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TISSUE DISTRIBUTION AND HEPATIC AND RENAL ONTOGENY OF THE MULTIDRUG RESISTANCE-ASSOCIATED PROTEIN (MRP) FAMILY IN MICE

Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas (J.M.M., A.L.S., X.C., C.D.K.); and Department of Pharmacology and Toxicology, College of Pharmacy, University of Arizona, Tucson, Arizona (N.J.C.)

(Received January 24, 2005; accepted March 30, 2005)

	Abstract

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Analysis of the mouse genome has revealed eight multidrug resistance-associated (Mrp) transporters, with mouse homologs for all human MRPs except MRP8. Whereas MRP expression in tissues of humans and rats has been examined, no characterization exists for mice. Furthermore, the ontogeny of mouse Mrps is unknown, and such knowledge may be helpful in understanding age-related pharmacokinetics. Therefore, the purpose of this study was to quantitatively determine 1) expression of the Mrp family in 12 different tissues, 2) gender variations in Mrp expression in liver and kidney, and 3) whether Mrp expression is altered during development. Highest expression of the Mrp family members is as follows: Mrp1 in testes, ovary, and placenta; Mrp2 in intestine, followed by liver and kidney; Mrp3 in large intestine; Mrp4 in kidney; Mrp5 in brain, followed by lung and stomach; Mrp6 in liver; Mrp7 in testes, intestine, and kidney; and Mrp9 solely in testes. Gender differences in Mrp expression were observed: Mrp1, 3, and 4 in kidney, as well as Mrp1 and 4 in liver were female-predominant. Ontogeny of the four Mrps expressed in liver was as follows: Mrp2 and Mrp4 were expressed at adult levels at birth; Mrp3 reached adult levels at day 30, and Mrp6 was not expressed until day 10. In kidney, Mrp1 and Mrp5 were expressed at adult levels at birth, whereas Mrp2, 3, 4, and 6 generally increased over time. In conclusion, marked differences in expression of the individual Mrp family members exist in various tissues, with age, and with gender.

As indicated by functional studies, MRPs are efflux transporters for structurally diverse amphipathic chemicals and organic anions. MRP1, 2, and 3 confer resistance to a variety of anticancer drugs including anthracyclines, vinca alkaloids, and methotrexate, and transport organic anions such as glutathione and glucuronide conjugates. Unlike MRP1, 2, and 3, MRP4 and 5 do not confer resistance to anthracyclines or vinca alkaloids. Overexpression of MRP4 and 5 is associated with increased cellular efflux of purine analogs (e.g., 6-mercaptopurine and thioguanine) and nucleoside-based antiviral drugs (e.g., adefovir) (Schuetz et al., 1999; Wielinga et al., 2002). MRP4 and 5 also transport cyclic nucleotides, such as cAMP and cGMP (Wielinga et al., 2002). Furthermore, several compounds of physiological and pharmacological importance, such as methotrexate, estradiol-17ß-glucuronide, bile acids, prostaglandins, and dehydroepiandrosterone-3-sulfate, were recently shown to be transported by MRP4 (Chen et al., 2001; Wielinga et al., 2002; Reid et al., 2003; Zelcer et al., 2003). Mutations in human MRP6 are associated with pseudoxanthoma elasticum, a hereditary disease characterized by progressive dystrophic mineralization of elastic fibers (Bergen et al., 2000). In vitro transport studies showed that MRP6 transports the anionic cyclopentapeptide and endothelin antagonist BQ-123 (Madon et al., 2000) and glutathione conjugates, and that MRP6 expression in tumor cells can confer weak resistance to some anticancer drugs (Belinsky et al., 2002). Recently, MRP7 was shown to transport estradiol-17ß-glucuronide, but not cyclic nucleotides, methotrexate, or bile acids (Chen et al., 2003), whereas MRP8 was reported to transport cyclic nucleotides (Guo et al., 2003). No functional studies have been reported for MRP9.

Neonatal sensitivity to various chemicals has always been of concern to clinicians. The liver serves a critical role in the pharmacokinetics of xenobiotics. It is known that expression of some drugmetabolizing enzymes is low in neonates, and this is often exacerbated in infants born premature. For example, development of UDP-glucuronosyltransferases plays a significant role in the metabolism and elimination of endogenous and exogenous chemicals, and insufficiency during development can lead to 1) hyperbilirubinemia, 2) kernicterus, and 3) gray-baby syndrome (Kawade and Onishi, 1981).

