Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs - PubMed (original) (raw)

. 2010 Aug;120(8):2942-52.

doi: 10.1172/JCI42168. Epub 2010 Jul 19.

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Organic anion transporting polypeptide 1a/1b-knockout mice provide insights into hepatic handling of bilirubin, bile acids, and drugs

Evita van de Steeg et al. J Clin Invest. 2010 Aug.

Abstract

Organic anion transporting polypeptides (OATPs) are uptake transporters for a broad range of endogenous compounds and xenobiotics. To investigate the physiologic and pharmacologic roles of OATPs of the 1A and 1B subfamilies, we generated mice lacking all established and predicted mouse Oatp1a/1b transporters (referred to as Slco1a/1b-/- mice, as SLCO genes encode OATPs). Slco1a/1b-/- mice were viable and fertile but exhibited markedly increased plasma levels of bilirubin conjugated to glucuronide and increased plasma levels of unconjugated bile acids. The unexpected conjugated hyperbilirubinemia indicates that Oatp1a/1b transporters normally mediate extensive hepatic reuptake of glucuronidated bilirubin. We therefore hypothesized that substantial sinusoidal secretion and subsequent Oatp1a/1b-mediated reuptake of glucuronidated compounds can occur in hepatocytes under physiologic conditions. This alters our perspective on normal liver functioning. Slco1a/1b-/- mice also showed drastically decreased hepatic uptake and consequently increased systemic exposure following i.v. or oral administration of the OATP substrate drugs methotrexate and fexofenadine. Importantly, intestinal absorption of oral methotrexate or fexofenadine was not affected in Slco1a/1b-/- mice. Further analysis showed that rifampicin was an effective and specific Oatp1a/1b inhibitor in controlling methotrexate pharmacokinetics. These data indicate that Oatp1a/1b transporters play an essential role in hepatic reuptake of conjugated bilirubin and uptake of unconjugated bile acids and drugs. Slco1a/1b-/- mice will provide excellent tools to study further the role of Oatp1a/1b transporters in physiology and drug disposition.

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Figures

Figure 1

Figure 1. Generation of Slco1a/1b–/– mice.

(A) To delete all the Slco1a and Slco1b genes, an inverted _Pgk_-neomycin cassette with loxP sequences was inserted, replacing exons 3 and 4 of Slco1a5. Additionally, a _Pgk_-thymidine kinase and a _Pgk_-hygromycin cassette with loxP sequences were inserted, replacing exon 4 of Slco1b2. The Slco1a/1b cluster was subsequently excised from the genome by Pgk-Cre recombinase transfection. Structure of the region after deletion of the Slco1a/1b cluster is shown as well. Probes (5′ and 3′) used for Southern blot analyses (positioned outside the respective targeting constructs) and forward (F1 and F2) and reverse (R1) primers used for PCR analyses are indicated by lines below the DNA backbone. Gene A and gene B represent _Slco1a_-like predicted genes EG435927 and EG625716 (according to the NCBI database), respectively. Triangles indicate loxP sequences, numbered boxes indicate exons, and arrows indicate transcriptional orientation. Centr, centromere; Tel, telomere; S, SacI; X, XbaI. (B) Southern blot analysis of tail DNA from WT and Slco1a/1b–/– mice digested with Asp718 and probed with partial Slco1a* or Slco1b2 cDNA. (C) RT-PCR analysis of Slco1a/1b genes in liver, kidney, and small intestine (SI) of WT and Slco1a/1b–/– mice. (D) Western blot analysis of crude membrane fractions of livers derived from WT and Slco1a/1b–/– mice. Blots were probed with either an antibody raised against mouse Oatp1a4 (sc-18436) or Oatp1b2 (sc-47270).

Figure 2

Figure 2. Analysis of plasma, bile, and urine from male WT and Slco1a/1b–/– mice.

Levels of total bilirubin, BMG, BDG, and UCB in plasma (A), bile (C), and urine (D) and levels of total bile acids, conjugated bile acids, and unconjugated bile acids in plasma (B) are shown. Bile was collected for 15 minutes after gallbladder cannulation and ligation of the common bile duct. Urine was collected by spot sampling from nonanesthetized mice. Data are presented as mean ± SD (n = 5–7; *P < 0.05; **P < 0.01; ***P < 0.001 when compared with WT). ND, not detectable; detection limits were 0.3 μM for plasma and urine and 2 μM for bile, i.e., ~5 pmol•min–1•g liver–1.

Figure 3

Figure 3. Role of hepatic Oatp1a/1b transporters in MTX disposition.

Pharmacokinetics of MTX after i.v. or oral administration of MTX (10 mg/kg) to female WT and Slco1a/1b–/– mice. MTX plasma concentration versus time curves after i.v. (A) or oral (B) administration, with the inset showing the semi-log plot of the i.v. data. (C) MTX liver levels (% of dose) versus time curves after i.v. administration. (D) MTX portal vein plasma concentration versus time curves after oral administration. All data are presented as mean ± SD (n = 4–8; *P < 0.05; **P < 0.01; ***P < 0.001 when compared with WT).

Figure 4

Figure 4. Urinary and fecal excretion of MTX (% of dose) in the first 24 hours after i.v. administration of MTX (10 mg/kg) to male WT and Slco1a/1b–/– mice.

All bars present mean ± SD (n = 6; **P < 0.01 when compared with WT).

Figure 5

Figure 5. Role of hepatic Oatp1a/1b transporters in FEX disposition.

Pharmacokinetics of FEX after i.v. or oral administration of [3H]FEX (1 mg/kg) to male WT and Slco1a/1b–/– mice. (A) [3H]FEX plasma concentration versus time curves after i.v. (A) or oral (B) administration, with the inset showing the semi-log plot of the i.v. data. (C) [3H]FEX liver levels (% of dose) versus time curves after i.v. administration. (D) [3H]FEX plasma concentration versus time curves after oral administration. All data are presented as mean ± SD (n = 4–5; *P < 0.05; **P < 0.01; ***P < 0.001 when compared with WT).

Figure 6

Figure 6. The effect of Oatp inhibition by rifampicin on MTX disposition in female WT and Slco1a/1b–/– mice.

MTX plasma concentration (A), MTX liver levels (% of dose) (B), and 7OH-MTX liver levels (% of dose) (C) 15 minutes after i.v. administration of MTX (10 mg/kg) with or without predosing of rifampicin (20 mg/kg). If applicable, rifampicin or vehicle was administered i.v. 3 minutes before MTX administration. All data are presented as mean ± SD (n = 5; **P < 0.01; ***P < 0.001 when compared with vehicle treatment).

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