BASIC RESEARCH

Organic Anion Transporter 3 Contributes to the Regulation of Blood Pressure

Volker Vallon, Satish A. Eraly, William R. Wikoff, Timo Rieg, Gregory Kaler, David M. Truong, Sun-Young Ahn, Nitish R. Mahapatra, Sushil K. Mahata, Jon A. Gangoiti, Wei Wu, Bruce A. Barshop, Gary Siuzdak and Sanjay K. Nigam
JASN September 2008, 19 (9) 1732-1740; DOI: https://doi.org/10.1681/ASN.2008020180

Abstract

Renal organic anion transporters (OAT) are known to mediate the excretion of many drugs, but their function in normal physiology is not well understood. In this study, mice lacking organic anion transporter 3 (Oat3) had a 10 to 15% lower BP than wild-type mice, raising the possibility that Oat3 transports an endogenous regulator of BP. The aldosterone response to a low-salt diet was blunted in Oat3-null mice, but baseline aldosterone concentration was higher in these mice, suggesting that aldosterone dysregulation does not fully explain the lower BP in the basal state; therefore, both targeted and global metabolomic analyses of plasma and urine were performed, and several potential endogenous substrates of Oat3 were found to accumulate in the plasma of Oat3-null mice. One of these substrates, thymidine, was transported by Oat3 expressed in vitro. In vivo, thymidine, as well as two of the most potent Oat3 inhibitors that were characterized, reduced BP by 10 to 15%; therefore, Oat3 seems to regulate BP, and Oat3 inhibitors might be therapeutically useful antihypertensive agents. Moreover, polymorphisms in human OAT3 might contribute to the genetic variation in susceptibility to hypertension.

The proximal tubule of the mammalian kidney efficiently secretes a large variety of organic anions (OA) leading to the rapid urinary excretion of those compounds. Basolateral uptake from plasma into the tubular cell, the first step in this secretory process, operates via anion exchange, coupling the entry of OA to the exit of dicarboxylates along their concentration gradient. Subsequent apical efflux from tubular cell into urine does not seem to be rate limiting and may occur via efflux pumps and/or anion exchange. Substrates for this secretory process include numerous important drugs (e.g., most nonsteroidal anti-inflammatory drugs, β-lactam antibiotics, diuretics, nucleoside analog antivirals). Accordingly, renal OA secretion has largely been studied from a pharmacologic perspective; however, physiologic substrates and functions are not well understood (for review, see references1–8).

Organic anion transporter 1 (Oat1),9,10 a gene originally identified as novel kidney transporter (NKT) and proposed to function in organic ion transport,11,12 and Oat3, a gene originally identified as Roct (reduced in oc transporter),13 seem to account largely for the basolateral uptake step of renal OA secretion: Both transporters have been immunolocalized to the basolateral membrane of the proximal tubule of human, rat, and mouse.14–21 Moreover, when expressed in Xenopus oocytes or epithelial cell lines, both Oat1 and Oat3 function as exchangers, coupling substrate entry to dicarboxylate exit.10,22,23

We, and subsequently others, recently characterized mice null for Oat1 and Oat3, finding specific defects in OA transport resulting in reduced renal excretion of diuretics and other drugs.24–28 In the course of performing these experiments, we discovered, unexpectedly, that the Oat3−/− mice, although otherwise grossly physiologically normal, manifested lowered BP. (Renal blood flow [as determined by paraaminohippuric acid clearance] and glomerular filtration [inulin clearance] were unaffected,26 indicating that altered renal hemodynamics do not account for the observed OA excretory defects in these mice.) BP was not affected in Oat1−/− mice or in those null for the related proximal tubular OAT, URAT1.29–31 These findings raised the possibility that Oat3 mediates the specific transport of an endogenous compound(s) involved in the regulation of BP. Moreover, they suggested the possibility that inhibition of Oat3 might also lower BP and, therefore, that Oat3 inhibitors might be useful in the treatment of hypertension. Here we describe experiments addressing these hypotheses.

RESULTS

Oat3 Knockout Mice Manifest Reduced BP

Tail-cuff measurements consistently revealed a 10 to 15% lowering of both systolic (Figure 1) and diastolic arterial BP (data not shown) in awake Oat3−/− compared with wild-type (WT) mice, in the absence of significant differences in heart rate (data not shown). This was confirmed when BP was directly measured via arterial catheterization in anesthetized mice (Figure 1); again, heart rate was not significantly different compared with WT mice (data not shown). Moreover, there were no differences between Oat3−/− and WT mice in general aspects of renal function (GFR, renal blood flow,26 and fractional excretion of fluid and electrolytes; data not shown). By contrast, BP was unaffected in mice null for the other major basolateral proximal tubular OAT, Oat1 (Figure 1), or in those null for mUrat1 (RST), an apical proximal tubular OAT involved in the renal reabsorption of urate.29–31 These results suggest the possibility of specific transport of an endogenous BP-regulating compound by Oat3.

Figure 1.
Figure 1.

Low BP in Oat3 knockout mice. Systolic BP was determined by tail-cuff method in awake mice, or mice were anesthetized and mean arterial BP was directly measured via an intra-arterial catheter. Data are means ± SEM of measurements in six to 20 mice of each genotype. BP measurements under anesthesia were previously reported for Oat1 knockout (−/−) mice.28 Mean arterial pressure was significantly reduced in Oat3−/− compared with WT mice (**P < 0.01) but not in mice lacking the related proximal tubular OAT Oat1 or mUrat1 (RST), raising the possibility that an endogenous BP-regulating compound is transported by Oat3.

