Decreased hepatic breast cancer resistance protein expression and function in multidrug resistance-associated protein 2-deficient (TR⁻) rats

Abstract

Multidrug resistance-associated protein (Mrp) 2-deficient (TR(-)) Wistar rats have been used to elucidate the role of Mrp2 in drug disposition. Decreased breast cancer resistance protein (Bcrp) levels were reported in sandwich-cultured hepatocytes (SCH) from TR(-) rats compared with those from wild-type (WT) rats. This study was designed to characterize hepatic Bcrp expression and function in TR(-) rats, using nitrofurantoin and pitavastatin as substrates. Bcrp was knocked down by RNA interference in rat SCH. Antibody BXP53, but not BXP21, specifically detected Bcrp knockdown in SCH. Bcrp protein levels were decreased markedly in TR(-) but not Mrp2-deficient Sprague-Dawley [Eisai hyperbilirubinemic rats (EHBR)] rats. Bcrp mRNA levels were decreased significantly in TR(-) livers as determined by TaqMan real-time reverse transcriptase-polymerase chain reaction. Biliary excretion of nitrofurantoin, a specific Bcrp substrate, was decreased significantly in SCH and isolated perfused livers from TR(-) rats compared with those from WT controls, indicating that hepatic Bcrp function is decreased in TR(-) rats. In Bcrp knockdown SCH, the biliary excretion index and in vitro biliary clearance of pitavastatin were decreased significantly to ∼ 58 and ∼ 52% of control, respectively, indicating that Bcrp plays a role in pitavastatin biliary excretion. Pitavastatin biliary excretion was decreased significantly in perfused livers from TR(-) compared with those from WT rats. In conclusion, expression and function of hepatic Bcrp are decreased significantly in TR(-) rats. The potential role of both Bcrp and Mrp2 should be considered when data generated in TR(-) rats are interpreted. TR(-) and EHBR rats in combination may be useful in differentiating the role of Mrp2 and Bcrp in drug/metabolite disposition.

Fig. 1.

Antibody BXP53 but not BXP21 specifically recognized rat Bcrp in rat SCH. A, Bcrp mRNA levels in Bcrp knockdown (Ad-siBcrp) and control (Ad-siNT) SCH. Two hours after seeding, rat hepatocytes were infected at multiplicity of infection of 20 with adenoviral vectors that expressed small interfering RNA (siRNA) targeting Bcrp (Ad-siBcrp) or nontarget control siRNA (Ad-siNT). Bcrp mRNA levels were determined by TaqMan real-time RT-PCR. Data are presented as the mean ± S.D.; n = 3 livers. *, p < 0.05, Ad-siBcrp versus Ad-siNT. B, immunoblot of Bcrp in Bcrp knockdown (Ad-siBcrp) and control (Ad-siNT) SCH using BXP53 or BXP21 antibodies. β-Actin was used as a loading control.

Fig. 2.

Decreased Bcrp protein levels in UNC TR⁻ and Harlan TR⁻ rat livers but not in EHBR rat livers. A, immunoblot of Bcrp in WT Wistar, UNC TR⁻, Harlan TR⁻, WT SD, and EHBR rat livers and freshly isolated WT rat hepatocytes (day 0) using BXP53 antibody (upper panel). β-Actin was used as a loading control (lower panel). Representative results of three to five rats per group are shown. B, relative Bcrp protein levels in TR⁻ and EHBR rat livers compared with those in WT Wistar or WT SD controls, respectively. Protein levels of Bcrp and β-actin were determined by densitometry. Total Bcrp protein levels (sum of the higher and lower molecular weight Bcrp) were normalized to β-actin. Data are presented as mean ± S.D.; n = 3 (WT Wistar), n = 5 (TR⁻), n = 4 (WT SD), and n = 4 (EHBR) rat livers. *, p < 0.05, TR⁻ or EHBR versus WT control. C, mRNA levels of Bcrp in WT Wistar (control) and TR⁻ rat livers. Bcrp mRNA levels were determined by TaqMan real-time RT-PCR. β-Actin was used as an internal control. Data are presented as mean ± S.D.; n = 8 (WT Wistar) and n = 11 (TR⁻, including two Harlan TR⁻) rat livers. *, p < 0.05, TR⁻ versus WT Wistar.

Fig. 3.

Bcrp expression, nitrofurantoin accumulation, BEI, and in vitro Cl_biliary in rat sandwich-cultured hepatocytes. A, mRNA levels of Bcrp in WT (control) and TR⁻ SCH. Bcrp mRNA levels in day 4 SCH were determined by TaqMan real-time RT-PCR. β-Actin was used as an internal control. Data are presented as the mean ± S.D.; n = 3 livers/group. *, p < 0.05, TR⁻ versus WT. B, nitrofurantoin accumulation, BEI, and in vitro Cl_biliary in cells plus bile (■) and cells (□) after a 10-min incubation with 5 μM nitrofurantoin in day 4 SCH from WT and UNC TR⁻ rats. Data are presented as the mean ± S.E.; n = 4 livers in triplicate. *, p < 0.05, TR⁻ versus WT. C, relationship between nitrofurantoin in vitro Cl_biliary and Bcrp protein levels expressed as a percentage of control values. Nitrofurantoin in vitro Cl_biliary in Ad-siBcrp-infected SCH from WT rats exhibiting extensive Bcrp knockdown (day 6; ●) and moderate Bcrp knockdown (day 4; ○), expressed as a percentage of control (Ad-siNT) values in day 6 and day 4 SCH, respectively, and in day 4 noninfected TR⁻ SCH (▴), expressed as a percentage of control (WT) values in day 4 SCH, were plotted on the y-axis. The in vitro Cl_biliary values were published previously in Fig. 3, B and C, of Yue et al. (2009) and have been replotted here for comparison. Bcrp protein levels (plotted on x-axis) were determined by immunoblot, followed by densitometry and normalized to β-actin, and drawn on the basis of the findings in Yue et al. (2009) and Abe et al. (2008). Data are presented as the mean ± S.D.

Fig. 4.

Decreased nitrofurantoin biliary excretion in TR⁻ rat IPLs. Cumulative biliary excretion of nitrofurantoin in recirculating WT (●) and UNC TR⁻ (○) rat IPLs after bolus administration of 8 μmol of nitrofurantoin to the perfusate reservoir. Data are presented as the mean ± S.D.; n = 3 to 5 livers/group. *, p < 0.05, TR⁻ versus WT.

Fig. 5.

Role of Bcrp in pitavastatin biliary excretion in rat SCH. Pitavastatin BEI and in vitro Cl_biliary (percentage of control) after a 10-min incubation with 5 μM pitavastatin were compared in noninfected, Ad-siNT-infected (control), and Ad-siBcrp-infected day 6 rat SCH. Data represent the mean ± S.D.; n = 3 livers. *, p < 0.05, Ad-siBcrp-infected versus Ad-siNT-infected. See Materials and Methods and the legend to Fig. 1A for details regarding adenoviral vector-mediated RNAi.

Fig. 6.

Decreased biliary excretion of pitavastatin in TR⁻ rat in situ IPLs. Cumulative biliary excretion of pitavastatin in WT (●) and UNC TR⁻ (○) rat in situ single-pass IPLs. Data are presented as mean ± S.D.; n = 3 livers/group; *p < 0.05, TR⁻ versus WT.