Organic anion-transporting polypeptides (rodents: Oatps; human: OATPs) mediate sodium-independent transport of various amphipathic organic solutes, such as bile acids, dyes (e.g., sulfobromophthalein), drugs (e.g., rifampicin, digoxin), toxins (e.g., microcystin), steroid conjugates, eicosanoids, and thyroid hormones into cells. Currently, 11 human OATPs, 13 rat Oatps, and 15 mouse Oatps have been identified. Hydropathy analysis predicts that all OATPs/Oatps contain 12-transmembrane domains. Additionally, all OATPs/Oatps contain the consensus sequence D-X-RW-(I,V)-GAWW-X-G-(F,L)-L at the border of extracellular loop 3 and transmembrane domain 6, termed the “OATP superfamily signature” (Hagenbuch and Meier, 2003).

Only a few of the OATPs/Oatps have been characterized at the functional, structural, and regulatory levels. Rat Oatp1a1, the first cloned member of the OATP/Oatp gene family, is expressed in liver and kidney, with broad substrate specificity (Jacquemin et al., 1994). Rodent Oatp1b2 and human OATP1B1 and 1B3 are selectively expressed in liver, where they are involved in hepatic uptake of albumin-bound compounds (Abe et al., 1999; Konig et al., 2000; Li et al., 2002). Oatp1a6, 1c1, and 4a1 are expressed mainly in kidney, brain, and placenta, respectively, and their physiological roles remain unclear. The recently cloned human and rat OATP4C1/Oatp4c1 transports digoxin across the basolateral membrane of renal proximal tubules (Mikkaichi et al., 2004). Recently, rat Oatp6b1 and 6d1, and human OATP6A1 were cloned (Suzuki et al., 2003). These three transporters are predominantly expressed in testes and transport dehydroepiandrosterone (DHEA) and DHEA sulfate into testes (Suzuki et al., 2003).

Neonates are more sensitive to the toxicity of some chemicals than are adults. This is an important factor to consider in drug therapy, and in chemical exposure. The increased susceptibility of immature rodents to some chemical exposures was first investigated in the late 1950s, and reduced hepatic biotransformation of some chemicals was shown to be responsible for the increased susceptibility (Jondorf et al., 1958; Fouts and Adamson, 1959). However, for a number of chemicals, it has been shown that newborns have a decreased ability to clear chemicals from blood. For example, ouabain is transported into hepatocytes by active transport and is excreted into bile largely without being biotransformed (Eaton and Klaassen, 1978). Newborn rodents are more sensitive to cardiac glycoside toxicity because of their low capacity to remove ouabain from the blood (Klaassen, 1972). In addition, Stacey and Klaassen (1979) showed that ouabain uptake into hepatocytes in newborns is low and increases with age. Liver and kidney are two major detoxication organs that eliminate chemicals from the body. Developmental changes of transporters in both organs can significantly influence the disposition of endo- and exogenous compounds.

Mice are becoming a more commonly used experimental animal model because of the availability of knockout mice. However, information on transporter systems, including Oatps, in mice is poorly characterized. Therefore, the purpose of this study is to determine 1) the constitutive expression of the 15 mouse Oatp genes by measuring their mRNA levels in 12 tissues, 2) whether there are gender differences in mouse Oatp expression, and 3) the ontogeny of mouse Oatps in mouse liver and kidney.

Materials and Methods

Tissue Distribution. Eight-week-old adult male and female C57BL/6 mice (n = 10/gender) were purchased from The Jackson Laboratory (Bar Harbor, ME) and housed according to the American Animal Association Laboratory Animal Care guidelines. Eleven tissues (liver, kidney, lung, stomach, duodenum, jejunum, ileum, large intestine, brain, testis, and ovary) were collected. Placenta was removed from pregnant mice on gestation day 17. The tissues were snap-frozen in liquid nitrogen. The intestine was longitudinally dissected, rinsed in saline, and divided into three equal-length sections (referred to as duodenum, jejunum, and ileum), before being snap-frozen in liquid nitrogen. All tissues were stored at –80°C.

