments using selective inhibitors demonstrated that extracellular adenosine synthesis and its subsequent binding to enterocytic A2B adenosine receptor (A2BAR) take place downstream GLP-2. Finally, studies in intestinal Caco-2 cells revealed the participation of A2BAR/cAMP/PKA intracellular pathway, ultimately leading to increased MRP2 localization in apical domains. CONCLUSION These findings reveal an on-demand, acute regulation of MRP2-associated barrier function, constituting a novel physiological mechanism of protection against the absorption of dietary xenobiotics in response to food intake.
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Affiliation(s)
- Guillermo N. Tocchetti
- Institute of Experimental Physiology (IFISE) National Scientific and Technical Research Council (CONICET)National University of Rosario (UNR) Rosario Argentina
- Department of Clinical Pharmacology and Pharmacoepidemiology University of Heidelberg Heidelberg Germany
| | - Camila J. Domínguez
- Institute of Experimental Physiology (IFISE) National Scientific and Technical Research Council (CONICET)National University of Rosario (UNR) Rosario Argentina
| | - Felipe Zecchinati
- Institute of Experimental Physiology (IFISE) National Scientific and Technical Research Council (CONICET)National University of Rosario (UNR) Rosario Argentina
| | - Maite R. Arana
- Institute of Experimental Physiology (IFISE) National Scientific and Technical Research Council (CONICET)National University of Rosario (UNR) Rosario Argentina
| | - Juan P. Rigalli
- Department of Clinical Pharmacology and Pharmacoepidemiology University of Heidelberg Heidelberg Germany
| | - María L. Ruiz
- Institute of Experimental Physiology (IFISE) National Scientific and Technical Research Council (CONICET)National University of Rosario (UNR) Rosario Argentina
| | - Silvina S. M. Villanueva
- Institute of Experimental Physiology (IFISE) National Scientific and Technical Research Council (CONICET)National University of Rosario (UNR) Rosario Argentina
| | - Aldo D. Mottino
- Institute of Experimental Physiology (IFISE) National Scientific and Technical Research Council (CONICET)National University of Rosario (UNR) Rosario Argentina
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11
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Domínguez CJ, Tocchetti GN, Rigalli JP, Mottino AD. Acute regulation of apical ABC transporters in the gut. Potential influence on drug bioavailability. Pharmacol Res 2020; 163:105251. [PMID: 33065282 DOI: 10.1016/j.phrs.2020.105251] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/13/2020] [Revised: 10/07/2020] [Accepted: 10/08/2020] [Indexed: 01/09/2023]
Abstract
The extensive intestinal surface offers an advantage regarding nutrient, ion and water absorptive capacity but also brings along a high exposition to xenobiotics, including drugs of therapeutic use and food contaminants. After absorption of these compounds by the enterocytes, apical ABC transporters play a key role in secreting them back to the intestinal lumen, hence acting as a transcellular barrier. Rapid and reversible modulation of their activity is a subject of increasing interest for pharmacologists. On the one hand, a decrease in transporter activity may result in increased absorption of therapeutic agents given orally. On the other hand, an increase in transporter activity would decrease their absorption and therapeutic efficacy. Although of less relevance, apical ABC transporters also contribute to disposition of drugs systemically administered. This review article summarizes the present knowledge on the mechanisms aimed to rapidly regulate the activity of the main apical ABC transporters of the gut: multidrug resistance protein 1 (MDR1), multidrug resistance-associated protein 2 (MRP2) and breast cancer resistance protein (BCRP). Regulation of these mechanisms by drugs, drug delivery systems, drug excipients and nutritional components are particularly considered. This information could provide the basis for controlled regulation of bioavailability of therapeutic agents and at the same time would help to prevent potential drug-drug interactions.
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Affiliation(s)
- Camila Juliana Domínguez
- Institute of Experimental Physiology, Faculty of Biochemical and Pharmaceutical Sciences, Rosario National University, Suipacha 570, 2000 Rosario, Argentina
| | - Guillermo Nicolás Tocchetti
- Institute of Experimental Physiology, Faculty of Biochemical and Pharmaceutical Sciences, Rosario National University, Suipacha 570, 2000 Rosario, Argentina; Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany
| | - Juan Pablo Rigalli
- Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120 Heidelberg, Germany; Department of Physiology, Radboud Institute for Molecular Life Sciences, Radboud University Medical Center, P.O. Box 9101, 6500 HB Nijmegen, the Netherlands
| | - Aldo Domingo Mottino
- Institute of Experimental Physiology, Faculty of Biochemical and Pharmaceutical Sciences, Rosario National University, Suipacha 570, 2000 Rosario, Argentina.
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12
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Spironolactone ameliorates lipopolysaccharide-induced cholestasis in rats by improving Mrp2 function: Role of transcriptional and post-transcriptional mechanisms. Life Sci 2020; 259:118352. [DOI: 10.1016/j.lfs.2020.118352] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/11/2020] [Revised: 08/14/2020] [Accepted: 08/24/2020] [Indexed: 12/15/2022]
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13
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Reduced Mrp2 surface availability as PI3Kγ-mediated hepatocytic dysfunction reflecting a hallmark of cholestasis in sepsis. Sci Rep 2020; 10:13110. [PMID: 32753644 PMCID: PMC7403153 DOI: 10.1038/s41598-020-69901-3] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2020] [Accepted: 06/30/2020] [Indexed: 12/14/2022] Open
Abstract
Sepsis-associated liver dysfunction manifesting as cholestasis is common during multiple organ failure. Three hepatocytic dysfunctions are considered as major hallmarks of cholestasis in sepsis: impairments of microvilli covering canalicular membranes, disruptions of tight junctions sealing bile-collecting canaliculae and disruptions of Mrp2-mediated hepatobiliary transport. PI3Kγ loss-of-function was suggested as beneficial in early sepsis. Yet, the PI3Kγ-regulated cellular processes in hepatocytes remained largely unclear. We analysed all three sepsis hallmarks for responsiveness to massive PI3K/Akt signalling and PI3Kγ loss-of-function, respectively. Surprisingly, neither microvilli nor tight junctions were strongly modulated, as shown by electron microscopical studies of mouse liver samples. Instead, quantitative electron microscopy proved that solely Mrp2 surface availability, i.e. the third hallmark, responded strongly to PI3K/Akt signalling. Mrp2 plasma membrane levels were massively reduced upon PI3K/Akt signalling. Importantly, Mrp2 levels at the plasma membrane of PI3Kγ KO hepatocytes remained unaffected upon PI3K/Akt signalling stimulation. The effect explicitly relied on PI3Kγ's enzymatic ability, as shown by PI3Kγ kinase-dead mice. Keeping the surface availability of the biliary transporter Mrp2 therefore is a cell biological process that may underlie the observation that PI3Kγ loss-of-function protects from hepatic excretory dysfunction during early sepsis and Mrp2 should thus take center stage in pharmacological interventions.
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14
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Ito K, Sjöstedt N, Brouwer KLR. Mechanistic Modeling of the Hepatic Disposition of Estradiol-17 β-Glucuronide in Sandwich-Cultured Human Hepatocytes. Drug Metab Dispos 2020; 48:116-122. [PMID: 31744810 PMCID: PMC6978695 DOI: 10.1124/dmd.119.088898] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2019] [Accepted: 11/12/2019] [Indexed: 11/22/2022] Open
Abstract
Estradiol-17β-glucuronide (E217G) is an estrogen metabolite that has cholestatic properties. In humans, circulating E217G is transported into hepatocytes by organic anion transporting polypeptides (OATPs) and is excreted into bile by multidrug-resistance associated protein 2 (MRP2). E217G is also a substrate of the basolateral efflux transporters MRP3 and MRP4, which translocate E217G from hepatocytes to blood. However, the contribution of basolateral efflux to hepatocyte disposition of E217G has not been evaluated previously. To address this question, E217G disposition was studied in sandwich-cultured human hepatocytes and mechanistic modeling was applied to calculate clearance values (mean ± S.D.) for uptake, intrinsic biliary excretion (CLint,bile) and intrinsic basolateral efflux (CLint,BL). The biliary excretion index of E217G was 45% ± 6%. The CLint,BL of E217G [0.18 ± 0.03 (ml/min)/g liver) was 1.6-fold higher than CLint,bile [0.11 ± 0.06 (ml/min)/g liver]. Simulations were performed to study the effects of increased CLint,BL and a concomitant decrease in CLint,bile on hepatic E217G exposure. Results demonstrated that increased CLint,BL can effectively reduce hepatocellular and biliary exposure to this potent cholestatic agent. Simulations also revealed that basolateral efflux can compensate for impaired biliary excretion and, vice versa, to avoid accumulation of E217G in hepatocytes. However, when both clearance processes are impaired by 90%, hepatocyte E217G exposure increases up to 10-fold. These data highlight the contribution of basolateral efflux transport, in addition to MRP2-mediated biliary excretion, to E217G disposition in human hepatocytes. This elimination route could be important, especially in cases where basolateral efflux is induced, such as cholestasis. SIGNIFICANCE STATEMENT: The disposition of the cholestatic estrogen metabolite estradiol-17β-glucuronide (E217G) was characterized in sandwich-cultured human hepatocytes. The intrinsic basolateral efflux clearance was estimated to be 1.6-fold higher than the intrinsic biliary excretion clearance, emphasizing the contribution of basolateral elimination in addition to biliary excretion. Simulations highlight how hepatocytes can effectively cope with increased E217G through the regulation of both basolateral and biliary transporters.
