Abstract
Organic anion transporters (Oats) mediate the initial step of active renal excretion, specifically substrate uptake into proximal tubule cells. Despite extensive characterization of rat Oats, mouse Oat expression patterns are virtually unknown. This study was designed to identify basal expression patterns of mouse Oat1 (Slc22a6), Oat2 (Slc22a7), and Oat3 (Slc22a8) mRNA, compare these patterns with those in rat, and characterize postnatal development of mouse Oat mRNA. Tissues were collected from adult male and female 129J and C57BL/6 mice, and male and female C57BL/6 mice 0 to 40 days of age. Oat mRNA levels were determined by branched DNA signal amplification. Mouse Oat1 mRNA was primarily expressed in kidney of both strains, with male predominance. Mouse Oat2 mRNA levels were highest in kidney of both strains without gender predominance. In both strains, Oat3 mRNA was highest in kidney, and liver expression was male-predominant. However, only 129J mice had higher Oat3 mRNA levels in female kidney than in male kidney. During postnatal development, both Oat1 and Oat2 mRNA levels began to rise after 25 days of age. Oat3 mRNA levels rose gradually from birth through 40 days of age. Oat2 mRNA increased 30-fold during the first 40 days, whereas Oat1 and Oat3 increased about 2-fold. The most notable species differences in Oat mRNA expression were a lack of Oat2 female predominance in mouse kidney and a less dramatic Oat3 male predominance in mouse liver. With the exception of a significant species difference in Oat2 expression, many similarities were found between rat and mouse Oat mRNA levels.
In kidney, organic anion transporters (Oats1) are generally accepted to function as uptake transporters that aid the passage of organic anions from interstitial fluid into proximal tubule cells. Organic anion transporters are important in pharmacology and toxicology because many xenobiotics are cleared from the body as anionic moieties. Additional processes and transporters aid the movement of anions through the cell and across the brush-border membrane. The importance Oats possess in determining half-life, clearance, and even efficacy or toxicity of xenobiotics demands a clear understanding of Oat expression patterns in commonly used laboratory animal models.
Oat expression has been extensively characterized in rat (Simonson et al., 1994; Sekine et al., 1997, 1998; Kusuhara et al., 1999; Nakajima et al., 2000; Buist et al., 2002; Kobayashi et al., 2002). However, limited data are available regarding mouse Oat expression. Mouse Oat1 (Slc22a6) was predominantly detected in kidney of male mouse tissues (Lopez-Nieto et al., 1997). Additionally, developmental expression of mouse Oat1 in kidney was examined at embryonic days 15 to 19. Greatest expression was found on embryonic day 19 (Lopez-Nieto et al., 1997). Pavlova et al. (2000) also determined prenatal developmental expression of mouse Oat1 mRNA during embryonic days 12 through 18. Oat1 mRNA was initially detected in kidney and brain on embryonic day 14. Kidney levels rose throughout development, whereas transcript levels in brain decreased between fetal and adult ages (Pavlova et al., 2000).
Oat2 (Slc22a7) expression was determined by in situ hybridization in adult mouse kidney and liver, where mRNA levels appeared to be highest in liver (Pavlova et al., 2000). In kidney, expression was localized to the cortex. Additionally, Oat2 expression was determined in mouse kidney, liver, lung, and cartilage at embryonic day 14 (Pavlova et al., 2000). Although Oat2 mRNA levels increased in kidney throughout development, liver levels were consistently higher than those in kidney (Pavlova et al., 2000).
Oat3 (Slc22a8) was first described in mouse by identification of differentially expressed genes in the juvenile polycystic kidney mouse model (Brady et al., 1999). Furthermore, expression of this gene was markedly decreased in kidney of the osteosclerosis mouse model, leading to the original name “reduced in osteosclerosis transporter” (Brady et al., 1999). In C57BL/6J mice, Oat3 mRNA was highly expressed in kidney, but not in any other tissue examined (Brady et al., 1999). Pavlova et al. (2000) observed limited expression of Oat3 mRNA in mouse brain and liver at embryonic day 12. Kidney expression of Oat3 mRNA was first observed on embryonic day 16 in the cortex. In adult mice, kidney levels of Oat3 mRNA were high, with lower expression observed in liver (Pavlova et al., 2000).