Identification of Potential Endogenous Oat3 Substrates

We performed analyses of Oat3−/− mouse urine and plasma so as to identify endogenous substrates of Oat3, among which might be included the aforementioned putative BP-regulating compound. We reasoned that such substrates should be increased in the plasma and possibly decreased in the urine of Oat3−/− mice, owing to loss of Oat3-mediated tubular secretion. We initially performed targeted gas chromatography/mass-spectrometry (GC/MS) as described previously,28 to determine the concentrations of approximately 30 of the most abundant small OA in the urine and plasma of WT and Oat3−/− mice, including various Krebs cycle intermediates and fatty and amino acid metabolites. No significant differences between WT and null mice were noted (Figure 2A, left), indicating that this set of compounds, although including several putative novel endogenous Oat1 substrates (Figure 2A, right) does not seem to contain any authentic substrates of Oat3. Accordingly, we performed global (metabolomic) rather than targeted mass spectrometric analyses of Oat3−/− plasma and urine, assaying the levels of several thousands of molecules simultaneously. These global analyses revealed that the metabolite composition of plasma and urine of Oat3−/− mice is indeed significantly different from that of WT mice, with 2% of the features in plasma and 14% of the features in urine significantly different in concentration between the genotypes (two-tailed t test, P < 0.01). Importantly, the observed differences were markedly skewed in the direction expected for endogenous substrates (i.e., increased in concentration in plasma and decreased in urine in Oat3−/− mice [Figure 2B]).

Figure 2.
Figure 2.

Detection of potential endogenous Oat3 substrates. (A) GC/MS analysis was performed to determine the levels of approximately 30 of the most abundant endogenous OA in plasma and urine. For each compound, the ratio of the concentration in urine (normalized to creatinine) to the concentration in plasma was calculated, representing a measure of the efficiency of renal excretion. Urine/plasma ratios of these compounds were largely unaffected in Oat3−/− mice (left). In comparison, several compounds manifested markedly lower urine/plasma ratios (i.e., lower apparent renal excretion) in Oat1−/− compared with WT mice, indicating the involvement of Oat1 in their tubular secretion (data taken from reference28). Notably, the urine/plasma ratio for α-ketoglutarate, which serves as a counterion for OA uptake, was greater in Oat1−/− compared with WT mice but unaffected in Oat3−/− mice. Data are means ± SEM (n = 4 per genotype). (B) A global mass-spectrometric (metabolomic) analysis was performed to compare the plasma and urine metabolite composition of Oat3−/− and WT mice. Data presented are the mean intensity values for the 100 compounds each in plasma and urine that were the most significantly different in intensity between Oat3−/− and WT mice. The majority (81%) of the significantly varying compounds in plasma were increased in Oat3−/− relative to WT, whereas the majority (64%) of those in urine were decreased in Oat3−/− relative to WT, consistent in each case with diminished renal excretion of the corresponding compounds.

The most significantly varying features were analyzed further; however, although the masses of the corresponding compounds were determined to an accuracy of 8 ppm, only a few could be matched to known metabolites (e.g., using the Metlin metabolite database). Plasma and urine intensity values in WT and Oat3−/− mice for two of the identified compounds, flavin mononucleotide (FMN) and thymidine, are presented in Figure 3A, along with data for an unknown compound of m/z 323.03. We subsequently tested the identified compounds for interaction with Oat3 in vitro and found that both FMN and thymidine inhibited tracer uptake in Oat3-expressing oocytes in a concentration-dependent manner (Figure 3B), as would be expected in the case of genuine Oat3 substrates. Because further experiments (see next paragraph) pointed to a particular role of thymidine, we directly assessed Oat3-mediated transport of this compound. We found that labeled thymidine was taken up into Oat3-expressing oocytes to a much greater degree than into control oocytes (Figure 3C), indicating that Oat3 can mediate the transport of thymidine. We also assessed thymidine handling by Oat1 both in vitro and in vivo. We determined that thymidine was not appreciably transported by Oat1 in vitro (Supplemental Figure 1A) and that plasma levels of thymidine were not elevated in the Oat1 knockout (Supplemental Figure 1B), indicating that thymidine does not undergo significant Oat1-mediated uptake/secretion in vivo.

Figure 3.
Figure 3.

Identification of potential endogenous Oat3 substrates. (A) Box plots of intensity values for two of the identified compounds from the metabolomic analysis, FMN and thymidine, and of an unknown compound of m/z of 323.03. **P < 0.01. (B) Tracer uptake into Oat3-expressing oocytes was determined in the presence of the indicated concentrations of the putative substrates, thymidine and FMN, and of the well-characterized Oat3 substrate estrone-3-sulfate and in their absence (control). Data are means ± SEM of measurements in four groups of four to five oocytes each, and the IC50 values were calculated by curve-fitting the points using nonlinear regression. (C) Uptake of labeled thymidine (0.097 μM) and ES (0.017 μM) was determined in oocytes microinjected with Oat3 and in control (noninjected) oocytes. Data are means ± SEM of measurements in five groups of four to eight oocytes each. Oat3-expressing oocytes manifested significantly greater uptake of both ES and thymidine than did control oocytes. #P < 0.001. (D) Intravenous application of thymidine induced a dosage-dependent reduction in BP in anesthetized WT and Oat3−/− mice. Data are means ± SEM in four mice. *P < 0.05, **P < 0.01, #P < 0.001 versus vehicle.