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Affiliation(s)
- Katsuaki Ito
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.I., N.S., K.L.R.B.); and DMPK Research Department, Teijin Pharma Limited, Hino, Tokyo, Japan (K.I.)
| | - Noora Sjöstedt
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.I., N.S., K.L.R.B.); and DMPK Research Department, Teijin Pharma Limited, Hino, Tokyo, Japan (K.I.)
| | - Kim L R Brouwer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina (K.I., N.S., K.L.R.B.); and DMPK Research Department, Teijin Pharma Limited, Hino, Tokyo, Japan (K.I.)
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15
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Marinelli RA, Vore M, Javitt NB. Hepatic Bile Formation: Canalicular Osmolarity and Paracellular and Transcellular Water Flow. J Pharmacol Exp Ther 2019; 371:713-717. [PMID: 31570498 DOI: 10.1124/jpet.119.261115] [Citation(s) in RCA: 15] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/08/2019] [Accepted: 09/25/2019] [Indexed: 03/08/2025] Open
Abstract
The purpose of this minireview is to show that a new paradigm is developing regarding hepatic bile flow. The focus thus far has been on carrier-mediated transport of bile acids and other solutes, such as glutathione, which create an osmotic gradient for the transcellular and paracellular flow of water into canaliculi. In addition to the physicochemical properties of bile acids, which govern the osmotic gradient, data now exist showing that the tight junctions governing paracellular water flow and Aquaporin-8 water channels governing transcellular water flow are regulated independently. Thus, the rate of water flow into the canaliculus in response to bile acid transport is variable and determines canalicular bile acid concentration, which affects the production and solubilization of cholesterol-lecithin vesicles. These new considerations modify thinking regarding the occurrence of cholestasis and its progression and reorient the design of experimental studies that can distinguish the different determinants of bile flow. SIGNIFICANCE STATEMENT: The paradigm that water flow into the canaliculus is determined only by the rate of carrier-mediated transport has been challenged recently by the changes that occur in hepatic bile composition in the Claudin-2 knockout mouse and with the cholestatic effect of estradiol 17β-d-glucuronide. Thus, a respective reduction in paracellular or transcellular canalicular water flow, probably via Aquaporin 8, has no significant effect on bile acid excretion.
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Affiliation(s)
- Raul A Marinelli
- Instituto de Fisiologia Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina (R.A.M.); Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, Kentucky (M.V.); and Department of Medicine and Pediatrics, New York University School of Medicine, New York, New York (N.B.J.)
| | - Mary Vore
- Instituto de Fisiologia Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina (R.A.M.); Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, Kentucky (M.V.); and Department of Medicine and Pediatrics, New York University School of Medicine, New York, New York (N.B.J.)
| | - Norman B Javitt
- Instituto de Fisiologia Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario, Rosario, Santa Fe, Argentina (R.A.M.); Department of Toxicology and Cancer Biology, College of Medicine, University of Kentucky, Lexington, Kentucky (M.V.); and Department of Medicine and Pediatrics, New York University School of Medicine, New York, New York (N.B.J.)
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16
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Deferm N, De Vocht T, Qi B, Van Brantegem P, Gijbels E, Vinken M, de Witte P, Bouillon T, Annaert P. Current insights in the complexities underlying drug-induced cholestasis. Crit Rev Toxicol 2019; 49:520-548. [PMID: 31589080 DOI: 10.1080/10408444.2019.1635081] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
Drug-induced cholestasis (DIC) poses a major challenge to the pharmaceutical industry and regulatory agencies. It causes both drug attrition and post-approval withdrawal of drugs. DIC represents itself as an impaired secretion and flow of bile, leading to the pathological hepatic and/or systemic accumulation of bile acids (BAs) and their conjugate bile salts. Due to the high number of mechanisms underlying DIC, predicting a compound's cholestatic potential during early stages of drug development remains elusive. A profound understanding of the different molecular mechanisms of DIC is, therefore, of utmost importance. Although many knowledge gaps and caveats still exist, it is generally accepted that alterations of certain hepatobiliary membrane transporters and changes in hepatocellular morphology may cause DIC. Consequently, liver models, which represent most of these mechanisms, are valuable tools to predict human DIC. Some of these models, such as membrane-based in vitro models, are exceptionally well-suited to investigate specific mechanisms (i.e. transporter inhibition) of DIC, while others, such as liver slices, encompass all relevant biological processes and, therefore, offer a better representation of the in vivo situation. In the current review, we highlight the principal molecular mechanisms associated with DIC and offer an overview and critical appraisal of the different liver models that are currently being used to predict the cholestatic potential of drugs.
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Affiliation(s)
- Neel Deferm
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Tom De Vocht
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Bing Qi
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Pieter Van Brantegem
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Eva Gijbels
- Entity of In Vitro Toxicology and Dermato-Cosmetology, Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, Brussels, Belgium
| | - Mathieu Vinken
- Entity of In Vitro Toxicology and Dermato-Cosmetology, Department of Pharmaceutical and Pharmacological Sciences, Vrije Universiteit Brussel, Brussels, Belgium
| | - Peter de Witte
- Laboratory for Molecular Biodiscovery, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, Leuven, Belgium
| | - Thomas Bouillon
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
| | - Pieter Annaert
- Department of Pharmaceutical and Pharmacological Sciences, Drug Delivery and Disposition, KU Leuven, Leuven, Belgium
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Anticholestatic mechanisms of ursodeoxycholic acid in lipopolysaccharide-induced cholestasis. Biochem Pharmacol 2019; 168:48-56. [DOI: 10.1016/j.bcp.2019.06.009] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/27/2019] [Accepted: 06/10/2019] [Indexed: 12/14/2022]
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18
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Improved hepatic MRP2/ABCC2 transport activity in LPS-induced cholestasis by aquaporin-1 gene transfer. Biochimie 2019; 165:179-182. [PMID: 31377196 DOI: 10.1016/j.biochi.2019.07.027] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2019] [Accepted: 07/30/2019] [Indexed: 12/23/2022]
Abstract
Multidrug resistance-associated protein 2 (MRP2/ABCC2), a hepatocyte canalicular transporter involved in bile secretion, is downregulated in cholestasis triggered by lipopolysaccharide. The human aquaporin-1 (hAQP1) adenovirus-mediated gene transfer to liver improves cholestasis by incompletely defined mechanisms. Here we found that hAQP1 did not affect MRP2/ABCC2 expression, but significantly increased its transport activity assessed in situ with endogenous and exogenous substrates, likely by a hAQP1-induced increase in canalicular membrane cholesterol amount. Our results suggest that hAQP1-induced MRP2/ABCC2 activation contributes to the cholestasis improvement.
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19
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Roma MG, Barosso IR, Miszczuk GS, Crocenzi FA, Pozzi EJS. Dynamic Localization of Hepatocellular Transporters: Role in Biliary Excretion and Impairment in Cholestasis. Curr Med Chem 2019; 26:1113-1154. [DOI: 10.2174/0929867325666171205153204] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2017] [Revised: 09/06/2017] [Accepted: 09/07/2017] [Indexed: 12/25/2022]
Abstract
Bile flow generation is driven by the vectorial transfer of osmotically active compounds from sinusoidal blood into a confined space, the bile canaliculus. Hence, localization of hepatocellular transporters relevant to bile formation is crucial for bile secretion. Hepatocellular transporters are localized either in the plasma membrane or in recycling endosomes, from where they can be relocated to the plasma membrane on demand, or endocytosed when the demand decreases. The balance between endocytic internalization/ exocytic targeting to/from this recycling compartment is therefore the main determinant of the hepatic capability to generate bile, and to dispose endo- and xenobiotics. Furthermore, the exacerbated endocytic internalization is a common pathomechanisms in both experimental and human cholestasis; this results in bile secretory failure and, eventually, posttranslational transporter downregulation by increased degradation. This review summarizes the proposed structural mechanisms accounting for this pathological condition (e.g., alteration of function, localization or expression of F-actin or F-actin/transporter cross-linking proteins, and switch to membrane microdomains where they can be readily endocytosed), and the mediators implicated (e.g., triggering of “cholestatic” signaling transduction pathways). Lastly, we discussed the efficacy to counteract the cholestatic failure induced by transporter internalization of a number of therapeutic experimental approaches based upon the use of compounds that trigger exocytic targetting of canalicular transporters (e.g., cAMP, tauroursodeoxycholate). This therapeutics may complement treatments aimed to transcriptionally improve transporter expression, by affording proper localization and membrane stability to the de novo synthesized transporters.