Ontogeny. Mice were bred in the animal facilities at the University of Kansas Medical Center. Liver and kidney from male and female C57BL/6 mice were collected at –2, 0, 5, 10, 15, 22, 30, 35, 40, and 45 days of age (n = 5/gender/age).

RNA Isolation. Total RNA was isolated using RNAzol Bee reagent (Tel-Test Inc., Friendswood, TX) as per the manufacturer's protocol. The concentration of total RNA in each sample was quantified spectrophotometrically at 260 nm. The integrity of each RNA sample was evaluated by formaldehydeagarose gel electrophoresis before analysis.

Branched DNA (bDNA) Signal Amplification Assay. The bDNA technique is a robust, high-throughput method used for characterizing mRNA expression. The mRNA for Oatp1a1, 1a4, 1a5, 1a6, 1b2, 1c1, 2a1, 2b1, 3a1, 4a1, 4c1, 5a1, 6b1, 6c1, and 6d1 was measured using the bDNA assay (QuantiGene bDNA signal amplification kit; Bayer Corp.-Diagnostics Div. (Tarrytown, NY), with modifications according to the method of Hartley and Klaassen (2000). The gene sequences of mouse Oatps were accessed from GenBank (Table 1). Multiple oligonucleotide probe sets (containing capture extenders, label extenders, and blockers) specific to a single mRNA transcript were designed using ProbeDesigner software, version 1.0 (Bayer Corp., Emeryville, CA). Probe sets for each Oatp are shown in Table 2. Each probe developed in ProbeDesigner was submitted to the National Center for Biotechnology Information (Bethesda, MD) for nucleotide comparison by the basic local alignment search tool (BLASTn) to ensure minimal cross-reactivity with other known mouse sequences and expressed sequence tags. Oligonucleotides with a high degree of similarity (>80%) to other mouse gene transcripts were eliminated from the design. Probes were designed with a melting temperature of approximately 63°C, enabling hybridization conditions to be held constant (i.e., 53°C) during each hybridization step, and for each probe set. All probes were synthesized (50-nmol synthesis scale) by QIAGEN Operon (Alameda, CA) and were obtained desalted and lyophilized. Total RNA (1 μg/μl) was added to each well (10 μl/well) of a 96-well plate containing 50 μl of capture hybridization buffer and 50 μl of diluted probe set. For each gene, total RNA was allowed to hybridize to the probe set overnight at 53°C. Subsequent hybridization steps were carried out per the manufacturer's protocol, and luminescence was measured with a Quantiplex 320 bDNA luminometer interfaced with Quantiplex data management software (version 5.02), for analysis of luminescence from 96-well plates. The luminescence for each well is reported as relative light units per 10 μg of total RNA.

Statistics. Differences between genders were determined by Student's t test. Statistical significance was considered at p < 0.05.

Results

Tissue Distribution of Mouse Oatps. mRNA expression of 15 mouse Oatps was quantified in 12 major tissues. Data are shown in Figs. 1, 2, 3, 4. Expression of Oatp1a1 mRNA (Fig. 1) was highest in liver and kidney of male mice, but its expression in other tissues was much lower. A gender difference in Oatp1a1 mRNA expression was observed in both liver and kidney, with higher levels in males. Expression of Oatp1a4 mRNA (Fig. 1) was highest in liver and brain, followed by testes and ovaries. A gender difference in Oatp1a4 expression in liver was observed, with higher levels being expressed in females. Expression of Oatp1a5 mRNA (Fig. 1) was highest in testes, moderate in ovary, and low in the other tissues. Oatp1a6 mRNA (Fig. 1) was almost exclusively expressed in kidney among the collected tissues. Oatp1a6 mRNA was expressed much less in liver than in kidney. However, a gender difference of Oatp1a6 was noted in liver, with higher levels in females.

Expression of Oatp1b2 mRNA (Fig. 2) was predominant in liver, with negligible expression in the other 11 tissues. Oatp1c1 (Fig. 2) was the only Oatp primarily expressed in brain. Oatp2a1, a prostaglandin transporter, was highest in placenta, moderate in lung and stomach, and lower in other tissues (Fig. 2). Oatp2b1 (Fig. 2) appeared to be ubiquitously expressed, with highest levels in liver and small intestine.