Effect on BP of Administration of Endogenous Oat3 Substrates

Thymidine or FMN could be directly involved in BP regulation, and/or as apparent Oat3 substrates, they could competitively inhibit the transport of other substrates, including that of the putative BP-regulating substrate. Accordingly, exogenous administration of these compounds might reproduce the phenotype of decreased BP observed in the Oat3−/− mice. FMN, thymidine, or vehicle (30 μl of water) was given as an intravenous bolus in anesthetized WT mice while BP was continuously monitored. We observed that whereas FMN at dosages up to 100 mg/kg intravenously and vehicle application were ineffective (data not shown), thymidine induced a dosage-dependent reduction in BP (Figure 3D) without lowering heart rate (data not shown). The BP response peaked within approximately 30 s and lasted for 3 to 5 min. The lower potency of thymidine versus FMN for the inhibition of Oat3-mediated uptake (Figure 3B) suggested that lowering of BP by this compound might not have been primarily due to Oat3 inhibition. In accordance with this inference, a very similar BP response to thymidine was observed in Oat3−/− mice (Figure 3D). Thus, the Oat3 substrate thymidine might accumulate in the plasma of mice lacking Oat3 and contribute to the lower BP in these mice.

Identification of Oat3 Inhibitors

Although thymidine and FMN might be insufficiently potent to inhibit Oat3 in vivo (see Figure 3B), more potent compounds might do so. Accordingly, we sought to identify other Oat3 inhibitors, testing the capacity for Oat3 inhibition of approximately 30 small OA of diverse chemical structures. Tested compounds encompassed fluorescein derivatives, steroid conjugates, nucleoside analog antivirals, and short-chain aliphatic dicarboxylates, among other small OA; data on the fluorescein derivatives and antivirals has previously been reported.32 Inhibition by each compound was determined as in the experiments presented in Figure 3B. The tested compounds varied widely in inhibitory potency, ranging from submicromolar to millimolar (Figure 4A). In general, the steroid conjugates were among the more potent of the compounds tested and the antivirals among those less potent

Figure 4.
Figure 4.

Reduced BP after administration of Oat3 inhibitors. (A) The inhibitory potency for Oat3 of each of approximately 30 OA was determined as in the experiments presented in Figure 3B. Among these, data on the fluorescein derivatives and antivirals has previously been reported.32 Data are means ± SEM of determinations of pIC50 (the negative log of IC50; thus, higher values indicate greater inhibitory potency). The chemical structures of the tested compounds are depicted above their corresponding pIC50 values. (B) Selected compounds from Figure 4A (▪) were intravenously infused into WT mice under anesthesia. These comprised the well-characterized OAT inhibitor probenecid and the fluorescent OA eosin-Y, fluorescein, 5CF, and 6- carboxyfluorescein, each administered at a dosage of 50 to 100 mg/kg. A consistent decrease in BP was noted after the infusion of the two higher potency compounds, eosin-Y and probenecid, but not after infusion of the three lower potency compounds (data not shown). Data are means ± SEM of measurements in three to four independent experiments each. *P < 0.05 versus vehicle.

Reduced BP after Administration of the Most Potent Oat3 Inhibitors

We tested the effects on BP of a subset of the previously mentioned compounds: The well-characterized OAT inhibitor probenecid6,7,24,28,33 and the fluorescent OA eosin-Y, fluorescein, 5-carboxyfluorescein (5CF), and 6-carboxyfluorescein (the latter compounds were chosen because they allowed for fluorometric determination of inhibitor plasma levels). Approximately 50 to 100 mg/kg of each of these compounds was intravenously infused into mice under anesthesia as described already (resulting in initial plasma concentrations of approximately 2 to 5 mM in the case of the fluorescent compounds), followed by recording of any changes in BP. We found that administration of the two highest potency compounds (of the five tested), eosin-Y and probenecid (Figure 4B), but not of the three lower potency compounds (data not shown) consistently caused an acute decrease in BP. These results are consistent with our hypothesis that inhibition of Oat3 by sufficiently potent inhibitors can lower BP. Nevertheless, it should be noted that given the relatively high dosages used and that each of the tested compounds interacts with Oat1 with comparable potency,33a nonspecific effects cannot be excluded.

Plasma Concentrations of Corticosterone, Adrenocorticotropic Hormone, Aldosterone, and Renin in Oat3 Knockout Mice

Given the high potency interaction of Oat3 with steroid conjugates demonstrated here (Figure 4A) as well as elsewhere (reviewed in reference6), we determined the concentrations of the BP-regulating adrenal steroids and of functionally related hormones in Oat3 knockout compared with WT plasma. We found lower BP in Oat3−/− mice to be associated with greater plasma adrenocorticotropic hormone (ACTH) but unchanged corticosterone concentrations (Figure 5A). ACTH stimulation increased plasma corticosterone by a factor of approximately 2.4 in both WT and Oat3−/− mice (Figure 5A), indicating intact acute ACTH-responsiveness in Oat3−/− mice. These results argue against an essential role for Oat3 in corticosterone release from the adrenal gland and against a primary role for defects in corticosterone handling in causing the lower BP in Oat3−/− mice.

Figure 5.
Figure 5.

Plasma vasoactive hormones and the response to low-salt diet in Oat3 knockout mice. (A) Lower BP in Oat3−/− mice was associated with increased plasma concentrations of ACTH, renin, and aldosterone, whereas plasma corticosterone concentrations were not different. The ACTH-induced increase in plasma corticosterone was unaffected in the knockouts, whereas the furosemide (FURO)-induced renin response was enhanced, and the low-NaCl diet–induced aldosterone increase was blunted (n = 6 to 9 per genotype). (B) The blunted aldosterone response to a low-NaCl diet in Oat3−/− compared with WT mice was associated with greater body weight loss and increased fluid intake but similar food intake (n = 6 per genotype). Data are means ± SEM. *P < 0.05, **P < 0.01 versus WT.