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Affiliation(s)
- Marcelo G. Roma
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Ismael R. Barosso
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Gisel S. Miszczuk
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Fernando A. Crocenzi
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
| | - Enrique J. Sánchez Pozzi
- Instituto de Fisiologia Experimental (IFISE) - Facultad de Ciencias Bioquimicas y Farmaceuticas (CONICET - U.N.R.), S2002LRL, Rosario, Argentina
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20
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Gijbels E, Vilas-Boas V, Deferm N, Devisscher L, Jaeschke H, Annaert P, Vinken M. Mechanisms and in vitro models of drug-induced cholestasis. Arch Toxicol 2019; 93:1169-1186. [PMID: 30972450 DOI: 10.1007/s00204-019-02437-2] [Citation(s) in RCA: 33] [Impact Index Per Article: 5.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2018] [Accepted: 04/02/2019] [Indexed: 12/12/2022]
Abstract
Cholestasis underlies one of the major manifestations of drug-induced liver injury. Drug-induced cholestatic liver toxicity is a complex process, as it can be triggered by a variety of factors that induce 2 types of biological responses, namely a deteriorative response, caused by bile acid accumulation, and an adaptive response, aimed at removing the accumulated bile acids. Several key events in both types of responses have been characterized in the past few years. In parallel, many efforts have focused on the development and further optimization of experimental cell culture models to predict the occurrence of drug-induced cholestatic liver toxicity in vivo. In this paper, a state-of-the-art overview of mechanisms and in vitro models of drug-induced cholestatic liver injury is provided.
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Affiliation(s)
- Eva Gijbels
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Vânia Vilas-Boas
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium
| | - Neel Deferm
- Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, O&N2, Herestraat 49, Bus 921, 3000, Leuven, Belgium
| | - Lindsey Devisscher
- Basic and Applied Medical Sciences, Gut-Liver Immunopharmacology Unit, Faculty of Medicine and Health Sciences, Ghent University, C. Heymanslaan 10, 9000, Ghent, Belgium
| | - Hartmut Jaeschke
- Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Boulevard, MS 1018, Kansas City, KS, 66160, USA
| | - Pieter Annaert
- Drug Delivery and Disposition, Department of Pharmaceutical and Pharmacological Sciences, KU Leuven, O&N2, Herestraat 49, Bus 921, 3000, Leuven, Belgium
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Laarbeeklaan 103, 1090, Brussels, Belgium.
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Meng LL, Qiu JW, Lin WX, Song YZ. [Clinical features and ABCC2 genotypic analysis of an infant with Dubin-Johnson syndrome]. ZHONGGUO DANG DAI ER KE ZA ZHI = CHINESE JOURNAL OF CONTEMPORARY PEDIATRICS 2019. [PMID: 30675866 DOI: 10.7499/j.issn.1008-8830.2019.01.012] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Dubin-Johnson syndrome (DJS) is an autosomal recessive disorder resulting from biallelic mutations of ABCC2 gene, with long-term or intermittent conjugated hyperbilirubinemia being the main clinical manifestation. This paper aims to report the clinical features and ABCC2 genotypes of an infant with DJS. A 9.5-month-old male infant was referred to the hospital due to abnormal liver function discovered over 9 months. The major clinical presentation was prolonged jaundice since neonatal period. A series of biochemistry analysis revealed markedly elevated total bilirubin, conjugated bilirubin and total bile acids. The patient had been managed in different hospitals, but the therapeutic effects were unsatisfactory due to undetermined etiology. Physical examination revealed jaundiced skin and sclera, and a palpable liver 3 cm below the right subcostal margin with medium texture. The spleen was not enlarged. Genetic analysis revealed a splice-site variant c.3988-2A>T and a nonsense variant c.3825C>G (p.Y1275X) in the ABCC2 gene of the infant, which were inherited from his mother and father respectively. The former had not been previously reported. Then ursodeoxycholic acid and phenobarbital were given orally. Half a month later, as a result, his jaundice disappeared and the biochemistry indices improved. However, the long-term outcome needs to be observed. Literature review revealed that neonates/infants with DJS presented with cholestatic jaundice soon after birth as the major clinical feature, and the ABCC2 variants exhibited marked heterogeneity.
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Affiliation(s)
- Lu-Lu Meng
- Department of Pediatrics, First Affiliated Hospital of Jinan University, Guangzhou 510632, China.
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22
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Abstract
Cholestasis can be defined as any situation of impaired bile secretion with concomitant accumulation of bile acids in the liver or in the systemic circulation. A variety of factors may evoke cholestasis, including genetic disorders, metabolic pathologies, infectious diseases, immunogenic stimuli, and drugs. Drug-induced cholestasis is a mechanistically complex process. At least three triggering factors of drug-induced cholestasis have been described, including effects on drug transporters, various hepatocellular changes, and altered bile canaliculi dynamics. These stimuli induce two cellular responses, each typified by a number of key events, namely a deteriorative response activated by bile acid accumulation and an adaptive response aimed at decreasing the uptake and increasing the export of bile acids into and from the liver, respectively. The mechanistic scenario of drug-induced cholestasis is described in this chapter.
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Affiliation(s)
- Eva Gijbels
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium
| | - Mathieu Vinken
- Department of In Vitro Toxicology and Dermato-Cosmetology, Vrije Universiteit Brussel, Brussels, Belgium.
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23
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In vivo antigenotoxic activity of Diplotaxis tenuifolia against cyclophosphamide-induced DNA damage: Relevance of modulation of hepatic ABC efflux transporters. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2018; 836:72-78. [PMID: 30442348 DOI: 10.1016/j.mrgentox.2018.06.006] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/05/2017] [Revised: 04/20/2018] [Accepted: 06/01/2018] [Indexed: 02/07/2023]
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24
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Fernández-Murga ML, Petrov PD, Conde I, Castell JV, Goméz-Lechón MJ, Jover R. Advances in drug-induced cholestasis: Clinical perspectives, potential mechanisms and in vitro systems. Food Chem Toxicol 2018; 120:196-212. [PMID: 29990576 DOI: 10.1016/j.fct.2018.07.017] [Citation(s) in RCA: 20] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/09/2018] [Revised: 07/05/2018] [Accepted: 07/06/2018] [Indexed: 12/12/2022]
Abstract
Despite growing research, drug-induced liver injury (DILI) remains a serious issue of increasing importance to the medical community that challenges health systems, pharmaceutical industries and drug regulatory agencies. Drug-induced cholestasis (DIC) represents a frequent manifestation of DILI in humans, which is characterised by an impaired canalicular bile flow resulting in a detrimental accumulation of bile constituents in blood and tissues. From a clinical point of view, cholestatic DILI generates a wide spectrum of presentations and can be a diagnostic challenge. The drug classes mostly associated with DIC are anti-infectious, anti-diabetic, anti-inflammatory, psychotropic and cardiovascular agents, steroids, and other miscellaneous drugs. The molecular mechanisms of DIC have been investigated since the 1980s but they remain debatable. It is recognised that altered expression and/or function of hepatobiliary membrane transporters underlies some forms of cholestasis, and this and other concomitant mechanisms are very likely in DIC. Deciphering these processes may pave the ways for diagnosis, prognosis and prevention, for which currently major gaps and caveats exist. In this review, we summarise recent advances in the field of DIC, including clinical aspects, the potential mechanisms postulated so far and the in vitro systems that can be useful to investigate and identify new cholestatic drugs.
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Affiliation(s)
- M Leonor Fernández-Murga
- Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain
| | - Petar D Petrov
- Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Madrid, Spain
| | - Isabel Conde
- Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain
| | - Jose V Castell
- Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Madrid, Spain; Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Spain
| | - M José Goméz-Lechón
- Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Madrid, Spain.
| | - Ramiro Jover
- Unidad de Hepatología Experimental, Instituto de Investigación Sanitaria La Fe (IIS La Fe), Valencia, Spain; Centro de Investigación Biomédica en Red de Enfermedades Hepáticas y Digestivas (CIBEREHD), Madrid, Spain; Departamento de Bioquímica y Biología Molecular, Facultad de Medicina, Universidad de Valencia, Spain.