The development of transporters is not well characterized, although some data suggest that lower expression of transporters in young animals is important in the disposition of drugs and other chemicals. For example, neonatal rats are sensitive to cardiac glycoside toxicity because expression of the uptake transporter Oatp2 is low in liver, thus delaying the elimination of cardiac glycosides and increasing toxicity (Guo et al., 2002). Similarly, neonatal jaundice may be due in part to poor expression of the hepatic canalicular transporter Mrp2 (Johnson et al., 2002; Huang et al., 2003).

Tissue distribution and ontogeny data are an important component of understanding and extrapolating pharmacokinetic data from mice to humans. Knowledge of the expression patterns of mouse Mrps is limited. Therefore, the purpose of this study is to determine 1) the mRNA expression of mouse Mrp transporters in 12 different tissues, 2) whether gender differences in Mrp mRNA expression exist between male and female mice in various tissues, and 3) Mrp mRNA expression in liver and kidney from prenatal day 2 to 45 days of age.

	Materials and Methods

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Animals. Male and female C57BL/6 mice (n = 10 per gender) were purchased from The Jackson Laboratory (Bar Harbor, ME), and tissues were collected at approximately 8 weeks of age. The following tissues were taken: liver, kidney, lung, stomach, duodenum, jejunum, ileum, large intestine, brain, testes, term placenta from pregnant dams (n = 5), and pooled ovaries (n = 5 samples; 1 sample = 10 ovary pairs from 10 individual mice). Ovaries were purchased from Charles River Breeding Laboratories (Portage, MI) from C57BL/6 mice undergoing ovariectomy for other experimental purposes. Tissues were snap-frozen in liquid nitrogen and stored at –80°C.

Ontogeny. Mice (C57BL/6) were bred at the University of Kansas Medical Center laboratory animal facilities, and livers and kidneys were collected from male and female mice at –2, 0, 5, 10, 15, 23, 30, 35, 40, and 45 days of age (n = 5/gender/age).

RNA Extraction. Total RNA was isolated using the RNA Bee reagent (Tel-Test Inc., Friendswood, TX) according to the manufacturer's instructions. RNA concentrations were determined spectrophotometrically, and quality of RNA was determined by gel electrophoresis.

Development of Specific Oligonucleotide Probe Sets for bDNA Analysis. The Mrp gene sequences were accessed from GenBank (Table 1). The target sequences were analyzed by ProbeDesigner Software Version 1.0 (Bayer Corp., Emeryville, CA), and the probe design and target regions are designated in Table 2. The oligonucleotide probes were specific for only one mRNA transcript. All oligonucleotide probes were designed with a T_m of approximately 63°C, enabling optimal hybridization conditions. Each probe set was submitted to the National Center for Biotechnology Information for nucleotide comparison by the basic local alignment search tool (BLASTn) to ensure minimal cross-reactivity with other mouse genomic sequences and expressed sequence tags.

Branched DNA Assay. The specific Mrp oligonucleotide probes were diluted in Tris-EDTA buffer, pH 8.0, according to instructions provided with the QuantiGene bDNA Signal Amplification Kit (GeneSpectra, Fremont, CA). Total RNA (1 µg/µl; 10 µl) was added to each well of a 96-well plate containing 50 µl of capture hybridization buffer and 50 µl of each diluted probe set. Total RNA was allowed to hybridize overnight at 53°C in a hybridization oven. Subsequent hybridization steps were carried out according to the manufacturer's protocol, and luminescence was measured with a Quantiplex 320 bDNA luminometer (Bayer Corp.-Diagnostics Div.), interfaced with Quantiplex Data Management Software Version 5.02 for analysis of luminescence from 96-well plates.

Statistical Analysis. Error bars represent standard errors of the mean. Data were analyzed by a two-tailed Student's t test. Asterisks represent statistical differences (P 0.05) in mRNA levels between male and female mice.