Oat3−/− mice had modestly greater plasma renin concentrations (Figure 5A) and greater renal expression of renin versus WT mice (data not shown). To test for a potential role of Oat3 in macula densa stimulation of renin secretion,16 we performed experiments with furosemide. Furosemide (40 mg/kg intraperitoneally) stimulates renal renin secretion by inhibiting the sensing of the luminal NaCl concentration at the macula densa,34 thereby increasing macula densa release of prostaglandin E2 (PGE2) across the basolateral membrane,35 a mechanism that may involve Oat3.16 On the basis of our previous studies,26 the applied dosage of furosemide is expected to induce similar maximal inhibition of NaCl reabsorption in thick ascending limb and macula densa in both genotypes; however, furosemide in fact increased renin levels to a greater degree in Oat3−/− than in WT mice (Figure 5A), indicating enhanced rather than reduced PGE2 signaling in the knockout; as further outlined in the Discussion, this may reflect impaired PGE2 reuptake in the knockouts.

In addition to increased renin, Oat3−/− mice manifested modestly greater plasma aldosterone (Figure 5A), which is expected to help stabilize BP by enhancing renal NaCl reabsorption. To test the efficiency of that system in Oat3−/− mice, we assessed the response to a low NaCl diet given for 7 d. Under basal conditions, no significant differences were observed between WT and Oat3−/− mice in body weight and food or fluid intake (data not shown). A low NaCl diet, however, induced a significantly greater loss of body weight in Oat3−/− compared with WT mice (Figure 5B), which could not be explained by lower food intake. This diet increased plasma renin to similar levels in both genotypes, but aldosterone upregulation was impaired in Oat3−/− mice (Figure 5A). It is possible that this impaired upregulation led to salt and fluid loss, lowering body weight and inducing thirst and increased fluid intake, as shown in Figure 5B.

Interaction of Aldosterone and Its Potential Precursors with Oat3 in Xenopus Oocytes

Given that Oat3 is expressed in adrenal tissue and is able to mediate transport of cortisol,36 aldosterone dysregulation in Oat3 knockouts might be due to loss of interaction of aldosterone or related compounds with Oat3. We found aldosterone and three of its potential precursors, progesterone, corticosterone, and desoxycorticosterone,37,38 to each dosage-dependently inhibit Oat3-mediated tracer uptake in vitro, with median inhibition concentration (IC50) values of 12, 29, 10, and 9 μM, respectively (Figure 6). Although these affinities are comparable to that of the prototypical Oat3 substrate estrone-3-sulfate (Figure 3B), they are much greater than physiologic free plasma concentrations of these compounds (this study)39,40 (although local concentrations around the adrenal glomerulosa cells might be considerably higher). Thus, the physiologic significance of these interactions is uncertain.

Figure 6.
Figure 6.

Interaction of aldosterone and its potential precursors, progesterone, corticosterone, and desoxycorticosterone, with Oat3. Uptake of 5-CF in mOat3-expressing Xenopus oocytes was determined in the presence of the indicated concentrations of the steroid hormones to calculate the respective IC50 values. Each compound caused a dosage-dependent decrease in uptake, consistent with competitive inhibition of uptake of the fluorescent tracer. Data are presented as percentage of control uptake and are the means ± SEM of measurements in four groups of four to five oocytes each. IC50 values were determined by curve-fitting the points using nonlinear regression.

DISCUSSION

Recent studies localized Oat3 to the basolateral membrane of macula densa cells, and it was proposed that Oat3 at that site might contribute to the local release or reuptake of PGE2.16,41 Basolateral release of PGE2 mediates the stimulation of renin release (in turn leading to aldosterone activation) that occurs in response to low salt concentrations at the macula densa or to inhibition of macula densa transport by furosemide.35 We find that the renin response to furosemide is enhanced in Oat3−/− mice, suggesting that Oat3 might contribute to the reuptake of PGE2 (loss of such reuptake might also have contributed to greater basal renin levels in Oat3−/− mice, helping ameliorate hypotension). Of note, the aldosterone response to salt deprivation was blunted in Oat3−/− mice; however, aldosterone dysregulation cannot explain the observed hypotension under basal conditions because basal plasma aldosterone concentrations were increased in mice lacking Oat3.

The aforementioned results raise the possibility that Oat3 mediates the transport of other endogenous regulators of BP. To test this possibility, we examined the effects of two of the compounds identified via metabolomic analyses and subsequent transport assays as potential endogenous Oat3 substrates: Thymidine and FMN. Whereas FMN displayed no effect, thymidine exhibited a consistent dosage-dependent lowering of BP. Normal serum concentrations of thymidine are approximately 1 μM, and there is a thymidine pool that is in rapid equilibrium with blood thymidine and that is at least 10 times larger than the pool of extracellular thymidine.42 It is difficult to know what the local concentrations of the exogenously administered thymidine were; however, assuming it was rapidly distributed into the aforementioned pools, a concentration of 20 μM would be achieved at 10 mg/kg injection, where the BP effect was first noted. Within a roughly 10-fold higher concentration, an effect comparable to that observed in the Oat3 knockout was observed.

Although a number of endogenous OA compounds transported by Oat3 may be involved in regulating BP, at the very least, these results suggest that some of them can, perhaps in concert, act to regulate BP under physiologic conditions. This also suggests that inhibitors of Oat3, regardless of whether they are actual substrates for the transporter, might lower BP. This could lead to a novel approach to the design of antihypertensive agents. We therefore tested a set of Oat3 inhibitors (selected from a larger screen) for effects on BP, finding that administration of two of the five tested compounds resulted in lowering of BP.

Together, the data suggest that (1) Oat3 regulates BP, (2) Oat3 affects the regulation of plasma aldosterone (although this cannot explain the hypotensive phenotype of Oat3-null mice), (3) at least some endogenous compounds that accumulate in the absence of Oat3 are substrates with the potential to regulate BP, and (4) administration of sufficiently potent inhibitors might result in lowering of BP. Although much more work needs to be done, the data suggest novel concepts regarding the physiologic role of Oat3 substrates and raise the possibilities that Oat3 inhibitors might be of use in the treatment of hypertension and that polymorphisms in human OAT3 might contribute to variability in the propensity for hypertension.

