Multidrug resistance associated protein 2 mediates transport of prostaglandin E2

BACKGROUND/AIM: Inactivation of prostaglandin E(2) (PGE(2)) in the liver is a rapid process and occurs mainly through beta-oxidation in the peroxisome of the hepatocyte. Biliary excretion of PGE(2) is also a means of elimination from the liver. We investigated the role of multidrug resistance-associated protein 2 (MRP2) in the transport of PGE(2). METHODS: Biliary PGE(2) elimination was measured in liver perfusions in Wistar and MRP2-deficient TR(-) rats. Furthermore, transport experiments were performed in membrane vesicles from human MRP2-infected Spodoptera frugiperda 21 (Sf21) insect cells. RESULTS: The liver perfusions showed a 3.5 times higher percentage of undegraded [(3)H]PGE(2) in bile of Wistar rats in comparison with MRP2 deficient (TR(-)) rats (3.6% vs. 1.1%, respectively; P <0.05). MRP2-mediated transport of the model substrate [(3)H]DNP-SG was inhibited by PGE(2). Half maximal inhibition was achieved at a concentration of approximately 15 microM PGE(2). In addition, [(3)H]PGE(2) uptake in these vesicles was detected, and determined to be ATP dependent. CONCLUSION: MRP2 mediates the transport of PGE(2) and its breakdown products. The biliary excretion of PGE(2) via MRP2 may contribute to rapid elimination of the prostaglandin but might also serve to relay prostaglandin signalling to the biliary tre

Actual Usage and Quality of Experimental Colitis Models in Preclinical Efficacy Testing: A Scoping Review

There is no standardized validated experimental model used to predict human drug response, and the choice of model is not based on systematic evidence. Therefore, we decided to systematically investigate which models are currently used by selecting studies from literature that use prescribed inflammatory bowel disease medication as a positive control. A search of PubMed was performed using terms describing experimental colitis models and the drugs used in corresponding clinical practice followed by the application of an animal filter. Each article was read and scored using a predesigned form describing the characteristics of the study (17 items), a quality assessment (10 items) completed by a meta-analysis. One hundred ninety-four unique articles were included that met the selection criteria. A large heterogeneity was found regarding the characteristics of the animals used, induction methods, treatment protocol, and reporting quality. If categorized by colitis model only a small number of studies used a chronic model (10/194). Almost all use acute chemical models that investigate a response to epithelial damage, rather than chronic colitis. Fifty-six percent used a TNBS model and 20% used a dextran sodium sulfate model. In these models, an ameliorating effect of 5-ASA and corticosteroids was demonstrated and also a difference in outcome when male or female animals are used. This scope describes a huge heterogeneity in study designs for preclinical drug efficacy. In addition, more than three-quarters of the studies used an acute model irrelevant for testing new treatment options for inflammatory bowel diseas

Mycophenolate Mofetil Impairs Transduction of Single-Stranded Adeno-Associated Viral Vectors

Adeno-associated virus (AAV) liver-directed gene therapy seems a feasible treatment for Crigler-Najjar syndrome type I, an inherited liver disorder characterized by severe unconjugated hyperbilirubinemia. Transient immunosuppression coupled with vector administration seems needed to overcome host immune responses that prevent long-term expression in patients. The immunosuppressive mycophenolate mofetil (MMF), which inhibits de novo synthesis of purines, is a promising candidate. To investigate the potential use of MMF in patients with Crigler-Najjar syndrome, we studied its effect on single-stranded AAV (ssAAV)-mediated correction of hyperbilirubinemia in the relevant preclinical model, the Gunn rat. Although MMF was well tolerated and effective it also impaired the efficacy of ssAAV. Subsequent in vitro studies showed that this effect is not specific for UGT1A deficiency. In fact, clinical relevant concentrations of mycophenolic acid (MPA), the active compound of MMF, also impair the transduction of HEK-293T cells by ssAAV. Because this effect was reversed by guanosine addition, it seems that intracellular levels of this nucleotide become limited, suggesting that MPA impairs second-strand DNA synthesis. This is corroborated by observations that MPA did not impair transduction of 293T cells by a self-complementary AAV (scAAV) vector and that MMF did not reduce the scAAV efficacy in the Gunn rat. In conclusion, MMF impairs ssAAV-mediated liver-directed gene therapy, which is relevant for the use of this immunosuppressive agent with single-stranded vectors. Furthermore, because this effect is due to impaired second-strand synthesis, the use of MMF with scAAV seems warrante

