|An experimental approach to evaluate the impact of impaired transport function on hepatobiliary drug disposition using Mrp2-deficient TR- rat sandwich-cultured hepatocytes in combination with Bcrp knockdown. |
Yang, K; Pfeifer, ND; Hardwick, RN; Yue, W; Stewart, PW; Brouwer, KL
Breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2) are members of the ATP binding cassette (ABC) transporter family located in the canalicular membrane of hepatocytes that mediate biliary excretion of many drugs and endogenous compounds. BCRP and MRP2 have overlapping substrate profiles. Predicting drug disposition in the setting of altered transport function has important clinical significance. This investigation was designed to establish an in vitro model system to evaluate the impact of impaired Mrp2 and Bcrp function on hepatobiliary drug disposition. To achieve Bcrp knockdown by RNA interference (RNAi), sandwich-cultured hepatocytes (SCH) from Mrp2-deficient (TR(-)) and wild-type (WT) rats were infected with adenoviral vectors to express shRNA targeting Bcrp (Ad-siBcrp) at multiplicity of infection (MOI) of 1-10. MOI of 5 was identified as optimal. At MOI of 5, viral infection as well as WT or TR(-) status was statistically significant predictors of the rosuvastatin (RSV) biliary excretion index (BEI), consistent with the known role of Bcrp and Mrp2 in the biliary excretion of RSV in vivo in rats. Relative to WT rat SCH, marginal mean BEI (%) of RSV in TR(-) rat SCH decreased by 28.6 (95% CI: 5.8-51.3). Ad-siBcrp decreased marginal mean BEI (%) of RSV by 13.3 (7.5-9.1) relative to SCH infected with adenoviral vectors expressing a nontargeting shRNA (Ad-siNT). The BEI of RSV was almost ablated in TR(-) rat SCH with Bcrp knockdown (5.9 ± 3.0%) compared to Ad-siNT-infected WT rat SCH (45.4 ± 6.6%). These results demonstrated the feasibility of Bcrp knockdown in TR(-) rat SCH as an in vitro system to assess the impact of impaired Bcrp and Mrp2 function. At MOI of 5, viral infection had minimal effects on RSV total accumulation, but significantly decreased marginal mean taurocholate total accumulation (pmol/mg of protein) and BEI (%) by 9.9 (7.0-12.8) and 7.5 (3.7-11.3), respectively, relative to noninfected SCH. These findings may be due to off-target effects on hepatic bile acid transporters, even though no changes in protein expression levels of the hepatic bile acid transporters were observed. This study established a strategy for optimization of the knockdown system, and demonstrated the potential use of RNAi in SCH as an in vitro tool to predict altered hepatobiliary drug disposition when canalicular transporters are impaired.
|Molecular and functional characterization of an organic anion transporting polypeptide cloned from human liver. |
Kullak-Ublick, G A, et al.
Gastroenterology, 109: 1274-82 (1995)
BACKGROUND & AIMS: Based on a recently cloned rat liver organic anion transporter, we attempted to clone the corresponding human liver organic anion transporting polypeptide. METHODS: A human liver complementary DNA library was screened with a specific rat liver complementary DNA probe. The human liver transporter was cloned by homology with the rat protein and functionally characterized in Xenopus laevis oocytes. RESULTS: The cloned human liver organic anion transporting polypeptide consists of 670 amino acids and shows a 67% amino acid identity with the corresponding rat liver protein. Injection of in vitro transcribed complementary RNA into frog oocytes resulted in the expression of sodium-independent uptake of [35S]bromosulfophthalein (Michaelis constant [Km], approximately 20 mumol/L), [3H]cholate (Km, approximately 93 mumol/L), [3H]taurocholate (Km, approximately 60 mumol/L), [14C]glycocholate, [3H]taurochenodeoxycholate, and [3H]tauroursodeoxycholate (Km, approximately 19 mumol/L). Northern blot analysis showed cross-reactivity with messenger RNA species from human liver, brain, lung, kidney, and testes. Polymerase chain reaction analysis of genomic DNA from a panel of human-rodent somatic cell hybrids mapped the cloned human organic anion transporter to chromosome 12. CONCLUSIONS: These studies show that the cloned human liver organic anion transporter is closely related to, but probably not identical to, the previously cloned rat liver transporter. Furthermore, its additional localization in a variety of extrahepatic tissues suggests that it plays a fundamental role in overall transepithelial organic anion transport of the human body.
|Expression cloning of a rat liver Na(+)-independent organic anion transporter. |
Jacquemin, E, et al.
Proc. Natl. Acad. Sci. U.S.A., 91: 133-7 (1994)
Using expression cloning in Xenopus laevis oocytes, we have isolated a cDNA encoding a rat liver organic anion-transporting polypeptide (oatp). The cloned oatp mediated Na(+)-independent uptake of sulfobromophthalein (BSP) which was Cl(-)-dependent in the presence of bovine serum albumin (BSA) at low BSP concentrations (e.g., 2 microM). Addition of increasing amounts of BSA had no effects on the maximal velocity of initial BSP uptake, but it increased the Km value from 1.5 microM (no BSA) to 24 microM (BSA/BSP molar ratio, 3.7) and 35 microM (BSA/BSP ratio, 18.4). In addition to BSP, the cloned oatp also mediated Na(+)-independent uptake of conjugated (taurocholate) and unconjugated (cholate) bile acids. Sequence analysis of the cDNA revealed an open reading frame of 2010 nucleotides coding for a protein of 670 amino acids (calculated molecular mass, 74 kDa) with four possible N-linked glycosylation sites and 10 putative transmembrane domains. Translation experiments in vitro indicated that the transporter was indeed glycosylated and that its polypeptide backbone had an apparent molecular mass of 59 kDa. Northern blot analysis with the cloned probe revealed crossreactivity with several mRNA species from rat liver, kidney, brain, lung, skeletal muscle, and proximal colon as well as from liver tissues of mouse and rabbit, but not of skate (Raja erinacea) and human.