CONCISE METHODS

Animals

Oat3−/− mice24 were back-crossed to C57BL/6J mice for eight generations. Heterozygous mice from the last back-cross were bred to each other to generate gene knockout and WT mice. All of the animals used in the experiments described descended from those mice. Mice were genotyped by PCR as described previously.24 Experimental protocols were in accordance with the Guide for Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee.

Materials

[3H]-estrone sulfate (ES) and [3H]-thymidine were purchased from Perkin-Elmer Life Sciences (Boston, MA), and 5CF, a fluorescent OA transported by mOat332 and other OA test compounds (all >98% pure and in the l-enantiomer) were purchased from Sigma-Aldrich (St. Louis, MO).

BP Measurements

Mice were anesthetized for terminal experiments with 100 mg/kg inactin intraperitoneally and 100 mg/kg ketamine intramuscularly, the jugular vein was cannulated for infusion of maintenance fluids and test compounds, and the femoral artery was cannulated for BP and heart rate measurement as described previously.43,44 In separate experiments, BP and heart rate in unanesthetized mice were determined by the tail-cuff method in trained awake mice as described previously.44–46

Urine and Plasma Samples

Mice were placed in metabolic cages (Tecniplast, Hohenpeissenberg, Germany) with free access to tap water and standard diet (0.44% Na+, 0.97% K+) for quantitative collection of urine, immediately followed by collection of plasma via puncture of the retrobulbar plexus as described previously.43

GC/MS

GC/MS was performed as described previously.28 Briefly, plasma and urine samples (100 μl or volumes equivalent to 0.25 to 1.00 μmol of creatinine, respectively) were reacted with pentafluoro-benzylhydroxylamine to form oximes of ketones, oxo-acids, and aldehydes. The reaction products were lyophilized and extracted in 42% t-amyl alcohol:chloroform over a column of silicic acid. The dried eluate was reacted with BSTFA/TRISIL to form trimethylsilyl derivatives. The latter were injected (0.5 to 1.0 μl) on a bonded phase (DB5) capillary column (30 m × 0.25 mm) in an Agilent 6890/5973 GC/MS. Electron impact mass spectra were obtained in scan mode (50 to 650 amu at 2.4 cycles/s). The species were quantified using calibrated response curves of selected ions and 4-nitrophenol and 2-oxocaproic acid as internal standards.

Global Metabolomics

Urine and plasma samples were extracted in methanol and lyophilized, and the equivalents of 0.4 to 1.4 μl of extracted urine (after normalization to the previously measured concentrations of creatinine) or 1 μl of extracted plasma were applied to a capillary reverse-phase column with dimensions 150 × 0.3 mm (diameter; Zorbax SB-300, Agilent) at a flow rate of 4 μl/min. Buffer A was H2O + 0.1% formic acid, and Buffer B was acetonitrile with 0.1% formic acid. Plasma samples were eluted with a gradient from 5 to 95% B over 45 min; for urine samples, the gradient was 5% B from 0 to 10 min then 5 to 95% from 10 to 55 min, then held at 95% to 60 min. The column was interfaced to an electrospray ionization time-of-flight mass spectrometer (Agilent). Data were collected from approximately 100 to 1000 m/z in continuum mode and converted from the instrument format (.wiff) to the common format (.cdf). Data were analyzed using the analysis and nonlinear alignment program XCMS,47 in which the integrated intensities of all observed ions were compared between the Oat3−/− and WT groups. These procedures resulted in the detection of approximately 4000 features in urine and approximately 5000 in plasma, which would include any metabolites with m/z values between 100 and 1000 that ionize under positive electrospray conditions and were within the detection limit of the instrument. The data were then analyzed using the recently developed software package XCMS, which performs a nonlinear alignment of the chromatograms and integration of the intensities for each ion.

Xenopus Oocyte Transport Experiments

Xenopus oocyte assays were performed as described previously.28 Briefly, capped cRNA was generated via in vitro transcription from linearized plasmid DNA, mOat3 (Image clone ID 4239544), or mOat1 (Image clone ID 4163278), using mMessage mMachine in vitro transcription kit (Ambion, Austin, TX). Stage V and VI Xenopus oocytes were isolated, maintained in Barth's growth medium, injected with approximately 20 nl/oocyte cRNA solution (1 μg/μl), and allowed to mature for 3 d after injection. Oat3-expressing and control noninjected oocytes were then incubated in Barth's buffer for 1 h at 25°C with Oat3 tracer substrate (50 μM 5CF, 0.017 μM 3H-ES, or 0.097 μM 3H-thymidine) in the presence of various concentrations of test compounds or in their absence. Transport was terminated by washes in ice-cold Barth's buffer, and the tracer content of groups of four to five oocytes was determined by fluorometry or scintillation counting in the case of experiments using 5CF and the radiolabeled tracers, respectively. The background uptake of tracer was determined in noninjected oocytes and subtracted from that in Oat3-injected oocytes to calculate the Oat3-mediated component of uptake. The IC50 were determined by nonlinear regression using Prism software 5.0 (GraphPad, San Diego, CA).

