Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

Since the late 1800s, scientists have recognized active transport in the kidney. Studies of p-aminohippurate (PAH) elimination, an analog of hippuric acid, eventually elucidated the tertiary active mechanism of renal organic anion excretion (Pritchard and Miller, 1993). The first step of PAH transport is cleavage of ATP, which provides the energy needed to create a Na⁺ gradient across the basolateral membrane of the proximal tubules. Second, the Na⁺ gradient drives inwardly directed cotransport of sodium and dicarboxylates, such as -ketoglutarate, creating a dicarboxylate gradient across the basolateral membrane. Finally, the dicarboxylate gradient fuels the exchange of dicarboxylates out of, and organic anions into, proximal tubule cells. Oat1 mediates the last step in this process.

Oat1 has been characterized in mouse (Lopez-Nieto et al., 1996), rat (Sekine et al., 1997; Sweet et al., 1997), and human (Hosoyamada et al., 1999; Lu et al., 1999; Race et al., 1999) kidney. Furthermore, an immunohistochemical study has localized Oat1 to the renal proximal tubule (Tojo et al., 1999). Comprised of approximately 550 amino acids, the molecular mass of Oat1 is about 56 to 64 kDa. A general characteristic of all of the Oats is that they are postulated to conform to a structure within the membrane that has twelve transmembrane domains (Lopez-Nieto et al., 1996; Sweet et al., 1997; Hosoyamada et al., 1999; Lu et al., 1999; Race et al., 1999). Additionally, there are four potential glycosylation and phosphorylation sites that may be involved in regulation of Oat1 activity (Sekine et al., 1997; Sweet et al., 1997). Substrates of Oat1 span a diverse range of chemical structures, demonstrating the multispecificity of this transporter. Regarding substrate identification, Ullrich and Rumrich (1988) and Fritzsch et al. (1988) have identified the general structural features of Oat1 substrates to include a negative or partial negative charge separated from a second charge by 6 Å and a hydrophobic region 8 Å in length.

Less is known regarding mechanisms and substrates for the other identified Oats, namely Oat2, Oat3, and OAT4. They were first isolated from rat kidney, rat brain, and human kidney, respectively (Simonson et al., 1994; Sekine et al., 1998; Kusuhara et al., 1999; Cha et al., 2000). In contrast to Oat1, these transporters are Na⁺ independent and cannot be trans-stimulated by -ketoglutarate or other substrates. Expression of Oat2 has been reported to be primarily localized in liver, whereas expression of both rat Oat3 and human OAT4 mRNA are highest in kidney (Simonson et al., 1994; Sekine et al., 1998; Kusuhara et al., 1999; Cha et al., 2000). Additionally, significant expression of Oat3 and human OAT4 mRNA was reported in brain and placenta, respectively (Kusuhara et al., 1999; Cha et al., 2000).

Renal organic anion transport is crucial in the excretion process of many xenobiotics and endogenous chemicals. Furthermore, age and gender are known to influence pharmacokinetic parameters, such as clearance and half-life of many drugs, as well as the extent of and sensitivity to drug toxicity. Reyes et al. (1998) demonstrated a gender difference in renal transport in which male rats transported more PAH than did females. Moreover, PAH half-life was extended in castrated male rats as compared with intact males. Typical half-life values for PAH were restored with testosterone replacement treatment in castrated rats. Transport differences based on age have also been observed, as in the case of cephalosporin -lactam antibiotics (Tune, 1975; Fanos and Dall'Agnola, 1999). Some cephalosporin therapies are dose-limited in adults because organic, anionic metabolites of this class of drugs have a high incidence of nephrotoxicity (Tune, 1975). However, toxicity is not apparent in immature human or rodent kidneys, possibly the result of organic anion transport differences between adult and immature kidneys.

Characterization of Oat expression has thus far been determined only in male tissue. Additionally, while Nakajima et al. (2000) have examined developmental expression of Oat1 primarily in embryonic kidney, expression of all three Oats in the postnatal developing kidney has not been examined in detail. Therefore, one goal of this study was to determine Oat1, Oat2, and Oat3 mRNA levels in many tissues of both male and female adult rats. The second objective was to describe Oat mRNA expression during the first 45 days of postnatal development in both the male and female kidney. The results of these experiments may aid in understanding the role of Oats in established gender- and age-specific drug responses and toxicities.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Animals

Tissue Expression Study. Adult male and female Sasco Sprague-Dawley (SD) rats (200-250 g; Charles River Laboratories Inc., Wilmington, MA) were housed according to American Animal Associations Laboratory Animal Care guidelines and acclimated for 1 week. Animals were given free access to water and rat-chow (Teklad; Harlan Sprague Dawley, Inc. Indianapolis, IN). Immediately following decapitation, tissues were removed, frozen in liquid nitrogen, and stored at 80°C.