Investigations into expression of mouse Oat1 and Oat3 mRNA levels in various adult tissues have been limited to male mice. Tissue distribution of Oat1 and Oat3 mRNA in female mice and Oat2 mRNA in both males and females remains to be determined. Furthermore, although embryonic expression of mouse Oat mRNA has been well documented, no data exist regarding postnatal developmental expression. Therefore, the primary goal of this study was to semiquantitatively determine Oat1, Oat2, and Oat3 mRNA levels in various tissues of male and female mice. Furthermore, Oat expression was determined as a function of postnatal development in male and female C57BL/6 mouse kidney. Additionally, these data were compared with Oat1, Oat2, and Oat3 mRNA levels in Sprague-Dawley rats to contribute to the understanding of species differences.
Materials and Methods
Materials. Sodium chloride, HEPES sodium salt, HEPES free acid, lithium lauryl sulfate, EDTA, and d-(+)-glucose were purchased from Sigma-Aldrich (St. Louis, MO). Micr-O-protect was purchased from Roche Diagnostics (Indianapolis, IN). Formaldehyde, 4-morpholinepropanesulfonic acid, sodium citrate, and NaHCO3 were purchased from Fischer Chemicals (Fairlawn, NJ). Chloroform, agarose, and ethidium bromide were purchased from AMRESCO Inc. (Solon, OH).
Animals. C57BL/6 mice were purchased from Charles River Laboratories Inc. (Wilmington, MA). Timed pregnant C57BL/6 females were purchased for the ontogeny study. 129J mice were purchased from The Jackson Laboratory (Bar Harbor, ME). Mice were maintained in a 12-h dark/light cycle, temperature- and humidity-controlled environment according to American Animal Associations Laboratory Animal Care guidelines and allowed water and rat chow (Teklad; Harlan, Indianapolis, IN) ad libitum. Mice were euthanized at 8 weeks of age (n = 5/gender). Kidney, liver, lung, small intestine, large intestine, and brain sections were immediately removed and snap-frozen in liquid nitrogen. Only kidney and liver were obtained from C57BL/6 females for analysis. The ontogeny study was conducted on kidney tissue from C57BL/6 mice euthanized every 5 days from birth (day 0) through 40 days of age (n = 5/gender/day).
Total RNA Isolation. Total RNA was isolated using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) according to the manufacturer's protocol. RNA pellets were resuspended in diethyl pyrocarbonate-treated deionized water. Total RNA concentrations were quantified spectrophotometrically at 260 nm. Integrity of RNA samples was analyzed by formaldehyde-agarose gel electrophoresis (1.2% agarose, 2.1 M formaldehyde in 1× MOPS, 210 μg of ethidium bromide/mg of sample) with visualization under ultraviolet light by ethidium bromide fluorescence.
Development of Specific Oligonucleotide Probe Sets for Branched DNA (bDNA) Analysis. Gene sequences of interest were accessed from GenBank (mouse Oat1, Oat2, and Oat3; Table 1). Target sequences were analyzed with ProbeDesigner software version 1.0 (Bayer Corp., Emeryville, CA) for suitability as capture, label, or blocker probes. Multiple specific probes were developed to each Oat mRNA transcript (Table 1). All oligonucleotide probes were designed with a melting temperature of approximately 63°C. This enabled hybridization conditions to be held constant (i.e., 53°C) during each step for each oligonucleotide probe set. Each probe designed in ProbeDesigner was submitted to the National Center for Biotechnology Information for nucleotide comparison by the Basic Local Alignment Search Tool (BLASTn; Altschul et al., 1997) to ensure minimal cross-reactivity with other mouse sequences. Oligonucleotides with a high degree of similarity (>80%) to other mouse gene transcripts were eliminated from the design. Together, the high melting temperature and sequence specificity to the gene of interest ensure detection of the specific mRNA of interest. Probes were synthesized by QIAGEN Operon (Alameda, CA).