Plasma Concentrations of Aldosterone, Renin, Corticosterone and ACTH

Mice were placed under short-term isoflurane anesthesia, and blood was taken from the retrobulbar plexus for determination of hematocrit and basal plasma concentrations of aldosterone (Diagnostic Systems Laboratories, Webster, TX; cross-reactivity with corticosterone 0.02%), renin (see below in this paragraph), corticosterone, and ACTH (both MP Biomedicals, Orangeburg, NY). To determine a potential role of Oat3 in corticosterone release from the adrenal gland,36 we performed an ACTH stimulation test. To this end, plasma corticosterone was determined 45 min after intraperitoneal application of 1 U of 1-24 ACTH (Cortrosyn®; Organon, Roseland, NJ).48 To determine a potential role of Oat3 in macula densa stimulation of renin secretion,16 we determined plasma renin concentration 45 min after intraperitoneal application of furosemide (40 mg/kg).34 In addition, the response in body weight and food and fluid intake as well as in plasma concentrations of renin and aldosterone to a low-NaCl diet for 8 d was assessed. Plasma renin concentration was measured by RIA (GammaCoat Plasma Renin Activity, CA-1533; DiaSorin, Stillwater, MN) as the generation of angiotensin I after addition of excess angiotensinogen substrate from bilaterally nephrectomized rats.49 Concentrations of Na+ and K+ in plasma and urine were determined using a flame photometer (Cole-Parmer Instrument Co., Vernon Hills, IL).

Statistical Analysis

Data are presented as means ± SEM. Statistical differences between gene-knockout and WT mice were analyzed by the unpaired t test. P < 0.05 was considered to be statistically significant.

DISCLOSURES

None.

Acknowledgments

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases grants DK56248 and DK28602 (to V.V.), DK064839 and DK075486 (to S.A.E.), and AI057695, DK079784, and HL35018 (to S.K.N.); the Department of Veterans Affairs (to V.V.); and Deutsche Forschungsgemeinschaft RI 1535/3-1 and RI 1535/3-2 (to T.R.).

We thank Duke A. Vaughn, Shamara Closson, Kerstin Richter, and Jana Schroth for expert technical assistance.

Footnotes

  • Supplemental information for this article is available online at http://www.jasn.org/.

    Published online ahead of print. Publication date available at www.jasn.org.

    V.V. and S.A.E. contributed equally to this work.

    N.R.M.'s current affiliation is Department of Biotechnology, IIT Madras, Chennai, India.