Developmental Expression Study. Pregnant Sasco SD rats were purchased at gestation day 13 (Charles River Laboratories Inc.) and housed in an American Animal Associations Laboratory Animal Care accredited facility. Dams were allowed to give birth, and pups remained with their respective dam and litter until weaned and separated at 21 days. Pups were decapitated at ages 0 (date of birth) through day 45 in 5-day increments (n = 5/gender/age). Kidneys were removed immediately following decapitation, frozen in liquid nitrogen, and stored at 80°C.

Total RNA Purification

Total RNA was isolated using RNAzol B reagent (Tel-Test Inc., Friendswood, TX) as per the manufacturer's protocol. RNA pellets were resuspended in diethyl pyrocarbonate-treated deionized water. The concentration of total RNA in each sample was quantified spectrophotometrically at 260 nm. Integrity of each RNA sample was analyzed by formaldehyde-agarose gel electrophoresis (1.2% agarose, 2.1 M formaldehyde in 1× MOPS, 0.5 µg/µl ethidium bromide). Each RNA sample was then visualized under ultraviolet light by ethidium bromide fluorescence.

Development of Specific Oligonucleotide Probe Sets for Branched-DNA (bDNA) Analysis of OATs

The Oat gene sequences of interest were accessed from GenBank and are listed in Table 1. Before development of each probe set, the nucleotide sequences were aligned using CLUSTAL W (Thompson et al., 1994) with software provided by OMIGA (Oxford Molecular Group PLC, Oxford, UK) to determine specific target regions (i.e., nucleotide regions of dissimilarity between Oat sequences) for oligonucleotide probe development. 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 hybridization step and for each oligonucleotide probe set. Each probe designed in ProbeDesigner was submitted to the National Center for Biotechnological Information for nucleotide comparison by the basic logarithmic alignment search tool (BLASTn; Altschul et al., 1997) to ensure minimal cross-reactivity with other rat sequences. Oligonucleotides with a high degree of similarity (80%) to other rat gene transcripts were eliminated from the design. Probes were synthesized by Operon Technologies (Almeda, CA).

bDNA Assay

Specific Oat oligonucleotide probes were diluted in lysis buffer supplied in the HV-QuantiGene bDNA signal amplification kit (Bayer Corp.-Diagnostics Div., Tarrytown, NY). All reagents required for RNA analysis (i.e., lysis buffer, amplifier/label probe buffer, capture hybridization buffer, and substrate solution) were supplied in the HV-QuantiGene bDNA signal amplification kit. Oat1, Oat2, and Oat3 mRNAs were analyzed according to the method of Hartley and Klaassen (2000). Briefly, total RNA (1 µg/µl; 10 µl) was added to each well of a 96-well plate containing capture hybridization buffer (50 µl) and 50 µl of each diluted probe set. Total RNA was allowed to hybridize to each probe set overnight at 53°C. Subsequent hybridization steps were carried out as per the manufacturer's protocol, and luminescence was measured with a Quantiplex 320 bDNA luminometer interfaced with Quantiplex data management software version 5.02 (Bayer Corp.-Diagnostics Div.) for analysis of luminescence from 96-well plates.

Northern Blot Analysis of Rat Oat2 mRNA

Probe Design. With the exception of probe 1, which was chosen from the 5'-untranslated region, the probes were selected from sequences in the 3'-untranslated region (probe sequences listed below). Four antisense oligonucleotide probes were used to enhance sensitivity of detection (University of Kansas Medical Center, Biotechnology Support Facility, Kansas City, KS). Probes used were 5'-GAC GGT TCA GTC TGC TTA CCA CAT GGA CCC-3'; 5'-CAG GAG CAG CTC CAT CCT TAG GTG GAG GAG-3'; 5'-GTG CTG CGG GAA GTT GTT CTG CTG CTG GAG-3'; and 5'-CCA TGA GCA ACC GTC TTT ATT TAT AGA AGC CCC C-3'.