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25
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Tocchetti GN, Domínguez CJ, Zecchinati F, Arana MR, Ruiz ML, Villanueva SSM, Mottino AD, Weiss J, Rigalli JP. Inhibition of multidrug resistance-associated protein 2 (MRP2) activity by the contraceptive nomegestrol acetate in HepG2 and Caco-2 cells. Eur J Pharm Sci 2018; 122:205-213. [DOI: 10.1016/j.ejps.2018.07.017] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2018] [Revised: 07/03/2018] [Accepted: 07/05/2018] [Indexed: 12/28/2022]
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26
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Park SW, Webster CRL, Anwer MS. Mechanism of inhibition of taurolithocholate-induced retrieval of plasma membrane MRP2 by cyclic AMP and tauroursodeoxycholate. Physiol Rep 2018; 5. [PMID: 29192063 PMCID: PMC5727282 DOI: 10.14814/phy2.13529] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/27/2017] [Revised: 11/03/2017] [Accepted: 11/07/2017] [Indexed: 12/26/2022] Open
Abstract
Taurolithocholate (TLC) produces cholestasis by inhibiting biliary solute secretion in part by retrieving MRP2 from the plasma membrane (PM). Tauroursodeoxycholate (TUDC) and cAMP reverse TLC‐induced cholestasis by inhibiting TLC‐induced retrieval of MRP2. However, cellular mechanisms for this reversal are incompletely understood. Recently, we reported that TLC decreases PM‐MRP2 by activating PKCε followed by phosphorylation of myristoylated alanine‐rich C kinase substrate (MARCKS). Thus, cAMP and TUDC may reverse TLC‐induced cholestasis by inhibiting the TLC/PKCε/MARCKS phosphorylation pathway. We tested this hypothesis by determining whether TUDC and/or cAMP inhibit TLC‐induced activation of PKCε and phosphorylation of MARCKS. Studies were conducted in HuH‐NTCP cell line and rat hepatocytes. Activation of PKCε was determined from the translocation of PKCε to PM using a biotinylation method. Phosphorylation of MARCKS was determined by immunoblotting with a phospho‐MARCKS antibody. TLC, but not cAMP and TUDC, activated PKCε and increased MARCKS phosphorylation in HuH‐NTCP as well in rat hepatocytes. Treatment with TUDC or cAMP inhibited TLC‐induced activation of PKCε and increases in MARCKS phosphorylation in both cell types. Based on these results, we conclude that the reversal of TLC‐induced cholestasis by cAMP and TUDC involves, at least in part, inhibition of TLC‐mediated activation of the PKCε/MARCKS phosphorylation pathway.
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Affiliation(s)
- Se Won Park
- Department of Biomedical Sciences, Cummings School of Veterinary Medicine at Tufts University, 200 Westboro Road, North Grafton, Massachusetts, USA
| | - Cynthia R L Webster
- Department of Clinical Sciences, Cummings School of Veterinary Medicine at Tufts University, 200 Westboro Road, North Grafton, Massachusetts, USA
| | - Mohammed S Anwer
- Department of Biomedical Sciences, Cummings School of Veterinary Medicine at Tufts University, 200 Westboro Road, North Grafton, Massachusetts, USA
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Crawford RR, Potukuchi PK, Schuetz EG, Schuetz JD. Beyond Competitive Inhibition: Regulation of ABC Transporters by Kinases and Protein-Protein Interactions as Potential Mechanisms of Drug-Drug Interactions. Drug Metab Dispos 2018; 46:567-580. [PMID: 29514827 PMCID: PMC5896366 DOI: 10.1124/dmd.118.080663] [Citation(s) in RCA: 40] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/26/2018] [Accepted: 03/02/2018] [Indexed: 12/14/2022] Open
Abstract
ATP-binding cassette (ABC) transporters are transmembrane efflux transporters mediating the extrusion of an array of substrates ranging from amino acids and lipids to xenobiotics, and many therapeutic compounds, including anticancer drugs. The ABC transporters are also recognized as important contributors to pharmacokinetics, especially in drug-drug interactions and adverse drug effects. Drugs and xenobiotics, as well as pathologic conditions, can influence the transcription of ABC transporters, or modify their activity or intracellular localization. Kinases can affect the aforementioned processes for ABC transporters as do protein interactions. In this review, we focus on the ABC transporters ABCB1, ABCB11, ABCC1, ABCC4, and ABCG2 and illustrate how kinases and protein-protein interactions affect these transporters. The clinical relevance of these factors is currently unknown; however, these examples suggest that our understanding of drug-drug interactions will benefit from further knowledge of how kinases and protein-protein interactions affect ABC transporters.
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Affiliation(s)
- Rebecca R Crawford
- Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - Praveen K Potukuchi
- Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - Erin G Schuetz
- Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee
| | - John D Schuetz
- Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee
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28
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Ursodeoxycholate Restores Biliary Excretion of Methotrexate in Rats with Ethinyl Estradiol Induced-Cholestasis by Restoring Canalicular Mrp2 Expression. Int J Mol Sci 2018; 19:ijms19041120. [PMID: 29642532 PMCID: PMC5979538 DOI: 10.3390/ijms19041120] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2018] [Revised: 03/29/2018] [Accepted: 04/08/2018] [Indexed: 12/12/2022] Open
Abstract
The in vivo relevance of ursodeoxycholate (UDCA) treatment (100 mg/kg/day, per oral tid for 5 days before cholestasis induction followed by the same dosing for 5 days) on hepatic function was investigated in rats with 17α-ethinylestradiol (EE, 10 mg/kg, subcutaneous for 5 days)-induced experimental cholestasis. The bile flow rate and the expression level of hepatic multidrug resistance-associated protein 2 (Mrp 2) that were decreased in cholestasis were restored after UDCA treatment. Consistent with this, the biliary excretion clearance (CLexc,bile) of a representative Mrp2 substrate—methotrexate (MTX)—was decreased in cholestatic rats but was restored after UDCA treatment. Consequently, the plasma concentrations of MTX, which were increased by cholestasis, were decreased to control levels by UDCA treatment. Thus, the restoration of CLexc,bile appears to be associated with the increase in Mrp2 expression on the canalicular membrane by UDCA treatment followed by Mrp2-mediated biliary excretion of MTX. On the other hand, the hepatic uptake clearance (CLup,liver) of MTX was unchanged by cholestasis or UDCA treatment, suggestive of the absence of any association between the uptake process and the overall biliary excretion of MTX. Since UDCA has been known to induce the expression of canalicular MRP2 in humans, UDCA treatment might be effective in humans to maintain or accelerate the hepatobiliary elimination of xenobiotics or metabolic conjugates that are MRP2 substrates.
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29
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Robichaux WG, Cheng X. Intracellular cAMP Sensor EPAC: Physiology, Pathophysiology, and Therapeutics Development. Physiol Rev 2018; 98:919-1053. [PMID: 29537337 PMCID: PMC6050347 DOI: 10.1152/physrev.00025.2017] [Citation(s) in RCA: 142] [Impact Index Per Article: 20.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/21/2017] [Revised: 09/05/2017] [Accepted: 09/06/2017] [Indexed: 12/13/2022] Open
Abstract
This review focuses on one family of the known cAMP receptors, the exchange proteins directly activated by cAMP (EPACs), also known as the cAMP-regulated guanine nucleotide exchange factors (cAMP-GEFs). Although EPAC proteins are fairly new additions to the growing list of cAMP effectors, and relatively "young" in the cAMP discovery timeline, the significance of an EPAC presence in different cell systems is extraordinary. The study of EPACs has considerably expanded the diversity and adaptive nature of cAMP signaling associated with numerous physiological and pathophysiological responses. This review comprehensively covers EPAC protein functions at the molecular, cellular, physiological, and pathophysiological levels; and in turn, the applications of employing EPAC-based biosensors as detection tools for dissecting cAMP signaling and the implications for targeting EPAC proteins for therapeutic development are also discussed.
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Affiliation(s)
- William G Robichaux
- Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center , Houston, Texas
| | - Xiaodong Cheng
- Department of Integrative Biology and Pharmacology, Texas Therapeutics Institute, The Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science Center , Houston, Texas
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30
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Wang L, Wang J, Cai W, Shi Y, Zhou X, Guo G, Guo C, Huang X, Han Z, Zhang S, Ma S, Zhou X, Fan D, Gershwin ME, Han Y. A Critical Evaluation of Liver Pathology in Humans with Danon Disease and Experimental Correlates in a Rat Model of LAMP-2 Deficiency. Clin Rev Allergy Immunol 2018; 53:105-116. [PMID: 28124283 DOI: 10.1007/s12016-017-8598-3] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/14/2023]
Abstract
Danon disease is a genetic deficiency in lysosome-associated membrane protein 2 (LAMP-2), a highly glycosylated constituent of the lysosomal membrane and characterized by a cardiomyopathy, skeletal muscle myopathy, and cognitive impairment. Patients, however, often manifest hepatic abnormalities, but liver function has not been well evaluated and the syndrome is relatively uncommon. Hence, we have taken advantage of a rat that has been deleted of LAMP-2 to study the relative role of LAMP-2 on liver function. Interestingly, rats deficient in LAMP-2 develop a striking increase in serum alkaline phosphatase (ALP) and a decrease in bile flow compared with wild-type littermates. Importantly and by ultrastructural analysis, deficient rats manifest dilated canaliculi that lack microvilli with evidence of bile-containing bodies. Moreover, following bile duct ligation, LAMP-2-deficient rats develop rapid and severe evidence of advanced cholestasis, with an increase in serum bilirubin, as early as 6 h later. In wild-type control rats, multidrug resistance-associated protein 2 (Mrp2) normally concentrates at the bile canalicular membranes to secrete conjugated bilirubin into bile. However, in LAMP-2y/- rats, Mrp2 was detected in hepatocytes compared with other canalicular proteins including P-glycoproteins, dipeptidyl peptidase IV (CD26), and aminopeptidase (CD13). Our data further suggest that LAMP-2 interacts with the membrane cytoskeletal proteins radixin and F-actin in determining the localization of integral membrane proteins.