The mechanism of ABCG5/ ABCG8 in biliary cholesterol secretion in mice

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8 1/1, Abcg8 1/2, and Abcg8 2/2 mice with hydrophilic and hydrophobic bile salts. In Abcg8 2/2 mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8 2/2 mice at a much lower infusion rate compared with Abc8 2/2 and Abcg8 1/2 mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45 % in cholesterol content under these conditions in Abcg8 2/2 mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.—Kosters,A.,C.Kunne,N.Looije,S.B.Patel, R. P. J. Oude Elferink, and A. K. Groen. The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice

Altered disposition of acetaminophen in mice with a disruption of the Mrp3 gene

MRP3 is an ABC transporter localized in the basolateral membrane of epithelial cells such as hepatocytes and enterocytes. In this study, the role of Mrp3 in drug disposition was investigated. Because Mrp3 preferentially transports glucuronide conjugates, we investigated the in vivo disposition of acetaminophen (APAP) and its metabolites. Mrp3+/+ and Mrp3-/- knockout mice received APAP (150 mg/kg), and bile was collected. Basolateral and canalicular excretion of APAP was also assessed in the isolated perfused liver. In separate studies, mice received 400 mg APAP/kg for assessment of hepatotoxicity. No differences were found in the biliary excretion of APAP, APAP-sulfate, and APAP-glutathione between Mrp3+/+ and Mrp3-/- mice. However, 20-fold higher accumulation of APAP-glucuronide (APAP-GLUC) was found in the liver of Mrp3-/- mice. Concomitantly, plasma APAP-GLUC content in Mrp3-/- mice was less than 10% of that in Mrp3+/+ mice. In addition, APAP-GLUC excretion in bile of Mrp3-/- mice was tenfold higher than in Mrp3+/+ mice. In the isolated perfused liver, we also found a strong decrease of APAP-GLUC secretion into the perfusate of Mrp3-/- livers. Plasma alanine aminotransferase (ALT), aspartate aminotransferase (AST), and histopathology showed that Mrp3-/- mice are more resistant to APAP hepatotoxicity than Mrp3+/+ mice, which is most likely a result of the faster repletion of hepatic GSH. In conclusion, basolateral excretion of APAP-GLUC in mice is nearly completely dependent on the function of Mrp3. In its absence, sufficient hepatic accumulation occurs to redirect some of the APAP-GLUC to bile. This altered disposition in Mrp3-/- mice is associated with reduced hepatotoxicit

Complex pharmacokinetic behavior of ezetimibe depends on abcc2, abcc3, and abcg2

Ezetimibe lowers plasma cholesterol levels by inhibiting the uptake of cholesterol in the intestine. Because of the extensive enterohepatic circulation of ezetimibe, relatively low doses are required to be effective. In blood and bile the majority of ezetimibe is present as a glucuronide conjugate, which is formed in the enterocyte. Presently, it is not clear which mechanisms are responsible for this efficient enterohepatic circulation. Abcc2, Abcc3, and Abcg2 are ATP-binding cassette (ABC) transporters that are expressed in both liver and intestine and are capable of transporting glucuronidated compounds. The aim of this study was to investigate the contribution of these transporters in the enterohepatic cycling of ezetimibe glucuronide (Ez-gluc). Transport studies were performed in plasma membrane vesicles from ABCC2-, ABCC3-, and ABCG2-expressing Sf21 insect cells. Furthermore, intestinal explants from wild-type and Abcc3(-/-) mice were used to study vectorial transport in a Ussing chamber setup. Finally, biliary excretion of Ez-gluc was measured in vivo after duodenal delivery of ezetimibe in wild-type, Abcc3(-/-), Abcc2(-/-), Abcg2(-/-), and Abcg2(-/-)/Abcc2(-/-) mice. ABCC3-, ABCC2-, and ABCG2-mediated transport was dose dependently inhibited by Ez-gluc. In the Ussing chamber Ez-gluc recovered from the basolateral side was significantly reduced in duodenal (2.2%), in jejunal (23%), and in ileal (23%) tissue of Abcc3(-/-) mice compared with that in tissues of wild-type mice. Biliary excretion of Ez-gluc was significantly reduced in Abcc3(-/-) (34%), Abcc2(-/-) (56%), and Abcg2(-/-)/Abcc2(-/-) (2.5%) compared with that in wild-type mice. These data demonstrate that the enterohepatic circulation of Ez-gluc strongly depends on the joint function of Abcc3, Abcc2, and Abcg