Blot Procedure. Antisense oligonucleotide probes were 3'-end labeled using Terminal Transferase (Roche Bioscience, Indianapolis, IN) resulting in an average specific activity of about 5 × 10⁸ cpm/µg DNA. Total RNA was isolated from pooled liver and kidney of approximately 9-week-old male and female SD rats (n = 3/male tissue; n = 5/female tissue), using TRIZOL (Invitrogen, Carlsbad, CA) per the manufacturer's protocol. Poly(A)⁺ mRNA was subsequently purified from total RNA using an Ambion Poly(A)Pure mRNA isolation kit (Ambion Inc., Austin, TX) per the manufacturer's protocol. Approximately 5 µg of poly(A)⁺-enriched RNA was electrophoretically resolved in 1% formaldehyde-agarose gel, capillary-blotted overnight onto Zeta-Probe nylon membrane (Bio-Rad, Hercules, CA), UV cross-linked and hybridized at 50°C overnight in ExpressHyb hybridization solution (CLONTECH, Palo Alto, CA). Posthybridization washes were executed at 55°C.

Statistical Analysis

Data were analyzed by the Student's t test (2-tailed, equal variance). Asterisks (*) indicate a statistical difference (p  0.05) between genders.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

A preliminary screen of pooled RNA (5 rats/gender) from rat adrenal, bladder, blood vessel, brain stem, caudate, cerebellum, cerebral cortex, duodenum, eye, heart, hippocampus, ileum, jejunum, kidney, large intestine, liver, lung, lymph node, mammary tissue, muscle, nasal epithelium, olfactory bulb, ovary, pancreas, pituitary, placenta, prostate, skin, spinal cord, spleen, stomach, testes, thalamus, thymus, thyroid, tongue, and uterus (data not shown) was performed to ensure the major tissues of expression were reported. The 10 tissues of highest expression (cerebellum, cortex, duodenum, ileum, jejunum, kidney, intestine, liver, lung, and stomach) were then examined individually (5 rats/gender). Predominant expression of Oat1 mRNA was in kidney in accordance with previous reports (Lopez-Nieto et al., 1996; Sekine et al., 1997; Sweet et al., 1997). All other tissues expressed minimal levels of this transporter (Fig. 1A). Furthermore, a trend emerged in which male kidney levels of Oat1 mRNA were higher than in female kidney, but the difference was not statistically significant. Likewise, highest Oat2 mRNA expression was also in kidney (Fig. 1B), contrary to previous reports (Simonson et al., 1994; Sekine et al., 1998) that identified Oat2 as liver specific. These data indicate that although Oat2 expression in males is highest in liver, expression in female kidney was considerably higher than in male liver. Northern blot analysis of Oat2 further confirmed these results (Fig. 2). This female predominance of Oat2 in kidney was not seen in other tissues. Moreover, Oat2 mRNA generally tended to be higher in male than in female tissues, with the exception of kidney. Finally, kidney also contained the highest levels of Oat3 mRNA in comparison to other tissues (Fig. 1C). Oat3 expression was also moderate in brain, lung, and male liver. Although a gender difference was not manifest in kidney, the primary tissue of expression, liver Oat3 mRNA levels were distinctly higher in male than in female rats.

View larger version (34K): [in this window] [in a new window]   Fig. 1.   Tissue expression of Oat mRNA. A, Oat1 expression was low in all tissues examined, with the exception of kidney. Additionally, a trend of higher expression in male than female kidney was observed; B, male expression of Oat2 mRNA was highest in liver. However, examination of female expression revealed markedly higher expression in female kidney than male liver; C, Oat3 mRNA was primarily expressed in kidney, with moderate expression in liver. However, a clear gender difference appeared with higher expression in male liver than female liver. *, p  0.05

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Oat2 mRNA expression in adult rat liver and kidney. This Northern blot verified Oat2 mRNA expression in female kidney. Four oligonucleotide probes specific to rat Oat2 mRNA were used. Lanes were loaded, from left to right, with poly(A)+-enriched RNA from male liver, male kidney, female liver, and female kidney.