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Affiliation(s)
- Lu Wang
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Jingbo Wang
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Weile Cai
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Yongquan Shi
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Xinmin Zhou
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Guanya Guo
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Changcun Guo
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Xiaofeng Huang
- Center of Electron Microscope, The Fourth Military Medical University, Xi'an, Shaanxi Province, 710032, China
| | - Zheyi Han
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Shuai Zhang
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Shuoyi Ma
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Xia Zhou
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - Daiming Fan
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China
| | - M Eric Gershwin
- Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, 451 Health Sciences Drive, Suite 6510, Davis, CA, 95616, USA.
| | - Ying Han
- Xijing Hospital of Digestive Diseases, The Fourth Military Medical University, 127 Changle West Road, Xi'an, Shaanxi Province, 710032, China.
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31
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Dzierlenga AL, Cherrington NJ. Misregulation of membrane trafficking processes in human nonalcoholic steatohepatitis. J Biochem Mol Toxicol 2018; 32:e22035. [PMID: 29341352 DOI: 10.1002/jbt.22035] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/19/2017] [Revised: 12/21/2017] [Accepted: 12/27/2017] [Indexed: 12/17/2022]
Abstract
Nonalcoholic steatohepatitis (NASH) remodels the expression and function of genes and proteins that are critical for drug disposition. This study sought to determine whether disruption of membrane protein trafficking pathways in human NASH contributes to altered localization of multidrug resistance-associated protein 2 (MRP2). A comprehensive immunoblot analysis assessed the phosphorylation, membrane translocation, and expression of transporter membrane insertion regulators, including several protein kinases (PK), radixin, MARCKS, and Rab11. Radixin exhibited a decreased phosphorylation and total expression, whereas Rab11 had an increased membrane localization. PKCδ, PKCα, and PKA had increased membrane activation, whereas PKCε had a decreased phosphorylation and membrane expression. Radixin dephosphorylation may activate MRP2 membrane retrieval in NASH; however, the activation of Rab11/PKCδ and PKA/PKCα suggest an activation of membrane insertion pathways as well. Overall these data suggest an altered regulation of protein trafficking in human NASH, although other processes may be involved in the regulation of MRP2 localization.
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Affiliation(s)
- Anika L Dzierlenga
- Department of Pharmacology & Toxicology, University of Arizona, Tucson, AZ, USA
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32
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Activation of insulin-like growth factor 1 receptor participates downstream of GPR30 in estradiol-17β-D-glucuronide-induced cholestasis in rats. Arch Toxicol 2017; 92:729-744. [PMID: 29090346 DOI: 10.1007/s00204-017-2098-3] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2017] [Accepted: 10/17/2017] [Indexed: 01/01/2023]
Abstract
Estradiol-17β-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.
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33
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Tocchetti GN, Arias A, Arana MR, Rigalli JP, Domínguez CJ, Zecchinati F, Ruiz ML, Villanueva SSM, Mottino AD. Acute regulation of multidrug resistance-associated protein 2 localization and activity by cAMP and estradiol-17β-D-glucuronide in rat intestine and Caco-2 cells. Arch Toxicol 2017; 92:777-788. [PMID: 29052767 DOI: 10.1007/s00204-017-2092-9] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/18/2017] [Accepted: 10/05/2017] [Indexed: 01/27/2023]
Abstract
Multidrug resistance-associated protein 2 (MRP2) is an ATP-dependent transporter expressed at the brush border membrane of the enterocyte that confers protection against absorption of toxicants from foods or bile. Acute, short-term regulation of intestinal MRP2 activity involving changes in its apical membrane localization was poorly explored. We evaluated the effects of dibutyryl-cAMP (db-cAMP), a permeable analog of cAMP, and estradiol-17β-D-glucuronide (E217G), an endogenous derivative of estradiol, on MRP2 localization and activity using isolated rat intestinal sacs and Caco-2 cells, a model of human intestinal epithelium. Changes in MRP2 localization were studied by Western blotting of plasma membrane (PM) vs. intracellular membrane (IM) fractions in both experimental models, and additionally, by confocal microscopy in Caco-2 cells. After 30 min of exposure, db-cAMP-stimulated sorting of MRP2 from IM to PM both in rat jejunum and Caco-2 cells at 10 and 100 µM concentrations, respectively, with increased excretion of the model substrate 2,4-dinitrophenyl-S-glutathione. In contrast, E217G (400 µM) induced internalization of MRP2 together with impairment of transport activity. Confocal microscopy analysis performed in Caco-2 cells confirmed Western blot results. In the particular case of E217G, MRP2 exhibited an unusual pattern of staining compatible with endocytic vesiculation. Use of selective inhibitors demonstrated the participation of cAMP-dependent protein kinase and classic calcium-dependent protein kinase C in db-cAMP and E217G effects, respectively. We conclude that localization of MRP2 in intestine may be subjected to a dynamic equilibrium between plasma membrane and intracellular domains, thus allowing for rapid regulation of MRP2 function.
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Affiliation(s)
- Guillermo Nicolás Tocchetti
- Instituto de Fisiología Experimental (IFISE-CONICET), Suipacha 570, 2000, Rosario, Argentina.,Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany
| | - Agostina Arias
- Instituto de Fisiología Experimental (IFISE-CONICET), Suipacha 570, 2000, Rosario, Argentina
| | - Maite Rocío Arana
- Instituto de Fisiología Experimental (IFISE-CONICET), Suipacha 570, 2000, Rosario, Argentina
| | - Juan Pablo Rigalli
- Instituto de Fisiología Experimental (IFISE-CONICET), Suipacha 570, 2000, Rosario, Argentina.,Department of Clinical Pharmacology and Pharmacoepidemiology, University of Heidelberg, Im Neuenheimer Feld 410, 69120, Heidelberg, Germany
| | | | - Felipe Zecchinati
- Instituto de Fisiología Experimental (IFISE-CONICET), Suipacha 570, 2000, Rosario, Argentina
| | - María Laura Ruiz
- Instituto de Fisiología Experimental (IFISE-CONICET), Suipacha 570, 2000, Rosario, Argentina
| | | | - Aldo Domingo Mottino
- Instituto de Fisiología Experimental (IFISE-CONICET), Suipacha 570, 2000, Rosario, Argentina.
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34
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Thakkar N, Slizgi JR, Brouwer KLR. Effect of Liver Disease on Hepatic Transporter Expression and Function. J Pharm Sci 2017; 106:2282-2294. [PMID: 28465155 DOI: 10.1016/j.xphs.2017.04.053] [Citation(s) in RCA: 73] [Impact Index Per Article: 9.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2017] [Revised: 04/20/2017] [Accepted: 04/21/2017] [Indexed: 12/27/2022]
Abstract
Liver disease can alter the disposition of xenobiotics and endogenous substances. Regulatory agencies such as the Food and Drug Administration and the European Medicines Evaluation Agency recommend, if possible, studying the effect of liver disease on drugs under development to guide specific dose recommendations in these patients. Although extensive research has been conducted to characterize the effect of liver disease on drug-metabolizing enzymes, emerging data have implicated that the expression and function of hepatobiliary transport proteins also are altered in liver disease. This review summarizes recent developments in the field, which may have implications for understanding altered disposition, safety, and efficacy of new and existing drugs. A brief review of liver physiology and hepatic transporter localization/function is provided. Then, the expression and function of hepatic transporters in cholestasis, hepatitis C infection, hepatocellular carcinoma, human immunodeficiency virus infection, nonalcoholic fatty liver disease and nonalcoholic steatohepatitis, and primary biliary cirrhosis are reviewed. In the absence of clinical data, nonclinical information in animal models is presented. This review aims to advance the understanding of altered expression and function of hepatic transporters in liver disease and the implications of such changes on drug disposition.
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Affiliation(s)
- Nilay Thakkar
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Jason R Slizgi
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - Kim L R Brouwer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599.