Oatp3a1 (Fig. 3) expression was highest in kidney and lung, with moderate expression in testes and ovaries. In kidney, Oatp3a1 mRNA was 3.3-fold higher in males than in females, as previously noted (Melia et al., 1998). Also shown in Fig. 3, mouse Oatp4a1, like rat Oatp4a1, was abundantly expressed only in placenta (Leazer and Klaassen, 2003). Oatp4c1 (Fig. 3) was mainly found in lung and kidney. A gender difference in renal expression of Oatp4c1 was observed, with higher levels in males than in females. Oatp5a1 (Fig. 3) expression was predominant in placenta, with lower levels in testes and lung. Oatp6b1, 6c1, and 6d1 (Fig. 4), three mouse homologs of the human gonad-specific transporter, were exclusively expressed in testes.

Ontogeny of Oatps in Male and Female Mouse Liver. The neonatal patterns of Oatp mRNA expression in male and female mouse liver are shown in Figs. 5 and 6. In adult mice, Oatp1a1 expression was mainly detected in liver and kidney, with higher levels in males than in females. As shown in Fig. 5, there was minimal expression of Oatp1a1 in mouse liver before 15 days of age. Thereafter, hepatic Oatp1a1 levels reached detectable levels at day 23 and reached adult levels by 30 days of age. Male-predominant expression was observed at all ages when Oatp1a1 was detectable (day 23 and thereafter). Figure 5 illustrates that the expression of Oatp1a4 was also low at birth but gradually increased to adult levels by approximately 23 days of age. The levels of Oatp1a4 mRNA in males decreased by 30 days of age, resulting in females expressing more Oatp1a4 than males. The ontogeny of Oatp1a6 expression is also shown in Fig. 5. Expression of Oatp1a6 mRNA was detected in liver of mice of all ages. Between days 5 and 10, there was an increase of Oatp1a6 mRNA. Similar to that observed for Oatp1a4, Oatp1a6 decreased in males between 15 and 45 days of age, resulting in higher Oatp1a6 expression in liver of adult females than that of males.

Oatp1b2 is a relatively liver-specific transporter, as indicated in Fig. 2. The expression of Oatp1b2 was minimal 2 days before birth, but its expression between birth and 10 days of age was about half that seen in adult mice (Fig. 6). Adult levels of Oatp1b2 were attained by 23 days of age. No gender difference in expression of Oatp1b2 was observed at any age. Oatp2a1 (prostaglandin transporter) expression in liver was similar at all ages. Oatp2b1 expression at different ages is also shown in Fig. 6. Before 15 days of age, Oatp2b1 expression was minimal, increased moderately at day 15, and reached adult levels by 23 days of age.

Ontogeny of Oatps in Male and Female Mouse Kidney. The postnatal developmental patterns of Oatp mRNA expression in male and female mouse kidney are shown in Figs. 7 and 8. As shown in Fig. 7, there was minimal expression of Oatp1a1 in mouse kidney at 22 days of age, followed by detectable levels at day 30, and reaching adult levels by 45 days of age. The male-predominant expression was observed at day 30 and 45. Figure 7 illustrates that the expression of Oatp1a4 was similar at all ages. In contrast to Oatp1a4 expression in liver, Oatp1a4 shows no gender difference in kidney. The expression of Oatp1a6 was low at birth (Fig. 7) and gradually increased until 15 days of age, when Oatp1a6 reached adult levels.

Oatp2a1 mRNA expression in both male and female mouse kidney was similar at all ages after birth, but was lower before birth (Fig. 8). Ontogenic expression of Oatp2b1 in kidney was similar to that of Oatp2a1 expression, being expressed at a low level 2 days before birth, but increasing to adult levels at birth. Oatp3a1 expression in mouse kidney was low before 30 days of age, but at 30 days of age, the expression of Oatp3a1 in males increased, whereas it remained low in the females.