Oligonucleotide probes generated for analysis of mouse Oat1, Oat2, and Oat3 expression by bDNA signal amplification
bDNA Assay. Reagents required for RNA analysis (i.e., lysis buffer, amplifier/label probe buffer, and substrate solution) were supplied in the HV-QuantiGene bDNA signal amplification kit (Bayer Corp.-Diagnostics Div., Tarrytown, NY). Oat1, Oat2, and Oat3 mRNA levels were analyzed according to the method of Hartley and Klaassen (2000). Briefly, specific mouse Oat oligonucleotide probes were diluted in lysis buffer. Total RNA (1 μg/μl; 10 μl) was added to each well of a 96-well plate containing 50 μl of capture hybridization buffer [0.05 M HEPES sodium salt, 0.05 M HEPES free acid, 0.037 M lithium lauryl sulfate, 0.5% (v/v) Micr-O-protect, 8 mM EDTA, 0.3% (w/v) nucleic acid blocking agent] and 50 μl of diluted probe set. Total RNA was allowed to hybridize to probe sets overnight at 53°C. Subsequent hybridization steps were carried out according to 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. Data are presented as relative light units (RLU) per 10 μg total RNA.
Statistical Analysis. Bars represent mean ± S.E.M. Data were analyzed by Student's t test. Asterisks indicate a statistical difference (p ≤ 0.05) between genders.
Results
Six tissues from C57BL/6 mice and 10 tissues from 129J mice were analyzed for Oat1, Oat2, and Oat3 mRNA levels (Fig. 1). Transcript levels for all three Oats were highest in kidney of both mouse strains. Oat1 mRNA levels were male-predominant in kidney of both C57BL/6 and 129J mice (Fig. 1, top left and right). In liver, Oat1 mRNA was nearly undetectable in both mouse strains. Oat2 mRNA levels were high in kidney with no apparent gender predominance (Fig. 1, middle left and right). Liver levels of Oat2 mRNA were low compared with kidney. Oat3 mRNA levels were similar between male and female C57BL/6 mice in most tissues examined (Fig. 1, bottom left). However, kidney levels of Oat3 mRNA were higher in 129J female mice than in males (Fig. 1, bottom right). Although Oat3 mRNA levels were low in liver as compared with kidney, there was a distinct male predominance in liver in both strains (Fig. 1, bottom insets).
Tissue expression of mouse Oat mRNA.
Top, Oat1 mRNA levels; middle, Oat2 mRNA levels; bottom, Oat3 mRNA levels. Left, C57BL/6 mouse strain; right, 129J mouse strain. Insets, enlargements of Oat3 mRNA levels in liver. Six and 10 tissues were analyzed for Oat mRNA levels in C57BL/6 and 129J mouse strains, respectively. Only kidney and liver were available from female C57BL/6 mice (n = 5), whereas all six tissues were analyzed from male C57BL/6 mice (n = 5). All 10 tissues were analyzed in both male and female 129J mice (n = 5). *, gender differences were identified by Student's t test (p ≤ 0.05). L. Intestine, large intestine.
The relative expression patterns of Oat mRNA in C57BL/6 and 129J mouse strains were compared with corresponding Oat mRNA in Sprague-Dawley rats by gender (Fig. 2; Buist et al., 2002). Rat and mouse both demonstrate Oat1 male predominance in kidney. However, the gender difference is more pronounced in both mouse strains as compared with rat (Fig. 2, top). Oat2, which is expressed at similar levels by males and females in kidney of both C57BL/6 and 129J mouse strains, is distinctly female-predominant in rat, with mRNA levels in male kidney being only 16% of those in female kidney (Fig. 2, middle). Oat3 mRNA expression in rat liver is male-predominant, exhibiting 11-fold higher levels than females. Oat3 mRNA levels are also expressed in a male-predominant manner in liver of both mouse strains; however, there is only an approximately 1.3- to 1.4-fold difference between males and females (Fig. 2, bottom).