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Dzierlenga AL, Clarke JD, Cherrington NJ. Nonalcoholic Steatohepatitis Modulates Membrane Protein Retrieval and Insertion Processes. Drug Metab Dispos 2016; 44:1799-1807. [PMID: 27604106 PMCID: PMC5074469 DOI: 10.1124/dmd.116.071415] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2016] [Accepted: 08/25/2016] [Indexed: 01/29/2023] Open
Abstract
Interindividual variability in drug response in nonalcoholic steatohepatitis (NASH) can be mediated by altered regulation of drug metabolizing enzymes and transporters. Among these is the mislocalization of multidrug resistance-associated protein (MRP2)/Mrp2 away from the canalicular membrane, which results in decreased transport of MRP2/Mrp2 substrates. The exact mechanism of this mislocalization is unknown, although increased activation of membrane retrieval processes may be one possibility. The current study measures the activation status of various mediators implicated in the active membrane retrieval or insertion of membrane proteins to identify which processes may be important in rodent methionine and choline deficient diet-induced NASH. The mediators currently known to be associated with transporter mislocalization are stimulated by oxidative stressors and choleretic stimuli, which play a role in the pathogenesis of NASH. The activation of protein kinases PKA, PKCα, PKCδ, and PKCε and substrates radixin, myristoylated alanine-rich C-kinase substrate, and Rab11 were measured by comparing the expression, phosphorylation, and membrane translocation between control and NASH. Many of the mediators exhibited altered activation in NASH rats. Consistent with membrane retrieval of Mrp2, NASH rats exhibited a decreased phosphorylation of radixin and increased membrane localization of PKCδ and PKCε, thought to be mediators of radixin dephosphorylation. Altered activation of PKCδ, PKA, and PKCα may impair the Rab11-mediated active insertion of Mrp2. Overall, these data suggest alterations in membrane retrieval and insertion processes that may contribute to altered localization of membrane proteins in NASH.
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Affiliation(s)
- A L Dzierlenga
- Department of Pharmacology & Toxicology, University of Arizona, Tucson, Arizona
| | - J D Clarke
- Department of Pharmacology & Toxicology, University of Arizona, Tucson, Arizona
| | - N J Cherrington
- Department of Pharmacology & Toxicology, University of Arizona, Tucson, Arizona
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Physiological and pathophysiological factors affecting the expression and activity of the drug transporter MRP2 in intestine. Impact on its function as membrane barrier. Pharmacol Res 2016; 109:32-44. [DOI: 10.1016/j.phrs.2016.04.014] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 02/01/2016] [Revised: 04/15/2016] [Accepted: 04/17/2016] [Indexed: 12/15/2022]
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Constrained spheroids for prolonged hepatocyte culture. Biomaterials 2016; 80:106-120. [DOI: 10.1016/j.biomaterials.2015.11.036] [Citation(s) in RCA: 23] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/11/2015] [Accepted: 11/29/2015] [Indexed: 12/15/2022]
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Canet MJ, Merrell MD, Hardwick RN, Bataille AM, Campion SN, Ferreira DW, Xanthakos SA, Manautou JE, A-Kader HH, Erickson RP, Cherrington NJ. Altered regulation of hepatic efflux transporters disrupts acetaminophen disposition in pediatric nonalcoholic steatohepatitis. Drug Metab Dispos 2015; 43:829-35. [PMID: 25788542 PMCID: PMC4429682 DOI: 10.1124/dmd.114.062703] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2014] [Accepted: 03/12/2015] [Indexed: 12/31/2022] Open
Abstract
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, representing a spectrum of liver pathologies that include simple hepatic steatosis and the more advanced nonalcoholic steatohepatitis (NASH). The current study was conducted to determine whether pediatric NASH also results in altered disposition of acetaminophen (APAP) and its two primary metabolites, APAP-sulfate and APAP-glucuronide. Pediatric patients with hepatic steatosis (n = 9) or NASH (n = 3) and healthy patients (n = 12) were recruited in a small pilot study design. All patients received a single 1000-mg dose of APAP. Blood and urine samples were collected at 1, 2, and 4 hours postdose, and APAP and APAP metabolites were determined by high-performance liquid chromatography. Moreover, human liver tissues from patients diagnosed with various stages of NAFLD were acquired from the Liver Tissue Cell Distribution System to investigate the regulation of the membrane transporters, multidrug resistance-associated protein 2 and 3 (MRP2 and MRP3, respectively). Patients with the more severe disease (i.e., NASH) had increased serum and urinary levels of APAP-glucuronide along with decreased serum levels of APAP-sulfate. Moreover, an induction of hepatic MRP3 and altered canalicular localization of the biliary efflux transporter, MRP2, describes the likely mechanism for the observed increase in plasma retention of APAP-glucuronide, whereas altered regulation of sulfur activation genes may explain decreased sulfonation activity in NASH. APAP-glucuronide and APAP-sulfate disposition is altered in NASH and is likely due to hepatic membrane transporter dysregulation as well as altered intracellular sulfur activation.
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Affiliation(s)
- Mark J Canet
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Matthew D Merrell
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Rhiannon N Hardwick
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Amy M Bataille
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Sarah N Campion
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Daniel W Ferreira
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Stavra A Xanthakos
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Jose E Manautou
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - H Hesham A-Kader
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Robert P Erickson
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
| | - Nathan J Cherrington
- Departments of Pharmacology and Toxicology (M.J.C., M.D.M., R.N.H., N.J.C.) and Pediatrics (H.A.K., R.P.E.), University of Arizona, Tucson, Arizona; School of Pharmacy, University of Connecticut, Storrs, Connecticut (A.M.B., D.W.F., J.E.M.); Drug Safety Research and Development, Pfizer, Inc., New York, New York (S.N.C.); Department of Pediatrics, Cincinnati Children's Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio (S.A.X.)
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Barosso IR, Zucchetti AE, Miszczuk GS, Boaglio AC, Taborda DR, Roma MG, Crocenzi FA, Sánchez Pozzi EJ. EGFR participates downstream of ERα in estradiol-17β-D-glucuronide-induced impairment of Abcc2 function in isolated rat hepatocyte couplets. Arch Toxicol 2015; 90:891-903. [PMID: 25813982 DOI: 10.1007/s00204-015-1507-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/27/2014] [Accepted: 03/16/2015] [Indexed: 11/28/2022]
Abstract
Estradiol-17β-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.
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Affiliation(s)
- Ismael R Barosso
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina
| | - Andrés E Zucchetti
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina
| | - Gisel S Miszczuk
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina
| | - Andrea C Boaglio
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina
| | - Diego R Taborda
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina
| | - Marcelo G Roma
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina
| | - Fernando A Crocenzi
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina
| | - Enrique J Sánchez Pozzi
- Instituto de Fisiología Experimental (IFISE), Facultad de Ciencias Bioquímicas y Farmacéuticas (CONICET - U.N.R.), Suipacha 570, S2002LRL, Rosario, Argentina.
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Dzierlenga AL, Clarke JD, Hargraves TL, Ainslie GR, Vanderah TW, Paine MF, Cherrington NJ. Mechanistic basis of altered morphine disposition in nonalcoholic steatohepatitis. J Pharmacol Exp Ther 2015; 352:462-70. [PMID: 25512370 PMCID: PMC4352592 DOI: 10.1124/jpet.114.220764] [Citation(s) in RCA: 42] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/15/2014] [Accepted: 12/12/2014] [Indexed: 01/21/2023] Open
Abstract
Morphine is metabolized in humans to morphine-3-glucuronide (M3G) and the pharmacologically active morphine-6-glucuronide (M6G). The hepatobiliary disposition of both metabolites relies upon multidrug resistance-associated proteins Mrp3 and Mrp2, located on the sinusoidal and canalicular membrane, respectively. Nonalcoholic steatohepatitis (NASH), the severe stage of nonalcoholic fatty liver disease, alters xenobiotic metabolizing enzyme and transporter function. The purpose of this study was to determine whether NASH contributes to the large interindividual variability and postoperative adverse events associated with morphine therapy. Male Sprague-Dawley rats were fed a control diet or a methionine- and choline-deficient diet to induce NASH. Radiolabeled morphine (2.5 mg/kg, 30 µCi/kg) was administered intravenously, and plasma and bile (0-150 or 0-240 minutes), liver and kidney, and cumulative urine were analyzed for morphine and M3G. The antinociceptive response to M6G (5 mg/kg) was assessed (0-12 hours) after direct intraperitoneal administration since rats do not produce M6G. NASH caused a net decrease in morphine concentrations in the bile and plasma and a net increase in the M3G/morphine plasma area under the concentration-time curve ratio, consistent with upregulation of UDP-glucuronosyltransferase Ugt2b1. Despite increased systemic exposure to M3G, NASH resulted in decreased biliary excretion and hepatic accumulation of M3G. This shift toward systemic retention is consistent with the mislocalization of canalicular Mrp2 and increased expression of sinusoidal Mrp3 in NASH and may correlate to increased antinociception by M6G. Increased metabolism and altered transporter regulation in NASH provide a mechanistic basis for interindividual variability in morphine disposition that may lead to opioid-related toxicity.