Discussion

The expression of mouse Oatps varies among tissues. The Oatps that have relatively high expression in liver are Oatp1a1, 1a4, 1b2, and 2b1, among which Oatp1b2 is almost exclusively expressed in liver. The Oatps that have relatively high mRNA expression in kidney are Oatp1a1, 1a6, 3a1, and 4c1. Furthermore, Oatp1a4 and 1c1 are highly expressed in brain. Oatp1a5, 6b1, 6c1, and 6d1 are predominantly expressed in testes, and Oatp2a1, 4a1, and 5a1 are predominantly expressed in placenta.

The tissue distribution of Oatp homologs in rats and mice is similar, but not identical. In both rats and mice, Oatp1a1 is highly expressed in liver and kidney (Li et al., 2002), Oatp1a4 in liver and brain (Noé et al., 1997; Guo et al., 2002a), Oatp1a6 in kidney (Ogura et al., 2000; Choudhuri et al., 2001), Oatp1b2 in liver (Li et al., 2002), Oatp1c1 in brain (Sugiyama et al., 2003), Oatp4a1 in placenta (Leazer and Klaassen, 2003), and Oatp6b1 and 6c1 in testes (Suzuki et al., 2003). The high expression of Oatp1a5 in mouse testes has not been reported previously, whereas in rats, it has been shown that Oatp1a5 is highly expressed in choroid plexus (Choudhuri et al., 2003), moderate in the retina (Abe et al., 1998), and slight in liver, kidney, ileum, and testes (Choudhuri et al., 2003; Augustine et al., 2005). Also, there is no mouse or human homolog of rat Oatp1a3 (Oat-K1), which is highly expressed in rat kidney (Saito et al., 1996). It should also be noted that humans have only one OATP1A subfamily member (OATP1A2), whereas rodents have five members (Oatplal, 1a3, 1a4, 1a5, and 1a6). Also, other Oatp subfamily members have comparable tissue distribution in humans and mice. For example, OATP1B/Oatp1b members (mouse Oatp1b2, human OATP1B1 and 1B3) are predominantly expressed in liver of both humans and mice (Abe et al., 1999; Li et al., 2002). OATP1C1/Oatp1c1 is detected in brain (Sugiyama et al., 2003). OATP2A1/Oatp2a1 and 2B1/2b1 are expressed ubiquitously (Lu et al., 1996a; Nishio et al., 2000). OATP6/Oatp6 subfamily members (mouse 6b1, 6c1, and 6d1, human OATP6A1) are exclusively expressed in testes of humans and mice (Suzuki et al., 2003).

Organ-specific expression of Oatps contributes to tissue-specific distribution of drugs and to tissue-specific toxicity. For example, pravastatin must enter the liver to exert its antilipidemic effect. Human OATP1B1 has been shown to transport pravastatin with high affinity (Hsiang et al., 1999). Phalloidin and microcystin both selectively accumulate in liver, leading to hepatotoxicity. Rat Oatp1b2, human OATP1B1, and OATP1B3 are known to transport phalloidin and microcystin (reviewed by Hagenbuch and Meier, 2003). Conversely, poor extraction of a drug by liver (due to a deficiency in uptake transporters) can cause elevated plasma levels of the drug, leading to toxicities in organs other than liver. A well defined example is the uptake of cardiac glycosides into neonatal rat liver, which is immature with respect to adult liver, resulting in a LD₅₀ for ouabain in newborns about 1/100th that in adult rats (Klaassen, 1972). This observation corresponds well with the low expression of Oatp1a4 in liver of young rats (Guo et al., 2002a) and the finding that Oatp1a4 transports cardiac glycosides with high affinity (Noé et al., 1997). Therefore, understanding tissue distribution and substrate specificity of various Oatps will help predict tissue distribution of Oatp substrates and provide information concerning which Oatp should be targeted or avoided when developing a new drug.