Comparison of rat and mouse kidney and liver Oat mRNA gender differences.
Male/female mRNA ratios are presented for Oat1 (top), Oat2 (middle), and Oat3 (bottom). Rat Oat differences were previously reported by Buist et al. (2002).
In addition to identifying differences in gender-predominant expression patterns, kidney/liver ratios were used to identify differences in relative tissue predominance (Fig. 3; Buist et al., 2002). The Oat1 relative tissue expression is similar between rat and mouse, the only difference being a slightly greater relative expression in kidney of male mice with respect to liver than was observed in male rats (Fig. 3, top). Oat2 mRNA tissue predominance was quite different between male rat and mouse (Fig. 3, middle). In male rat, Oat2 mRNA is lower in kidney than in liver, whereas in mouse, Oat2 mRNA is much higher in kidney than in liver. There was not a dramatic difference in relative tissue expression of Oat2 between female rats and mice. The Oat3 mRNA kidney/liver ratio in male rats was 6.9, whereas in C57BL/6 and 129J mice the ratio was 52 and 33, respectively (Fig. 3, bottom). This second species difference is due to much lower levels of Oat3 mRNA in male mouse liver than was observed in male rat liver as compared with respective kidney levels. As with the female Oat2 ratios, Oat3 mRNA kidney/liver ratios in female rats and mice were similar.
Comparison of kidney and liver Oat mRNA levels in male and female rats and mice.
Kidney/liver mRNA ratios are presented for Oat1 (top), Oat2 (middle), and Oat3 (bottom). Rat Oat data were previously reported by Buist et al. (2002).
Postnatal development of Oat mRNA expression was examined in kidney of male and female C57BL/6 mice. Oat1 mRNA levels declined slightly from the day of birth through 10 days of age, at which point mRNA expression reached a plateau (Fig. 4, top). Between 25 and 30 days of age, male levels of Oat1 mRNA began to climb and continued to do so through 40 days of age, whereas female levels remained relatively constant. Male levels of Oat1 mRNA increased 2.5-fold from birth to 40 days of age, whereas female levels did not change (Fig. 4, top). Additionally, male-predominant expression of Oat1 mRNA becomes apparent at 30 days of age. Oat2 mRNA was low from birth through 25 days of age in kidney (Fig. 4, middle). Between 25 and 30 days of age, Oat2 mRNA levels began to rise, with male levels rising more sharply than female levels. By 40 days of age, male and female Oat2 mRNA levels had increased 38- and 25-fold from birth, respectively. By 40 days of age, Oat3 mRNA levels increased 3-fold above levels at birth, with a similar expression pattern in male and female kidney throughout development (Fig. 4, bottom).
Developmental expression of Oat mRNA in C57BL/6 mouse kidney.
Top, Oat1 mRNA levels; middle, Oat2 mRNA levels; bottom, Oat3 mRNA levels. Kidneys were removed from male and female C57BL/6 mice from birth (day 0) through 40 days of age (n = 5/age/gender) and analyzed for Oat mRNA levels. Gender differences at each time point were identified by Student's t test (p ≤ 0.05).
Discussion
Understanding species differences carries a 2-fold importance as regards xenobiotic disposition. First, knowledge of species differences with respect to drug metabolism, clearance, and toxicity at the molecular level enhances characterization of commonly used laboratory animal models. Second, information regarding species differences increases the ability to apply animal data describing metabolism, clearance, and toxicity to other species, such as humans. Oats are known to participate in clearance by transporting a wide range of xenobiotics, including nonsteroidal anti-inflammatory drugs, β-lactam antibiotics, vitamins, and antineoplastic drugs, thus influencing the pharmacokinetics of such substrates. In this respect, knowledge of Oat expression in commonly used animal models is necessary to interpret data and extrapolate results across species.