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Affiliation(s)
- Anika L Dzierlenga
- Departments of Pharmacology and Toxicology (A.L.D., J.D.C., T.L.H., N.J.C.) and Pharmacology (T.W.V.), University of Arizona, Tucson, Arizona; Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina (G.R.A., M.F.P.); and Section of Experimental and Systems Pharmacology, Washington State University, Spokane, Washington (G.R.A., M.F.P.)
| | - John D Clarke
- Departments of Pharmacology and Toxicology (A.L.D., J.D.C., T.L.H., N.J.C.) and Pharmacology (T.W.V.), University of Arizona, Tucson, Arizona; Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina (G.R.A., M.F.P.); and Section of Experimental and Systems Pharmacology, Washington State University, Spokane, Washington (G.R.A., M.F.P.)
| | - Tiffanie L Hargraves
- Departments of Pharmacology and Toxicology (A.L.D., J.D.C., T.L.H., N.J.C.) and Pharmacology (T.W.V.), University of Arizona, Tucson, Arizona; Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina (G.R.A., M.F.P.); and Section of Experimental and Systems Pharmacology, Washington State University, Spokane, Washington (G.R.A., M.F.P.)
| | - Garrett R Ainslie
- Departments of Pharmacology and Toxicology (A.L.D., J.D.C., T.L.H., N.J.C.) and Pharmacology (T.W.V.), University of Arizona, Tucson, Arizona; Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina (G.R.A., M.F.P.); and Section of Experimental and Systems Pharmacology, Washington State University, Spokane, Washington (G.R.A., M.F.P.)
| | - Todd W Vanderah
- Departments of Pharmacology and Toxicology (A.L.D., J.D.C., T.L.H., N.J.C.) and Pharmacology (T.W.V.), University of Arizona, Tucson, Arizona; Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina (G.R.A., M.F.P.); and Section of Experimental and Systems Pharmacology, Washington State University, Spokane, Washington (G.R.A., M.F.P.)
| | - Mary F Paine
- Departments of Pharmacology and Toxicology (A.L.D., J.D.C., T.L.H., N.J.C.) and Pharmacology (T.W.V.), University of Arizona, Tucson, Arizona; Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina (G.R.A., M.F.P.); and Section of Experimental and Systems Pharmacology, Washington State University, Spokane, Washington (G.R.A., M.F.P.)
| | - Nathan J Cherrington
- Departments of Pharmacology and Toxicology (A.L.D., J.D.C., T.L.H., N.J.C.) and Pharmacology (T.W.V.), University of Arizona, Tucson, Arizona; Curriculum in Toxicology, University of North Carolina, Chapel Hill, North Carolina (G.R.A., M.F.P.); and Section of Experimental and Systems Pharmacology, Washington State University, Spokane, Washington (G.R.A., M.F.P.)
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Ding L, Zhang B, Zhan C, Yang L, Wang Z. Danning tablets attenuates α-naphthylisothiocyanate-induced cholestasis by modulating the expression of transporters and metabolic enzymes. BMC COMPLEMENTARY AND ALTERNATIVE MEDICINE 2014; 14:249. [PMID: 25033983 PMCID: PMC4223591 DOI: 10.1186/1472-6882-14-249] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 01/14/2014] [Accepted: 07/09/2014] [Indexed: 01/01/2023]
Abstract
Background The Danning tablets (DNts) is commonly prescribed in China as a cholagogic formula. Our previous studies showed that DNts exerted the protective effect on α-naphthylisothiocyanate (ANIT)-induced liver injury with cholestasis in a dose-dependent mannar. However, the detailed molecular mechanisms of DNts against ANIT-induced cholestasis are still not fully explored. Methods Danning tablet (3 g/kg body weight/day) was administered orally to experimental rats for seven days before they were treated with ANIT (60 mg/kg daily via gastrogavage) which caused cholestasis. Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), total bilirubin (T-Bil), direct bilirubin (D-Bil) and total bile acid (TBA) were measured to evaluate the protective effect of Danning tablet at 12, 24 and 48h after ANIT treatment. Meanwhile, total bilirubin or total bile acid in the bile, urine and liver were also measured at 48h after ANIT treatment. Furthermore, the hepatic or renal mRNA and protein levels of metabolic enzymes and transports were investigated to elucidate the protective mechanisms of Danning tablet against ANIT-induced cholestasis. Results In this study, we found that DNts significantly attenuated translocation of multidrug resistance-associated protein 2 (Mrp2) from the canalicular membrane into an intracellular and up-regulated the hepatic mRNA and protein expressions of metabolic enzymes including cytochrome P450 2b1(Cyp2b1) and uridine diphosphate-5¢- glucuronosyltransferase (Ugt1a1)) and transporters including bile salt export pump (Bsep) and multidrug resistance protein 2 (Mdr2)) as well as renal organic solute transporter beta (Ostβ), accompanied by further increase in urinary and biliary excretion of bile acid and bilirubin. Conclusions DNts might promote bile acid and bilirubin elimination by regulating the expressions of hepatic and renal transporters as well as hepatic metabolic enzymes.
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Miszczuk GS, Barosso IR, Zucchetti AE, Boaglio AC, Pellegrino JM, Sánchez Pozzi EJ, Roma MG, Crocenzi FA. Sandwich-cultured rat hepatocytes as an in vitro model to study canalicular transport alterations in cholestasis. Arch Toxicol 2014; 89:979-90. [PMID: 24912783 DOI: 10.1007/s00204-014-1283-x] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/24/2014] [Accepted: 05/20/2014] [Indexed: 12/28/2022]
Abstract
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 17β-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.
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Affiliation(s)
- Gisel S Miszczuk
- Instituto de Fisiología Experimental (IFISE) - Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Facultad de Ciencias Bioquímicas y Farmacéuticas - Universidad Nacional de Rosario (UNR), Suipacha 570, S2002LRL, Rosario, Argentina
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Erlinger S, Arias IM, Dhumeaux D. Inherited disorders of bilirubin transport and conjugation: new insights into molecular mechanisms and consequences. Gastroenterology 2014; 146:1625-38. [PMID: 24704527 DOI: 10.1053/j.gastro.2014.03.047] [Citation(s) in RCA: 141] [Impact Index Per Article: 12.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/23/2013] [Revised: 03/12/2014] [Accepted: 03/23/2014] [Indexed: 12/11/2022]
Abstract
Inherited disorders of bilirubin metabolism might reduce bilirubin uptake by hepatocytes, bilirubin conjugation, or secretion of bilirubin into bile. Reductions in uptake could increase levels of unconjugated or conjugated bilirubin (Rotor syndrome). Defects in bilirubin conjugation could increase levels of unconjugated bilirubin; the effects can be benign and frequent (Gilbert syndrome) or rare but severe, increasing the risk of bilirubin encephalopathy (Crigler-Najjar syndrome). Impairment of bilirubin secretion leads to accumulation of conjugated bilirubin (Dubin-Johnson syndrome). We review the genetic causes and pathophysiology of disorders of bilirubin transport and conjugation as well as clinical and therapeutic aspects. We also discuss the possible mechanisms by which hyperbilirubinemia protects against cardiovascular disease and the metabolic syndrome and the effects of specific genetic variants on drug metabolism and cancer development.
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Affiliation(s)
| | | | - Daniel Dhumeaux
- Henri Mondor Hospital, Créteil, University of Paris-Est, Créteil, France
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Cuperus FJC, Claudel T, Gautherot J, Halilbasic E, Trauner M. The role of canalicular ABC transporters in cholestasis. Drug Metab Dispos 2014; 42:546-60. [PMID: 24474736 DOI: 10.1124/dmd.113.056358] [Citation(s) in RCA: 56] [Impact Index Per Article: 5.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022] Open
Abstract
Cholestasis, a hallmark feature of hepatobiliary disease, is characterized by the retention of biliary constituents. Some of these constituents, such as bile acids, inflict damage to hepatocytes and bile duct cells. This damage may lead to inflammation, fibrosis, cirrhosis, and eventually carcinogenesis, sequelae that aggravate the underlying disease and deteriorate clinical outcome. Canalicular ATP-binding cassette (ABC) transporters, which mediate the excretion of individual bile constituents, play a key role in bile formation and cholestasis. The study of these transporters and their regulatory nuclear receptors has revolutionized our understanding of cholestatic disease. This knowledge has served as a template to develop novel treatment strategies, some of which are currently already undergoing phase III clinical trials. In this review we aim to provide an overview of the structure, function, and regulation of canalicular ABC transporters. In addition, we will focus on the role of these transporters in the pathogenesis and treatment of cholestatic bile duct and liver diseases.