Ontogeny of Oat mRNA was subsequently investigated in kidney because this was the tissue in which all three family members were primarily expressed. Oat1 mRNA expression gradually increased in kidney during the first 30 days of life (Fig. 3A). An Oat1 gender divergence, male expression exceeding that in females, became apparent on days 40 and 45, coinciding with the trend previously observed in the tissue expression study (Fig. 1A). Distinct from Oat1, the amount of Oat2 mRNA remained low through the first 30 postnatal days (Fig. 3B). However, Oat2 expression markedly increased from days 35 through 45 in female rats, without an analogous increase in male expression. Oat3 mRNA expression matured earlier than that of Oat1 or Oat2 in kidney (Fig. 3C). In fact, developmental expression was characterized by a marked increase in Oat3 mRNA during the first 10 days of life without an apparent gender difference during any stage of development.

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Developmental expression of Oat mRNA in kidney. A, Oat1 mRNA levels were low at birth (day 0) and increased gradually through day 35. Slightly higher expression in male kidney first became apparent at day 40; B, expression of Oat2 was minimal until day 35, at which time female expression began to increase through day 45. Male expression was constant throughout development; C, Oat3 expression in kidney matured earliest of the three genes, nearing adult levels as early as day 10. *, p  0.05

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Numerous factors influence toxicity including gender and age. Some xenobiotics follow a gender-specific disposition profile. Diflunisal (a nonsteroidal anti-inflammatory drug) and nilvadipine (a calcium channel antagonist) are two such examples. Clearance of diflunisal is more rapid in males, and females more actively excrete nilvadipine (Macdonald et al., 1990; Zieleniewski et al., 1998). Such differences in clearance can alter the extent of both drug efficacy and toxicity. Also, ages ranging from infancy to senescence may produce a wide spectrum of responses to xenobiotics. Specifically, neonates may experience increased sensitivity to some xenobiotics, whereas other xenobiotics lead to a decreased susceptibility to toxicity as compared with adults. For example, some cephalosporins (e.g., cephaloridine) are nephrotoxic to adults but not infants (Tune, 1975).

During the past century, considerable research efforts have focused on determining the mechanism of active organic anion transport in renal proximal tubules. The model substrate for these studies was PAH because of the nearly complete clearance of this anion after a single pass through the kidneys. In 1997, several groups concurrently cloned and characterized the protein responsible for PAH transport in the kidney (Lopez-Nieto et al., 1996; Sekine et al., 1997; Sweet et al., 1997; Wolff et al., 1997). This protein was named Oat1. An extensive list of substrates has since emerged based primarily on their ability to inhibit radiolabeled PAH transport by oocytes expressing Oat1. Additionally, Oat1 tissue and developmental expression was briefly described. However, tissue expression studies failed to consider female tissues, and developmental characterization was incomplete. Therefore, the aims of this study were to quantitatively describe relative expression of Oat1 mRNA in multiple tissues (of which 10 are shown in Fig. 1) of both male and female SD rats, as well as to determine developmental expression in male and female kidney. Quantification of relative Oat mRNA expression was determined with the bDNA signal amplification method because of the established sensitivity, reproducibility, and wide range of linear response of this method (Hartley and Klaassen, 2000). Furthermore, a direct comparison between DNA polymerase chain reaction and bDNA in the identification of plasma human immunodeficiency virus-1 demonstrated comparable specificity and sensitivity (Rouet et al., 2001).

In agreement with previous reports, kidney expressed more Oat1 mRNA than any other tissue (Lopez-Nieto et al., 1996; Sekine et al., 1997; Sweet et al., 1997; Wolff et al., 1997). Furthermore, renal Oat1 mRNA expression in male rats tended to be higher than in females. This trend paralleled data reported by Reyes et al. (1998) that identified more active PAH transport in male rat renal cortical slices when compared with female slices. Noting that the Oat1 mRNA expression was principally in kidney in this study (Fig. 1A), the next objective was to determine the presence of Oat1 in immature kidneys at several stages of development. A gradually increasing pattern of Oat1 mRNA in male and female kidney was detected (Fig. 3A). Additionally, the possible gender divergence in adult kidneys became apparent at ages 40 and 45 days. A correlation may be drawn between low Oat1 mRNA expression at birth and lack of susceptibility to cephalosporin-induced nephrotoxicity in infants (Fanos and Dall'Agnola, 1999). The hallmark of cephalosporin nephrotoxicity is accumulation of the drug in proximal tubule cells (Tune, 1975). Accumulation is the result of a highly efficient Oat1 cellular influx of cephalosporins from blood coupled with a less efficient efflux transport across the luminal membrane. The developmental pattern of Oat1 mRNA expression may be involved in age-specific nephrotoxicity of drugs such as cephaloridine. Thus, specific gender- and age-dependent patterns of Oat1 mRNA expression directly correlate to the variability of male and female PAH transport in kidneys, as well as to the age-dependent nephrotoxicity of cephalosporins.