REFERENCES

  1. 1.↵
    Robertson EE, Rankin GO: Human renal organic anion transporters: Characteristics and contributions to drug and drug metabolite excretion. Pharmacol Ther 109 : 399 –412, 2006
    OpenUrlCrossRefPubMed
  2. 2.
    Eraly SA, Blantz RC, Bhatnagar V, Nigam SK: Novel aspects of renal organic anion transporters. Curr Opin Nephrol Hypertens 12 : 551 –558, 2003
    OpenUrlCrossRefPubMed
  3. 3.
    Eraly SA, Bush KT, Sampogna RV, Bhatnagar V, Nigam SK: The molecular pharmacology of organic anion transporters: From DNA to FDA? Mol Pharmacol 65 : 479 –487, 2004
    OpenUrlAbstract/FREE Full Text
  4. 4.
    Koepsell H, Endou H: The SLC22 drug transporter family. Pflugers Arch 447 : 666 –676, 2004
    OpenUrlCrossRefPubMed
  5. 5.
    Sweet DH, Bush KT, Nigam SK: The organic anion transporter family: From physiology to ontogeny and the clinic. Am J Physiol Renal Physiol 281 : F197 –F205, 2001
    OpenUrlPubMed
  6. 6.↵
    Burckhardt BC, Burckhardt G: Transport of organic anions across the basolateral membrane of proximal tubule cells. Rev Physiol Biochem Pharmacol 146 : 95 –158, 2003
    OpenUrlCrossRefPubMed
  7. 7.↵
    Wright SH, Dantzler WH: Molecular and cellular physiology of renal organic cation and anion transport. Physiol Rev 84 : 987 –1049, 2004
    OpenUrlCrossRefPubMed
  8. 8.↵
    Nigam SK, Bush KT, Bhatnagar V: Drug and toxicant handling by the OAT organic anion transporters in the kidney and other tissues. Nat Clin Pract Nephrol 3 : 443 –448, 2007
    OpenUrlCrossRefPubMed
  9. 9.↵
    Burckhardt G, Bahn A, Wolff NA: Molecular physiology of renal p-aminohippurate secretion. News Physiol Sci 16 : 114 –118, 2001
    OpenUrlPubMed
  10. 10.↵
    Sweet DH, Wolff NA, Pritchard JB: Expression cloning and characterization of ROAT1: The basolateral organic anion transporter in rat kidney. J Biol Chem 272 : 30088 –30095, 1997
    OpenUrlAbstract/FREE Full Text
  11. 11.↵
    Lopez-Nieto CE, You G, Bush KT, Barros EJ, Beier DR, Nigam SK: Molecular cloning and characterization of NKT, a gene product related to the organic cation transporter family that is almost exclusively expressed in the kidney. J Biol Chem 272 : 6471 –6478, 1997
    OpenUrlAbstract/FREE Full Text
  12. 12.↵
    Lopez-Nieto CE, You G, Barros EJ, Beier DR, Nigam SK: Molecular cloning and characterization of a novel transport protein with very high expression in the kidney [Abstract]. J Am Soc Nephrol 7 : 1301 , 1996
    OpenUrl
  13. 13.↵
    Brady KP, Dushkin H, Fornzler D, Koike T, Magner F, Her H, Gullans S, Segre GV, Green RM, Beier DR: A novel putative transporter maps to the osteosclerosis (oc) mutation and is not expressed in the oc mutant mouse. Genomics 56 : 254 –261, 1999
    OpenUrlCrossRefPubMed
  14. 14.↵
    Hosoyamada M, Sekine T, Kanai Y, Endou H: Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney. Am J Physiol 276 : F122 –F128, 1999
    OpenUrlPubMed
  15. 15.
    Ljubojevic M, Herak-Kramberger CM, Hagos Y, Bahn A, Endou H, Burckhardt G, Sabolic I: Rat renal cortical OAT1 and OAT3 exhibit gender differences determined by both androgen stimulation and estrogen inhibition. Am J Physiol Renal Physiol 287 : F124 –F138, 2004
    OpenUrlCrossRefPubMed
  16. 16.↵
    Bahn A, Ljubojevic M, Lorenz H, Schultz C, Ghebremedhin E, Ugele B, Sabolic I, Burckhardt G, Hagos Y: Murine renal organic anion transporter 1 (mOAT1) and organic anion transporter 3 (mOAT3) facilitate the transport of neuroactive tryptophan metabolites. Am J Physiol Cell Physiol 289 : C1075 –C1084, 2005
    OpenUrlCrossRefPubMed
  17. 17.
    Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, Inui K: Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J Am Soc Nephrol 13 : 866 –874, 2002
    OpenUrlAbstract/FREE Full Text
  18. 18.
    Kojima R, Sekine T, Kawachi M, Cha SH, Suzuki Y, Endou H: Immunolocalization of multispecific organic anion transporters, OAT1, OAT2, and OAT3, in rat kidney. J Am Soc Nephrol 13 : 848 –857, 2002
    OpenUrlAbstract/FREE Full Text
  19. 19.
    Hasegawa M, Kusuhara H, Sugiyama D, Ito K, Ueda S, Endou H, Sugiyama Y: Functional involvement of rat organic anion transporter 3 (rOat3; Slc22a8) in the renal uptake of organic anions. J Pharmacol Exp Ther 300 : 746 –753, 2002
    OpenUrlAbstract/FREE Full Text
  20. 20.
    Cha SH, Sekine T, Fukushima JI, Kanai Y, Kobayashi Y, Goya T, Endou H: Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol Pharmacol 59 : 1277 –1286, 2001
    OpenUrlAbstract/FREE Full Text
  21. 21.↵
    Tojo A, Sekine T, Nakajima N, Hosoyamada M, Kanai Y, Kimura K, Endou H: Immunohistochemical localization of multispecific renal organic anion transporter 1 in rat kidney. J Am Soc Nephrol 10 : 464 –471, 1999
    OpenUrlAbstract/FREE Full Text
  22. 22.↵
    Sweet DH, Chan LM, Walden R, Yang XP, Miller DS, Pritchard JB: Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na+ gradient. Am J Physiol Renal Physiol 284 : F763 –F769, 2003
    OpenUrlCrossRefPubMed
  23. 23.↵
    Bakhiya A, Bahn A, Burckhardt G, Wolff N: Human organic anion transporter 3 (hOAT3) can operate as an exchanger and mediate secretory urate flux. Cell Physiol Biochem 13 : 249 –256, 2003
    OpenUrlCrossRefPubMed
  24. 24.↵
    Sweet DH, Miller DS, Pritchard JB, Fujiwara Y, Beier DR, Nigam SK: Impaired organic anion transport in kidney and choroid plexus of organic anion transporter 3 (Oat3 (Slc22a8)) knockout mice. J Biol Chem 277 : 26934 –26943, 2002
    OpenUrlAbstract/FREE Full Text
  25. 25.
    Vanwert AL, Bailey RM, Sweet DH: Organic anion transporter 3 (Oat3/Slc22a8) knockout mice exhibit altered clearance and distribution of penicillin G. Am J Physiol Renal Physiol 293 : F1332 –F1341, 2007
    OpenUrlCrossRefPubMed
  26. 26.↵
    Vallon V, Rieg T, Ahn SY, Wu W, Eraly SA, Nigam SK: Overlapping in vitro and in vivo specificities of the organic anion transporters OAT1 and OAT3 for loop and thiazide diuretics. Am J Physiol Renal Physiol 294 : F867 –F873, 2008
    OpenUrlCrossRefPubMed