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Affiliation(s)
- Frans J C Cuperus
- Hans Popper Laboratory of Molecular Hepatology, Division of Gastroenterology and Hepatology, Department of Internal Medicine III, Medical University of Vienna, Vienna, Austria
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Abstract
Bile acids, synthesized from cholesterol, are known to produce beneficial as well as toxic effects in the liver. The beneficial effects include choleresis, immunomodulation, cell survival, while the toxic effects include cholestasis, apoptosis and cellular toxicity. It is believed that bile acids produce many of these effects by activating intracellular signaling pathways. However, it has been a challenge to relate intracellular signaling to specific and at times opposing effects of bile acids. It is becoming evident that bile acids produce different effects by activating different isoforms of phosphoinositide 3-kinase (PI3K), Protein kinase Cs (PKCs), and mitogen activated protein kinases (MAPK). Thus, the apoptotic effect of bile acids may be mediated via PI3K-110γ, while cytoprotection induce by cAMP-GEF pathway involves activation of PI3K-p110α/β isoforms. Atypical PKCζ may mediate beneficial effects and nPKCε may mediate toxic effects, while cPKCα and nPKCδ may be involved in both beneficial and toxic effects of bile acids. The opposing effects of nPKCδ activation may depend on nPKCδ phosphorylation site(s). Activation of ERK1/2 and JNK1/2 pathway appears to mediate beneficial and toxic effects, respectively, of bile acids. Activation of p38α MAPK and p38β MAPK may mediate choleretic and cholestatic effects, respectively, of bile acids. Future studies clarifying the isoform specific effects on bile formation should allow us to define potential therapeutic targets in the treatment of cholestatic disorders.
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Affiliation(s)
- Mohammed Sawkat Anwer
- Department of Biomedical Sciences, Cummings School of Veterinary Medicine at Tufts University, 200 Westboro Road, North Grafton, MA, USA
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Cressman AM, Petrovic V, Piquette-Miller M. Inflammation-mediated changes in drug transporter expression/activity: implications for therapeutic drug response. Expert Rev Clin Pharmacol 2014; 5:69-89. [DOI: 10.1586/ecp.11.66] [Citation(s) in RCA: 69] [Impact Index Per Article: 6.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
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Abstract
Bile is a unique and vital aqueous secretion of the liver that is formed by the hepatocyte and modified down stream by absorptive and secretory properties of the bile duct epithelium. Approximately 5% of bile consists of organic and inorganic solutes of considerable complexity. The bile-secretory unit consists of a canalicular network which is formed by the apical membrane of adjacent hepatocytes and sealed by tight junctions. The bile canaliculi (∼1 μm in diameter) conduct the flow of bile countercurrent to the direction of portal blood flow and connect with the canal of Hering and bile ducts which progressively increase in diameter and complexity prior to the entry of bile into the gallbladder, common bile duct, and intestine. Canalicular bile secretion is determined by both bile salt-dependent and independent transport systems which are localized at the apical membrane of the hepatocyte and largely consist of a series of adenosine triphosphate-binding cassette transport proteins that function as export pumps for bile salts and other organic solutes. These transporters create osmotic gradients within the bile canalicular lumen that provide the driving force for movement of fluid into the lumen via aquaporins. Species vary with respect to the relative amounts of bile salt-dependent and independent canalicular flow and cholangiocyte secretion which is highly regulated by hormones, second messengers, and signal transduction pathways. Most determinants of bile secretion are now characterized at the molecular level in animal models and in man. Genetic mutations serve to illuminate many of their functions.
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Affiliation(s)
- James L Boyer
- Department of Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut, USA.
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Pfeifer ND, Hardwick RN, Brouwer KLR. Role of hepatic efflux transporters in regulating systemic and hepatocyte exposure to xenobiotics. Annu Rev Pharmacol Toxicol 2013; 54:509-35. [PMID: 24160696 DOI: 10.1146/annurev-pharmtox-011613-140021] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/18/2022]
Abstract
Hepatic efflux transporters include numerous well-known and emerging proteins localized to the canalicular or basolateral membrane of the hepatocyte that are responsible for the excretion of drugs into the bile or blood, respectively. Altered function of hepatic efflux transporters due to drug-drug interactions, genetic variation, and/or disease states may lead to changes in xenobiotic exposure in the hepatocyte and/or systemic circulation. This review focuses on transport proteins involved in the hepatocellular efflux of drugs and metabolites, discusses mechanisms of altered transporter function as well as the interplay between multiple transport pathways, and highlights the importance of considering intracellular unbound concentrations of transporter substrates and/or inhibitors. Methods to evaluate hepatic efflux transport and predict the effects of impaired transporter function on systemic and hepatocyte exposure are discussed, and the sandwich-cultured hepatocyte model to evaluate comprehensively the role of hepatic efflux in the hepatobiliary disposition of xenobiotics is characterized.
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Affiliation(s)
- Nathan D Pfeifer
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599; ,
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YANG KYUNGHEE, KÖCK KATHLEEN, SEDYKH ALEXANDER, TROPSHA ALEXANDER, BROUWER KIML. An updated review on drug-induced cholestasis: mechanisms and investigation of physicochemical properties and pharmacokinetic parameters. J Pharm Sci 2013; 102:3037-57. [PMID: 23653385 PMCID: PMC4369767 DOI: 10.1002/jps.23584] [Citation(s) in RCA: 78] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2013] [Revised: 04/13/2013] [Accepted: 04/16/2013] [Indexed: 12/15/2022]
Abstract
Drug-induced cholestasis is an important form of acquired liver disease and is associated with significant morbidity and mortality. Bile acids are key signaling molecules, but they can exert toxic responses when they accumulate in hepatocytes. This review focuses on the physiological mechanisms of drug-induced cholestasis associated with altered bile acid homeostasis due to direct (e.g., bile acid transporter inhibition) or indirect (e.g., activation of nuclear receptors, altered function/expression of bile acid transporters) processes. Mechanistic information about the effects of a drug on bile acid homeostasis is important when evaluating the cholestatic potential of a compound, but experimental data often are not available. The relationship between physicochemical properties, pharmacokinetic parameters, and inhibition of the bile salt export pump among 77 cholestatic drugs with different pathophysiological mechanisms of cholestasis (i.e., impaired formation of bile vs. physical obstruction of bile flow) was investigated. The utility of in silico models to obtain mechanistic information about the impact of compounds on bile acid homeostasis to aid in predicting the cholestatic potential of drugs is highlighted.
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Affiliation(s)
- KYUNGHEE YANG
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - KATHLEEN KÖCK
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - ALEXANDER SEDYKH
- Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - ALEXANDER TROPSHA
- Chemical Biology and Medicinal Chemistry, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
| | - KIM L.R. BROUWER
- Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina 27599
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Schonhoff CM, Webster CRL, Anwer MS. Taurolithocholate-induced MRP2 retrieval involves MARCKS phosphorylation by protein kinase Cϵ in HUH-NTCP Cells. Hepatology 2013; 58:284-92. [PMID: 23424156 PMCID: PMC3681903 DOI: 10.1002/hep.26333] [Citation(s) in RCA: 29] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/31/2012] [Accepted: 02/01/2013] [Indexed: 12/28/2022]
Abstract
UNLABELLED Taurolithocholate (TLC) acutely inhibits the biliary excretion of multidrug-resistant associated protein 2 (Mrp2) substrates by inducing Mrp2 retrieval from the canalicular membrane, whereas cyclic adenosine monophosphate (cAMP) increases plasma membrane (PM)-MRP2. The effect of TLC may be mediated via protein kinase Cϵ (PKCϵ). Myristoylated alanine-rich C kinase substrate (MARCKS) is a membrane-bound F-actin crosslinking protein and is phosphorylated by PKCs. MARCKS phosphorylation has been implicated in endocytosis, and the underlying mechanism appears to be the detachment of phosphorylated myristoylated alanine-rich C kinase substrate (pMARCKS) from the membrane. The aim of the present study was to test the hypothesis that TLC-induced MRP2 retrieval involves PKCϵ-mediated MARCKS phosphorylation. Studies were conducted in HuH7 cells stably transfected with sodium taurocholate cotransporting polypeptide (HuH-NTCP cells) and in rat hepatocytes. TLC increased PM-PKCϵ and decreased PM-MRP2 in both HuH-NTCP cells and hepatocytes. cAMP did not affect PM-PKCϵ and increased PM-MRP2 in these cells. In HuH-NTCP cells, dominant-negative (DN) PKCϵ reversed TLC-induced decreases in PM-MRP2 without affecting cAMP-induced increases in PM-MRP2. TLC, but not cAMP, increased MARCKS phosphorylation in HuH-NTCP cells and hepatocytes. TLC and phorbol myristate acetate increased cytosolic pMARCKS and decreased PM-MARCKS in HuH-NTCP cells. TLC failed to increase MARCKS phosphorylation in HuH-NTCP cells transfected with DN-PKCϵ, and this suggested PKCϵ-mediated phosphorylation of MARCKS by TLC. In HuH-NTCP cells transfected with phosphorylation-deficient MARCKS, TLC failed to increase MARCKS phosphorylation or decrease PM-MRP2. CONCLUSION Taken together, these results support the hypothesis that TLC-induced MRP2 retrieval involves TLC-mediated activation of PKCϵ followed by MARCKS phosphorylation and consequent detachment of MARCKS from the membrane.
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Affiliation(s)
| | - Cynthia R. L. Webster
- Department of Clinical Sciences, Tufts Cummings School of Veterinary Medicine, 200 Westboro Road, North Grafton, MA, USA
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