Unlike Oat1, only a few studies have addressed the extent of Oat2 expression or identification of substrates for this transporter, which was first cloned in 1994 (Simonson et al., 1994). Previous studies consistently described Oat2 as a liver-specific transporter (Simonson et al., 1994; Sekine et al., 1998). However, these studies were deficient in that they lacked analysis of Oat2 mRNA expression in both male and female tissues, and developmental expression of Oat2 was not addressed in detail. In response to this deficiency, this study was the first to quantitatively identify relative Oat2 mRNA expression in 37 male and female tissues. Surprisingly, Oat2 expression in female kidney was markedly higher than that of all other tissues, including male liver. A Northern blot of male and female Oat2 expression further supported this observation. Therefore, while predominant male expression of Oat2 mRNA was indeed in liver, female Oat2 expression in kidney was substantially higher than that of male liver. The developmental study of Oat2 mRNA further verified this striking gender difference. Analysis of kidney tissue throughout development revealed minimal Oat2 mRNA expression in kidney for the first 30 days, irrespective of gender. However, at the onset of female sexual maturity (approximately day 35), Oat2 mRNA levels in kidney increased dramatically and continued to rise with increasing age. Furthermore, male expression remained low, never changing significantly from day 0 expression levels. Interestingly, pregnant rat Oat2 expression in kidney at gestation day 18 further exceeded adult nonpregnant female expression 1.7-fold (data not shown). Although few Oat2 substrates have been identified, the remarkable gender divergence of renal Oat2 expression suggests an important role in clearance mechanisms of the female kidney. One function of Oat2 could be excretion of conjugated estrogen. Although this has not yet been investigated for Oat2, estrone sulfate was identified as a substrate of Oat3 (Kusuhara et al., 1999). Additionally, anionic metabolites of the antihypertensive drug, nilvadipine, are actively transported by female proximal tubules, whereas male clearance relies primarily on glomerular filtration (Zieleniewski et al., 1998). Together with data reported here, Oat2 may be implicated in proximal tubule transport of this drug.

Oat3, the third member of the rat Oat family, was first isolated from rat choroid plexus (Kusuhara et al., 1999). As with Oat2, there is little knowledge regarding Oat3 substrates, transport mechanism, or biological significance. Also similar to Oat2 is the availability of only cursory information addressing tissue and developmental gene expression. This quantitative analysis of Oat3 mRNA expression throughout the body confirmed the previous report that Oat3 is predominately in kidney (Kusuhara et al., 1999). However, Oat3 transcripts are also moderately expressed in the liver and some regions of the brain. Although expressed to a lesser extent than in kidney, Oat3 may nonetheless be biologically significant in these other tissues. Interestingly, a clear gender difference emerged in hepatic Oat3 expression. Male Oat3 mRNA expression in liver was much higher than in female liver. Unique among the Oats was the early maturation (10 days) of Oat3 transcript levels. Further investigations of the gender-divergent liver expression and the early elevation of OAT3 during kidney development are required to determine the importance of these observations.

In summary, predominant expression of the Oat family members was in kidney. Furthermore, gender differences became apparent in renal Oat1 and Oat2 mRNA levels, as well as hepatic Oat3 mRNA expression. Expression of Oat1 was slightly higher in male kidney than in female kidney, whereas the presence of Oat2 mRNA was considerably higher in female kidney than male kidney or liver, negating the presumed liver specificity of Oat2. Conversely, the Oat3 gender difference occurred in liver, rather than kidney, where male mRNA levels were higher than female levels. Ontogeny data supported the tissue expression results in kidney and identified three distinct maturation patterns. Oat3 matured earliest, whereas Oat1 mRNA expression did not reach adult levels until about day 30. Finally, Oat2 expression was low until the onset of sexual maturity at day 35, when transcript levels began to increase dramatically in females without a similar increase in male expression.