Nephrotoxicity associated with α2u-globulin illustrates the importance of clearly characterizing gender and species differences. Nephropathy occurs in male rats following exposure to certain chemicals such as d-limonene (Lehman-McKeeman et al., 1989) or unleaded gasoline (Short et al., 1987). However, this phenomenon does not occur in female rats or male or female mice. Male rats accumulate α2u-globulin-rich protein droplets in kidney following exposure to certain chemicals, whereas females do not (Short et al., 1987). The accumulation of these droplets initiates a mechanism ultimately leading to nephropathy (Short et al., 1989). Thus, α2u-globulin-associated nephropathy is both gender- and species-specific. Knowledge of the species-specific nature of α2u-globulin nephropathy has been critical to determining human risk assessment guidelines. Likewise, as substrates of Oats continue to be identified, knowledge of species differences will be critical in evaluating the relevance or applicability of identified toxicities between species.
C57BL/6 and 129J mice are commonly used as background strains in the development of knockout (KO) mice. Numerous KO mouse models are available that might be used to delineate mechanisms of differential expression. For example, Stat5b KO mice can be used to investigate the role of growth hormone (GH) in gender-specific expression. However, to use such models, it is necessary to determine whether the gene of interest is differentially expressed in the background, or wild-type, strain. In the case of Oats, Buist et al. (2002) identified female-predominant expression of Oat2 mRNA in kidney and male-predominant expression of Oat3 in liver of Sprague-Dawley rats. These expression patterns were subsequently related to GH regulation (Buist and Klaassen, 2003). The next goal was to examine the mechanisms of differential Oat2 and Oat3 expression in various mouse models of GH disruption. Therefore, mouse Oat expression in C57BL/6 and 129J mice was determined to identify the applicability of KO mouse models to investigate these gender-predominant expression phenomena.
Gender and species (rat versus mouse) differences in Oat mRNA expression are summarized in Figs. 1, 2, 3. Most notably, Oat2 mRNA levels are markedly higher in female (125 RLU) than in male (25 RLU) rat kidney (Buist et al., 2002). However, Oat2 expression is not gender-predominant in mouse kidney. Interestingly, perfluoro-octanoic acid (PFOA), a chemical used in the production of nonstick cookware and protective finishes on carpets, is excreted into urine by female rats at a greater rate than by male rats (Griffith and Long, 1980; Hanhijärvi et al., 1982; Kudo et al., 2002). Additionally, PFOA induces liver toxicity in male rats, but not in female rats, presumably due to differences in the urinary excretion rate (Griffith and Long, 1980). Moreover, mice exhibit no sex-related difference in toxicity following PFOA exposure (Sohlenius et al., 1992). Similarly, there is not a significant difference in Oat2 mRNA levels in kidney between male and female mice. Provided PFOA is a substrate of Oat2, urinary excretion may be similar in male and female mice and may occur at a rate similar to that in female rats, thus avoiding the liver toxicity observed in male rats. Hence, Oat2 expression may be an important determinant in the excretion and toxicity of PFOA and should be examined as a possible factor in PFOA-related hepatotoxicity.
Distribution of Oat mRNA between kidney and liver varies between species. The Oat2 mRNA kidney/liver ratio is higher in mouse than in rat (Fig. 3, middle). Additionally, the Oat3 mRNA kidney/liver ratio in male mice is much higher than in male rats. The differences in distribution of Oats may translate into species differences in the pharmacokinetics of Oat substrates if protein expression and transport activity are similar to mRNA expression. Thus, species differences are important considerations in determining the most appropriate experimental animal models to use in answering questions regarding toxicity or urinary excretion following xenobiotic exposure.
Development of KO animals, and ensuing experiments, will help to specifically determine the role individual Oats play in organic anion excretion. An Oat3 KO mouse has already been developed (Sweet et al., 2002). This model has no known morphological defects. However, transport of taurocholate, estrone sulfate, and para-aminohippurate by renal slices of Oat3 KO mice is lower than that by wild-type controls. This model will ultimately help to define the role of Oat3 in mouse kidney. Additionally, the data will likely be useful in understanding the handling of organic anions by rat kidney.