	Results

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Tissue Distribution of the Mouse Mrp Family. Mouse Mrp1 mRNA expression was quite low in most of the tissues examined, but was highly expressed in gonads and placenta (Fig. 1). Expression of Mrp1 in liver was very low, yet detectable, with higher levels in livers from female than from male mice. Expression of Mrp2 was highest in small intestine, decreasing from duodenum to ileum (Fig. 1). High expression of Mrp2 was also observed in liver and kidney. No gender differences in Mrp2 mRNA expression were observed for the tissues analyzed. Mrp3 was highest in the large intestine, with expression detected throughout the gastrointestinal tract (Fig. 1). Mrp3 mRNA was moderately expressed in liver and kidney. Marked gender-specific expression was observed in kidney, with Mrp3 expressed about 14-fold higher in female than in male kidney. Mrp4 expression was highest in kidney, with moderate expression in ovary, lung, stomach, and liver (Fig. 1). Female predominant expression of Mrp4 was observed in liver and kidney, where it was approximately 2- to 2.5-fold higher, respectively, in female than in male mice.

View larger version (25K): [in this window] [in a new window]   FIG. 1. Tissue distribution of Mrp1-4 mRNA in 12 mouse tissues: liver, kidney, lung, stomach, duodenum, jejunum, ileum, large intestine, brain, testes, ovary, and placenta. Data are quantified by the bDNA assay and expressed as mean relative light units ± S.E.M. Asterisks represent statistically significant differences between male and female mice (P < 0.05).

View larger version (24K): [in this window] [in a new window]   FIG. 2. Tissue distribution of Mrp5, 6, 7, and 9 mRNA in 12 mouse tissues: liver, kidney, lung, stomach, duodenum, jejunum, ileum, large intestine, brain, testes, ovary, and placenta. Data are quantified by the bDNA assay and expressed as mean relative light units ± S.E.M. Asterisks represent statistically significant differences between male and female mice (P < 0.05).

View larger version (27K): [in this window] [in a new window]   FIG. 3. Ontogeny of Mrp2, 3, 4, and 6 mRNA expression in male and female mouse liver. Livers from male and female mice were removed on prenatal day –2 and postpartum days 0, 5, 10, 15, 23, 30, 35, 40, and 45. All data are expressed as mean relative light units ± S.EM. for a minimum of five male and five female mice in each age group. Asterisks denote a statistical significance between male and female mice (P < 0.05).

View larger version (25K): [in this window] [in a new window]   FIG. 4. Ontogeny of Mrp1, 2, and 3 expression in male and female mouse kidney. Kidneys from male and female mice were removed on prenatal day –2 and postpartum days 0, 5, 10, 15, 22, 30, and 45. All data are expressed as mean relative light units ± S.EM. for a minimum of five male and five female mice in each age group. Asterisks denote a statistical significance between male and female mice (P < 0.05).

View larger version (26K): [in this window] [in a new window]   FIG. 5. Ontogeny of Mrp4, 5, and 6 expression in male and female mouse kidney. Kidneys from male and female mice were removed on prenatal day –2 and postpartum days 0, 5, 10, 15, 22, 30, and 45. All data are expressed as mean relative light units ± S.EM. for a minimum of five male and five female mice in each age group. Asterisks denote a statistical significance between male and female mice (P < 0.05).

	Discussion

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Mrp1 is highly expressed in reproductive tissues such as testes, ovaries, and placenta in mice (Fig. 1). Humans have high expression of MRP1 in lung, bladder, spleen, testes, thyroid, and adrenal glands (Zaman et al., 1994; Kruh et al., 1995); however, the data from humans (Northern blotting) indicates little variation among these tissues, and it is thus difficult to compare these studies to the present data obtained in mice. Contrasting data between species exist, with modest MRP1 expression in ovary and placenta of humans, yet high expression in mice. Rats have the highest expression of Mrp1 in large intestine and stomach, but reproductive tissues were not examined (Cherrington et al., 2002). Marked expression of mouse Mrp1 in testes, ovary, and placenta is suggestive of a physiological function other than efflux of xenobiotics and chemotherapeutic drugs. The functional involvement of Mrp1 as an efflux transporter in testes has been demonstrated, and Mrp1 serves to protect the testes from chemotherapeutic agents, such as etoposide (Wijnholds et al., 1997). MRP1 has been shown to transport steroid conjugates such as 17ß-estradiol-glucuronide with high affinity (Jedlitschky et al., 1996); however, high expression of Mrp1 in ovary/testes could suggest involvement in unconjugated steroid transport.