Lipotoxicity and steatohepatitis in an overfed mouse model for non-alcoholic fatty liver disease

The major risk factors for non-alcoholic fatty liver disease (NAFLD) are obesity, insulin resistance and dyslipidemia. The cause for progression from the steatosis stage to the inflammatory condition (non-alcoholic steatohepatitis (NASH)) remains elusive at present. Aim of this study was to test whether the different stages of NAFLD as well as the associated metabolic abnormalities can be recreated in time in an overfed mouse model and study the mechanisms underlying the transition from steatosis to NASH. Male C57BI/6J mice were subjected to continuous intragastric overfeeding with a high-fat liquid diet (HFLD) for different time periods. Mice fed a solid high-fat diet (HFD) ad libitum served as controls. Liver histology and metabolic characteristics of liver, white adipose tisue (WAT) and plasma were studied. Both HFD-fed and HFLD-overfed mice initially developed liver steatosis, but only the latter progressed in time to NASH. NASH coincided with obesity, hyperinsulinemia, loss of liver glycogen and hepatic endoplasmatic reticulum stress. Peroxisome proliferator-activated receptor gamma (Ppary), fibroblast growth factor 21 (Fgf21), fatty acid binding protein (Fabp) and fatty acid translocase (CD36) were induced exclusively in the livers of the HFLD-overfed mice. Inflammation, reduced adiponectin expression and altered expression of genes that influence adipogenic capacity were only observed in WAT of HFLD-overfed mice. In conclusion: this dietary mouse model displays the different stages and the metabolic settings often found in human NAFLD. Lipotoxicity due to compromised adipose tissue function is likely associated with the progression to NASH, but whether this is cause or consequence remains to be established. (C) 2011 Elsevier B.V. All rights reserve

Bone marrow-derived multidrug resistance protein ABCB4 protects against atherosclerotic lesion development in LDL receptor knockout mice

OBJECTIVE: Several members of the ATP binding cassette (ABC)-transporter super family expressed in macrophages protect against atherosclerosis by promoting macrophage cholesterol and phospholipid efflux. Systemic disruption of ABCB4 in mice results in a virtual absence of phospholipids in bile and a strongly impaired biliary cholesterol secretion, indicating that ABCB4 plays an essential role in cellular lipid efflux. The aim of the current study was to determine the role of bone marrow-derived ABCB4 in atherosclerotic lesion development. METHODS: Chimeras were created that specifically lack ABCB4 in bone marrow-derived cells, including macrophages, by performing a bone marrow transplantation on LDL receptor knockout (LDLr-/-) mice. Atherosclerotic lesion development was induced by feeding a high-cholesterol diet (15% fat and 0.25% cholesterol). RESULTS: Serum cholesterol levels were significantly lower in mice reconstituted with ABCB4 knockout bone marrow as a result of reduced VLDL and LDL cholesterol levels. Despite the lower serum cholesterol levels, ABCB4 deficiency in bone marrow-derived cells resulted in a 1.8-fold (p=0.005) increase in lesion size. In vitro foam cell formation, induced with acetylated LDL (AcLDL) in peritoneal macrophages, was increased in the absence of ABCB4, possibly due to a 2-fold (p <0.05) increased association of AcLDL, while the efflux of cholesterol was unaffected. CONCLUSION: Bone marrow-derived ABCB4 has an important anti-atherosclerotic function, probably by limiting macrophage foam cell formatio

Cyclosporin A induces peritoneal fibrosis and angiogenesis during chronic peritoneal exposure to a glucose-based, lactate-buffered dialysis solution in the rat

BACKGROUND/AIMS: Cyclosporin A (CsA) stimulates the development of fibrosis. We investigated whether CsA contributes to peritoneal alterations induced by long-term exposure to dialysis solutions. METHODS: Ten rats received peritoneal infusion of dialysis solution and oral CsA for 8 weeks. Eight received only the dialysis solution (controls). Peritoneal function was assessed at 8 weeks followed by sacrifice. The number of vessels was counted, fibrosis was assessed and hydroxyproline was determined. PCR was performed for vascular endothelial growth factor (VEGF), connective tissue growth factor (CTGF) and transforming growth factor-beta (TGF-beta). RESULTS: Histology revealed more fibrosis, hydroxyproline and vessels (thick walled) in CsA-exposed animals. Peritoneal transport was not different. The mRNA content of TGF-beta, CTGF and VEGF was higher in CsA. CONCLUSION: CsA combined with exposure to dialysis solutions was associated with increased peritoneal fibrosis and angiogenesi