Transport by hepatic slices in the Oat3 KO mouse model may not be directly correlated to rat, due to higher expression of Oat3 in rat liver with respect to kidney than is found in mice (Fig. 3, bottom). For example, hepatic uptake of taurocholate by liver slices from Oat3 KO mice does not differ from wild-type mouse uptake (Sweet et al., 2002). Yet, male rat liver has relatively higher levels of Oat3 mRNA than have male mice. Therefore, Oat3 may play a larger role in hepatic transport of taurocholate in male rat. However, other transporters such as sodium taurocholate polypeptide likely transport a larger load of bile salts in liver than does Oat3 in most species. Thus, the expression of Oat3 in liver may not be of critical importance until faced with a disease state, when Oat3 may act as a backup system of bile salt uptake. Nonetheless, species differences bear consideration, especially if a KO model should also be developed for Oat2. Dramatically higher expression of Oat2 mRNA in female rat kidney will likely dictate a female-predominant participation of Oat2 in renal organic anion transport that would not be observed in mice. Development of an Oat2-deficient rat model would be an excellent method for determining the importance of the marked gender difference in rat kidney expression of Oat2.
Expression of Oats during postnatal development was examined in the present study because infants often process xenobiotics differently from adults. Differences may be due to age-divergent expression of metabolic enzymes and/or transport systems necessary for absorption or excretion. para-Aminohippurate, a classic substrate for the organic anion transport system, and penicillin, another Oat substrate, are excreted less efficiently by newborns than by adults (West et al., 1948; Barnett et al., 1949). Furthermore, in humans, the capacity to transport organic anions does not mature until 7 to 8 months of age (West et al., 1948). Rat Oat transcripts in kidney are known to require 20 to 45 days after birth to reach adult levels of expression (Buist et al., 2002). Additionally, the current data demonstrate that renal Oats in mice are also immature at birth and require approximately 25 days to mature to adult levels. In particular, Oat1 and Oat2 mRNA remain low through 25 days of age. Expression of both begins to rise at 30 days of age, whereas Oat3 expression matures more gradually. Additionally, Oat1 and Oat3 mRNA levels are increased 2- and 3-fold during the first 40 days of postnatal development, respectively. In contrast, Oat2 mRNA levels increase 30-fold during the same time frame. These data may be related to the fact that there is minimal tubular development, despite a full complement of nephrons, at birth (West et al., 1948). In fact, the length of human proximal tubules is not fully mature by 3.5 years of age (Fetterman et al., 1965). Furthermore, the increase in Oat expression during postnatal development may be linked to lengthening of the tubular structure by a common developmental trigger. Most importantly, recognition of the fact that organic anion transport is less developed in newborns than in adults sets the stage to consider the age-dependent expression of Oats as a mechanism for age-related changes in renal excretion and toxicity.
Overall, rat and mouse relative Oat mRNA levels share many similarities. However, a significant species difference was identified in the expression of Oat2 mRNA. Rats express Oat2 mRNA at high levels in female kidney and low levels in male kidney, whereas mice express high levels of Oat2 in kidney, without gender specificity. Additionally, whereas rats and mice demonstrate a male-predominant expression pattern of Oat3 in liver, the relative expression between liver and kidney is different in male rat as compared with male mouse. Specifically, the level of Oat3 mRNA in liver is higher in male rat than it is in male mouse with respect to kidney. These differences indicate that species differences in Oats exist and may be important in the interpretation of data regarding pharmacokinetics and toxicity of organic anions.
Acknowledgments
We thank Jon Maher, Mindy Shelby, and Drs. Tyra Leazer and Angela Slitt for their assistance in this project.
Footnotes
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↵1 Abbreviations used are: Oat, organic anion transporter; bDNA, branched DNA; GH, growth hormone; KO, knockout; PFOA, perfluoro-octanoic acid; RLU, relative light unit(s).
- Received December 1, 2003.
- Accepted March 2, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