Mrp2 expression in mice is highest in small intestine, followed by liver and kidney. This parallels data from humans and rats, in which expression of MRP2 is also high in liver and intestine (Kool et al., 1997; Cherrington et al., 2002). In liver, MRP2 serves as the canalicular efflux pump for many organic anions that are transported across the canalicular membrane into bile. Mutations in the MRP2 gene in humans (Dubin-Johnson syndrome) and rats (TR^–, EHBR) results in conjugated hyperbilirubinemia, due to a decreased ability to excrete bilirubin-glucuronides (Ito et al., 1997; Toh et al., 1999). Prominent expression of Mrp2 in the small intestine suggests that Mrp2 is involved in excretion of xenobiotics into the intestinal lumen. Deficiencies in intestinal Mrp2 expression have been linked to alterations in intestinal transport (Dietrich et al., 2001).

MRP3 expression is highest in large intestine in humans, mice, and rats. In mice, Mrp3 is highly expressed in colon, with significant expression throughout the digestive tract, liver, and kidney (Fig. 1). Studies using isolated basolateral membrane vesicles from rat intestine suggest that Mrp3 is involved in intestinal transport (Shoji et al., 2004).

Mrp3 is a highly inducible, retrograde transporter that can efflux organic anions from hepatocytes into blood for eventual excretion into urine (Slitt et al., 2003; Trauner and Boyer, 2003). Mrp3 is moderately expressed in livers of mice (Fig. 1) and humans (Kool et al., 1997; Cherrington et al., 2002), but in rat is barely detectable.

In mice, Mrp4 is predominantly expressed in kidney, with moderate expression in stomach and ovary (Fig. 1). In rats and humans, Mrp4 is most highly expressed in kidney and lung (Kool et al., 1997; Chen and Klaassen, 2004). In rat kidney, Mrp4 is expressed in the apical membrane of proximal tubules and has been functionally described as important in urinary efflux of cAMP and cGMP, while also playing an important role in the blood-brain barrier (van Aubel et al., 2002; Leggas et al., 2004). High expression in kidney and low expression in liver suggest that the role of Mrp4 in hepatic transport is minor, under naive conditions.

Mrp5 in mice is predominantly expressed in brain with significant expression in gonads, placenta, lung, and stomach. In humans, MRP5 is ubiquitously expressed, with highest expression in skeletal muscle, followed by brain (Kool et al., 1997). Rat Mrp5 expression has not been fully characterized; however, efflux of adefovir, an antiviral compound, from rat brain has been correlated with Mrp5 expression (Dallas et al., 2004).

In mouse, highest expression of Mrp6 is in liver, with minor expression in proximal portions of the intestine as well (Fig. 2). Similarly, humans and rats have high MRP6 expression in liver, as well as kidney and intestine (Kool et al., 1999; Madon et al., 2000). Whereas expression of MRP6 is consistently high in liver of all three species, the functional significance of this expression is not understood. Furthermore, the significance of MRP6 expression in intestine and kidney has not been revealed, yet mutations in MRP6 are known to be associated with pseudoxanthoma elasticum (Bergen et al., 2000). MRP6 is functionally quite different from other Mrps, with poor affinity for other Mrp substrates (Madon et al., 2000).

Mrp7 expression in mice was high in testes, with significant intestinal expression, and moderate expression in ovary and placenta. Humans also have highest expression of MRP7 in testes, with moderate expression in skin (Hopper et al., 2001). MRP7 was only detectable in these two human tissues by reverse transcription-polymerase chain reaction, suggesting minimal overall expression (Hopper et al., 2001; Chen et al., 2003). Much like other MRPs, MRP7 transports anticancer compounds, estrogen-glucuronides, and leukotrienes (Hopper et al., 2001; Chen et al., 2003).

MRP9 is highly expressed in mouse and human testes, yet its expression is unknown for rats. Tissue distribution of mouse Mrp9 indicates sole expression in testes, with virtually no mRNA expression observed in other tissues (Fig. 2). This is in agreement with previous work showing Mrp9 to be highly expressed in seminiferous tubules in mice (Shimizu et al., 2003). Functional aspects of MRP9 have not been elucidated, but expression of MRP9 was shown to be high in human testes and in breast tissue (Bera et al., 2002).