The present study indicates that there is relatively poor expression of mouse Oatps in the gastrointestinal tract, namely the stomach, duodenum, jejunum, ileum, and large intestine. Of the Oatps, only Oatp2b1 has relatively high expression in intestine. The relatively poor expression of Oatps in the intestine suggests that Oatps probably play a relatively minor role in intestinal absorption of chemicals. Actually, it is generally believed that most xenobiotics are mainly absorbed from the intestine by simple diffusion (Schwenk, 1987). Instead, Oatps appear to play an important role in the distribution and elimination of chemicals, as suggested by the high expression of Oatps in liver, kidney, lung, brain, testes, and placenta. As previously stated, Oatp1b2 is responsible for transport of microcystin and phalloidin into liver to produce hepatotoxicity, as well as uptake of sulfobromophthalein (BSP) into liver for subsequent excretion into bile.

Gender differences in Oatp1a1 expression were observed in mouse liver and kidney, with higher expression in males than in females. It has been shown that in rat and mouse kidney, male-predominant expression of Oatp1a1 is androgen-dependent (Lu et al., 1996; Isern et al., 2001). This gender-specific pattern may explain some physiological/toxicological phenomena. For instance, Oatp1a1 is localized to the apical membrane domain of proximal tubules in kidney, where it reabsorbs organic anions from the lumen. The male-predominant expression of Oatp1a1 may be responsible for the 250-fold higher rate of urinary excretion of exogenously administered radioactive estradiol-17β-d-glucuronide in females as compared with male rats (Gotoh et al., 2002).

In contrast to the lack of gender differences in the expression of Oatp1a4 and 1a6 in rats, gender differences in the expression of these two transporters were observed in mouse liver, with higher expression in females than in males, the opposite of Oatp1a1 (Li et al., 2002). Oatp3a1 and 4c1 were expressed at higher levels in female kidney than in males. These gender differences might be due to sex hormones or due to gender-related secretion patterns of growth hormone (Noshiro and Negishi, 1986; Waxman et al., 1991; Aida and Negishi, 1993; Buist et al., 2003). Furthermore, investigation is needed to elucidate the regulatory mechanisms of these gender-related differences in Oatp expression.

The ontogenic expression of the various Oatps in mouse liver exhibited different developmental patterns of expression. The low expression of Oatp1a4 in development may result in a higher toxicity of ouabain in newborn mice. In newborn rats, low expression of Oatp1a4 results in less uptake of ouabain into liver, and thus, high amounts of ouabain remain in blood and other tissues, causing toxicity (Klaassen, 1972; Guo et al., 2002a). The importance of Oatp1a4 has been further illustrated by the finding that pregnenalone-16α-carbonitrile (PCN), a prototypical rodent pregnane X receptor ligand, stimulates hepatic clearance of cardiac glycosides in newborn rats, resulting in decreased toxicity of cardiac glycosides (Klaassen, 1974a,b). Oatp1a4 is a sinusoidal hepatic uptake transporter, with high affinity for cardiac glycosides (Noé et al., 1997). PCN treatment dramatically accelerates the maturation of hepatic Oatp1a4 mRNA and protein levels in neonatal rats (Guo et al., 2002a). Thus, newborn rats are more sensitive to cardiac glycoside toxicity because of their low capacity to remove ouabain from the blood, and PCN protects newborns from cardiac glycoside toxicity because it induces Oatp1a4 and the uptake of ouabain into liver.

Kidney is another important organ for chemical disposition. The kidney in newborns is immature and renal functions are limited. The present data indicate that renal expression of Oatps is minimal at or before birth, and then gradually increases to adult levels. Low renal expression of Oatps in newborns may protect the kidney from toxicity of Oatp substrates.

Oatp6b1, 6c1, and 6d1 are mouse homologs of human and rat gonad-specific transporters and are thought to be responsible for testicular uptake of DHEA and DHEA sulfate, precursors of in vivo androgen and, thus, estrogen biosynthesis (Suzuki et al., 2003). In this study, Oatp6b1, 6c1, and 6d1 were found to be exclusively expressed in testes.

In conclusion, the present study shows that mouse Oatp expression is highly variable among tissues. There are also gender- and age-related differences in the expression of these uptake transporters. These variances most likely result in tissue-, gender-, and age-related differences in the pharmaco- and toxicokinetic profiles of xenobiotics. Furthermore, the similarities and differences in Oatp expression among rat, mouse, and human will aid in extrapolation of rodent pharmacokinetic data to humans.