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Electron-microscopic demonstration of multidrug resistance protein 2 (Mrp2) retrieval from the canalicular membrane in response to hyperosmolarity and lipopolysaccharide.

ABSTRACT: Immunohistochemical studies suggest that canalicular secretion via multidrug resistance protein 2 (Mrp2), a conjugate export pump encoded by the Mrp2 gene, is regulated by rapid transporter retrieval from/insertion into the canalicular membrane. The present study was undertaken in order to investigate this suggestion by means of immunogold electron microscopy. Therefore the effects of lipopolysaccharide (LPS) and osmolarity on Mrp2 localization were studied following immunogold labelling in the perfused rat liver by quantitative electron microscopy and morphometric analyses, and by confocal laser scanning microscopy. Mrp2 activity was assessed in the isolated perfused rat liver by measuring the excretion of dinitrophenyl-S-glutathione as a substrate of Mrp2. Both LPS and hyperosmolarity resulted in a statistically significant decrease in immunogold-labelled Mrp2 in the canalicular membrane and canalicular villi, and an increase in labelling in the pericanalicular cytoplasm. Canalicular morphometric parameters were unchanged under these conditions compared with controls. Under hyperosmolar perfusion Mrp2, but not the canalicular protein dipeptidylpeptidase IV, was found inside the cells, as shown by double immunofluorescence and confocal laser scanning microscopy. The findings suggest a selective retrieval of Mrp2 from the canalicular membrane under the influence of hyperosmolarity and LPS, whereas canalicular morphology remains unchanged.

SUBMITTER: Dombrowski F

PROVIDER: S-EPMC1221052 | biostudies-other | 2000 May

REPOSITORIES: biostudies-other

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Project description:In perfused rat liver, hyperosmolarity induces Mrp2- (Kubitz, R., D'urso, D., Keppler, D., and Häussinger, D. (1997) Gastroenterology 113, 1438-1442) and Bsep retrieval (Schmitt, M., Kubitz, R., Lizun, S., Wettstein, M., and Häussinger, D. (2001) Hepatology 33, 509-518) from the canalicular membrane leading to cholestasis. The aim of this study was to elucidate the underlying signaling events. Hyperosmolarity-induced retrieval of Mrp2 and Bsep from the canalicular membrane in perfused rat liver was accompanied by an activating phosphorylation of the Src kinases Fyn and Yes but not of c-Src. Both hyperosmotic transporter retrieval and Src kinase activation were sensitive to apocynin (300 μmol/liter), N-acetylcysteine (NAC; 10 mmol/liter), and SU6656 (1 μmol/liter). Also PP-2 (250 nmol/liter), which inhibited hyperosmotic Fyn but not Yes activation, prevented hyperosmotic transporter retrieval from the canalicular membrane, suggesting that Fyn but not Yes mediates hyperosmotic Bsep and Mrp2 retrieval. Neither hyperosmotic Fyn activation nor Bsep/Mrp2 retrieval was observed in livers from p47(phox) knock-out mice. Hyperosmotic activation of JNKs was sensitive to apocynin and NAC but insensitive to SU6656 and PP-2, indicating that JNKs are not involved in transporter retrieval, as also evidenced by experiments using the JNK inhibitors L-JNKI-1 and SP6001255, respectively. Hyperosmotic transporter retrieval was accompanied by a NAC and Fyn knockdown-sensitive inhibition of biliary excretion of the glutathione conjugate of 1-chloro-2,4-dinitrobenzene in perfused rat liver and of cholyl-L-lysyl-fluorescein secretion into the pseudocanaliculi formed by hepatocyte couplets. Hyperosmolarity triggered an association between Fyn and cortactin and increased the amount of phosphorylated cortactin underneath the canalicular membrane. It is concluded that the hyperosmotic cholestasis is triggered by a NADPH oxidase-driven reactive oxygen species formation that mediates Fyn-dependent retrieval of the Mrp2 and Bsep from the canalicular membrane, which may involve an increased cortactin phosphorylation.

Project description:BACKGROUND AND OBJECTIVE:Tacrolimus is an immunosuppressive drug used for the prevention of the allograft rejection in kidney transplant recipients. It exhibits a narrow therapeutic index and large pharmacokinetic variability. Tacrolimus is mainly metabolized by cytochrome P450 (CYP) 3A4 and 3A5 and effluxed via ATP-binding cassette (ABC) transporters such as P-glycoprotein (P-gp), encoded by ABCB1 gene. The influence of CYP3A5*3 on the pharmacokinetics of tacrolimus has been well characterized. On the other hand, the contribution of polymorphisms in other genes is controversial. In addition, the involvement of other efflux transporters than P-gp in tacrolimus disposition is uncertain. The present study was designed to investigate the effects of genetic polymorphisms of CYP3As and efflux transporters on the pharmacokinetics of tacrolimus. SUBJECTS AND METHODS:A total of 500 blood concentrations of tacrolimus from 102 adult stable kidney transplant recipients were included in the analyses. Genetic polymorphisms in CYP3A4 and CYP3A5 genes were determined. In addition, the genes of efflux transporters including P-gp (ABCB1), multidrug resistance-associated protein (MRP2/ABCC2) and breast cancer resistance protein (BCRP/ABCG2) were genotyped. For ABCC2 gene, haplotypes were determined as follows: H1 (wild type), H2 (1249G>A), H9 (3972C>T) and H12 (-24C>T and 3972C>T). Population pharmacokinetic analysis was performed using nonlinear mixed effects modeling. RESULTS:Analyses revealed that the CYP3A5 expressers (CYP3A5*1 carriers) and MRP2 high-activity group (ABCC2 H2/H2 and H1/H2) showed a decreased dose-normalized trough concentration of tacrolimus by 2.3-fold (p < 0.001) and 1.5-fold (p = 0.007), respectively. The pharmacokinetics of tacrolimus were best described using a two-compartment model with first order absorption and an absorption lag time. In the population pharmacokinetic analysis, CYP3A5 expressers and MRP2 high-activity groups were identified as the significant covariates for tacrolimus apparent clearance expressed as 20.7 × (age/50)(-0.78) × 2.03 (CYP3A5 expressers) × 1.40 (MRP2 high-activity group). No other CYP3A4, ABCB1 or ABCG2 polymorphisms were associated with the apparent clearance of tacrolimus. CONCLUSIONS:This is the first report showing that MRP2/ABCC2 has a crucial impact on the pharmacokinetics of tacrolimus in a haplotype-specific manner. Determination of the ABCC2 as well as CYP3A5 genotype may be useful for more accurate tacrolimus dosage adjustment.

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Electron-microscopic demonstration of multidrug resistance protein 2 (Mrp2) retrieval from the canalicular membrane in response to hyperosmolarity and lipopolysaccharide.

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