Several genes involved in drug disposition exhibit gender-predominant expression patterns. For example, a 1-fold higher expression of Mdr1b in female, as compared to male, kidney was observed previously (Schinkel et al., 1994). Furthermore, gender-specific Mrp expression patterns and alterations in drug disposition have not been reported. However, examples of gender differences in renal excretion of organic anions or cations are numerous, including para-aminohippuric acid and furosemide (Cerrutti et al., 2002). Thus, gender-specific Mrp expression could lead to altered efflux into urine.

Female-predominant expression of some Mrp transporters was observed in mice. Most notable were female-predominant expression of Mrp4 in liver and kidney, as well as marked differences in Mrp3 expression in liver, where expression was 14-fold higher in females than in males (Fig. 1). Although higher hepatic expression of Mrp1 was noted in females, the functional significance is questionable because of its low expression in liver.

The ontogeny study illustrates expression patterns in liver as a function of developmental age (Fig. 3). In liver, Mrp2 was expressed at adult levels at birth; however, Mrp3 and Mrp6 expression increased during the first few weeks of life. In contrast, rat Mrp2 and Mrp4 mRNA and protein increase gradually over time, with rat Mrp4 exhibiting male-predominant expression in liver (Johnson et al., 2002; Chen and Klaassen, 2004). Mouse Mrp4 also has a unique pattern of development in liver, with maximal expression at birth, then decreasing during the first 10 days of age to adult levels. Mrp6 is not expressed until 10 days of age, when the mRNA levels are almost 3-fold higher than in adult mice.

There are several Mrps expressed in kidney, including Mrps 1-6. The renal ontogeny of these transporters can be divided into three expression patterns: 1) Mrp1 expression remains relatively constant from birth to adulthood, 2) expression of Mrp2, 3, and 4 increases during the first few weeks of age, and 3) highest expression of Mrp5 is seen at birth, and expression decreases during the first few weeks (Figs. 4 and 5). The majority of Mrps were not expressed at adult levels in newborn animals, thus suggesting that Mrp substrates would not be readily excreted by the kidney.

Several gender-predominant patterns of expression were observed in kidney. Mrp1, 3, and 4 all showed female-predominant expression by 6 weeks of age (Figs. 4 and 5). Male expression of renal Mrp1 and Mrp3 was similar to adult female levels at 3 weeks of age, but then expression in males decreased markedly (Fig. 5). In contrast, Mrp4 expression increased in female kidneys after 30 days of age, whereas in males, it remained relatively constant.

In conclusion, highest expression of the Mrp family members was observed as follows: Mrp1 in testes, ovary, and placenta; Mrp2 in intestine, followed by liver and kidney; Mrp3 in large intestine; Mrp4 in kidney; Mrp5 in brain, followed by lung and stomach; Mrp6 in liver; Mrp7 in testes, intestine, and kidney; and Mrp9 solely in testes. Expression of several Mrps in reproductive tissues was high, suggesting a role in transport of hormones or other endogenous substrates. Furthermore, significant gender differences in Mrp1, Mrp3, and Mrp4 expression may lead to altered disposition of chemicals in kidney. The ontogeny data demonstrate that several Mrps do not exhibit mature expression until 1 month of age or later, suggesting slower xenobiotic elimination during postnatal development. Taken together, these data create a foundation that describes tissue distribution, ontogeny, and gender-specific expression of Mrps in mouse, which will be useful in understanding pharmacokinetic data in mice, and in extrapolation of data from mice to humans.

	Acknowledgments

 
We thank Drs. Tyra Leazer, Yuji Tanaka, Susan Buist, Jay Petrick, Matt Dieter, Chuan Chen, Hong Lu, and Jim Brady for their technical expertise. Furthermore, we thank Mindy Shelby and Dave Buckley for help in collecting tissues.

	Footnotes

 
Grant support was provided by National Institute of Health Grants ES-09716 and ES-07079.

Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.105.003780.

ABBREVIATIONS: Mrp, multidrug resistance-associated protein; ABC, ATP-binding cassette transporter; ABCC, ABC subfamily C; TMD, transmembrane domain; BQ-123, cyclo(L-Leu-D-Trp-D-Asp-L-Pro-D-Val); bDNA, branched DNA.

Address correspondence to: Dr. Curtis Klaassen, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7417. E-mail: cklaasse{at}kumc.edu

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