  27. 27.
    Vanwert AL, Sweet DH: Impaired clearance of methotrexate in organic anion transporter 3 (Slc22a8) knockout mice: A gender specific impact of reduced folates. Pharm Res 25 : 453 –462, 2008
    OpenUrlCrossRefPubMed
  28. 28.↵
    Eraly SA, Vallon V, Vaughn DA, Gangoiti JA, Richter K, Nagle M, Monte JC, Rieg T, Truong DM, Long JM, Barshop BA, Kaler G, Nigam SK: Decreased renal organic anion secretion and plasma accumulation of endogenous organic anions in OAT1 knock-out mice. J Biol Chem 281 : 5072 –5083, 2006
    OpenUrlAbstract/FREE Full Text
  29. 29.↵
    Mori K, Ogawa Y, Ebihara K, Aoki T, Tamura N, Sugawara A, Kuwahara T, Ozaki S, Mukoyama M, Tashiro K, Tanaka I, Nakao K: Kidney-specific expression of a novel mouse organic cation transporter-like protein. FEBS Lett 417 : 371 –374, 1997
    OpenUrlCrossRefPubMed
  30. 30.
    Enomoto A, Kimura H, Chairoungdua A, Shigeta Y, Jutabha P, Cha SH, Hosoyamada M, Takeda M, Sekine T, Igarashi T, Matsuo H, Kikuchi Y, Oda T, Ichida K, Hosoya T, Shimokata K, Niwa T, Kanai Y, Endou H: Molecular identification of a renal urate anion exchanger that regulates blood urate levels. Nature 417 : 447 –452, 2002
    OpenUrlPubMed
  31. 31.↵
    Eraly SA, Vallon V, Rieg T, Gangoiti JA, Wikoff WR, Siuzdak G, Barshop BA, Nigam SK: Multiple organic anion transporters contribute to net renal excretion of uric acid. Physiol Genomics 33 : 180 –192, 2008
    OpenUrlCrossRefPubMed
  32. 32.↵
    Truong DM, Kaler G, Khandelwal A, Swaan PW, Nigam SK: Multi-level analysis of organic anion transporters 1, 3, and 6 reveals major differences in structural determinants of antiviral discrimination. J Biol Chem 283 : 8654 –8663, 2008
    OpenUrlAbstract/FREE Full Text
  33. 33.↵
    Dantzler WH, Wright SH: The molecular and cellular physiology of basolateral organic anion transport in mammalian renal tubules. Biochim Biophys Acta 1618 : 185 –193, 2003
    OpenUrlPubMed
  34. 33a.↵
    Kaler G, Truong DM, Khandelwal A, Nagle M, Eraly SA, Swaan PW, Nigam SK: Structural variation governs substrate specificity for organic anion transporter (OA1) homologs. J Biol Chem 282 : 23841 –23853, 2007
    OpenUrlAbstract/FREE Full Text
  35. 34.↵
    Kim SM, Mizel D, Huang YG, Briggs JP, Schnermann J: Adenosine as a mediator of macula densa-dependent inhibition of renin secretion. Am J Physiol Renal Physiol 290 : F1016 –F1023, 2006
    OpenUrlCrossRefPubMed
  36. 35.↵
    Harris RC, Zhang MZ, Cheng HF: Cyclooxygenase-2 and the renal renin-angiotensin system. Acta Physiol Scand 181 : 543 –547, 2004
    OpenUrlCrossRefPubMed
  37. 36.↵
    Asif AR, Steffgen J, Metten M, Grunewald RW, Muller GA, Bahn A, Burckhardt G, Hagos Y: Presence of organic anion transporters 3 (OAT3) and 4 (OAT4) in human adrenocortical cells. Pflugers Arch 450 : 88 –95, 2005
    OpenUrlCrossRefPubMed
  38. 37.↵
    Vinson GP: Glomerulosa function and aldosterone synthesis in the rat. Mol Cell Endocrinol 217 : 59 –65, 2004
    OpenUrlCrossRefPubMed
  39. 38.↵
    Ishimura K, Fujita H: Light and electron microscopic immunohistochemistry of the localization of adrenal steroidogenic enzymes. Microsc Res Tech 36 : 445 –453, 1997
    OpenUrlCrossRefPubMed
  40. 39.↵
    Schneider JS, Stone MK, Wynne-Edwards KE, Horton TH, Lydon J, O'Malley B, Levine JE: Progesterone receptors mediate male aggression toward infants. Proc Natl Acad Sci U S A 100 : 2951 –2956, 2003
    OpenUrlAbstract/FREE Full Text
  41. 40.↵
    Vincent M, Gomez-Sanchez CE, Bataillard A, Sassard J: Steroids during development of genetic hypertension in rats of Lyon strain. Am J Physiol 257 : H506 –H510, 1989
    OpenUrlPubMed
  42. 41.↵
    Nilwarangkoon S, Anzai N, Shiraya K, Yu E, Islam R, Cha SH, Onozato ML, Miura D, Jutabha P, Tojo A, Kanai Y, Endou H: Role of mouse organic anion transporter 3 (mOat3) as a basolateral prostaglandin E(2) transport pathway. J Pharmacol Sci 103 : 48 –55, 2007
    OpenUrlCrossRefPubMed
  43. 42.↵
    Lee DJ, Prensky W, Krause G, Hughes WL: Blood thymidine level and iododeoxyuridine incorporation and reutilization in DNA in mice given long-acting thymidine pellets. Cancer Res 36 : 4577 –4583, 1976
    OpenUrlAbstract/FREE Full Text
  44. 43.↵
    Vallon V, Grahammer F, Volkl H, Sandu CD, Richter K, Rexhepaj R, Gerlach U, Rong Q, Pfeifer K, Lang F: KCNQ1-dependent transport in renal and gastrointestinal epithelia. Proc Natl Acad Sci U S A 102 : 17864 –17869, 2005
    OpenUrlAbstract/FREE Full Text
  45. 44.↵
    Rieg T, Bundey RA, Chen Y, Deschenes G, Junger W, Insel PA, Vallon V: Mice lacking P2Y2 receptors have salt-resistant hypertension and facilitated renal Na+ and water reabsorption. FASEB J 21 : 3717 –3726, 2007
    OpenUrlCrossRefPubMed
  46. 45.
    Vallon V, Wyatt AW, Klingel K, Huang DY, Hussain A, Berchtold S, Friedrich B, Grahammer F, Belaiba RS, Gorlach A, Wulff P, Daut J, Dalton ND, Ross J Jr, Flogel U, Schrader J, Osswald H, Kandolf R, Kuhl D, Lang F: SGK1-dependent cardiac CTGF formation and fibrosis following DOCA treatment. J Mol Med 84 : 396 –404, 2006
    OpenUrlCrossRefPubMed
  47. 46.↵
    Vallon V, Huang DY, Grahammer F, Wyatt AW, Osswald H, Wulff P, Kuhl D, Lang F: SGK1 as a determinant of kidney function and salt intake in response to mineralocorticoid excess. Am J Physiol Regul Integr Comp Physiol 289 : R395 –R401, 2005
    OpenUrlCrossRefPubMed
  48. 47.↵
    Smith CA, Want EJ, O'Maille G, Abagyan R, Siuzdak G: XCMS: Processing mass spectrometry data for metabolite profiling using nonlinear peak alignment, matching, and identification. Anal Chem 78 : 779 –787, 2006
    OpenUrlCrossRefPubMed
  49. 48.↵
    Meiner VL, Cases S, Myers HM, Sande ER, Bellosta S, Schambelan M, Pitas RE, McGuire J, Herz J, Farese RV Jr: Disruption of the acyl-CoA:cholesterol acyltransferase gene in mice: Evidence suggesting multiple cholesterol esterification enzymes in mammals. Proc Natl Acad Sci U S A 93 : 14041 –14046, 1996
    OpenUrlAbstract/FREE Full Text
  50. 49.↵
    Rieg T, Schnermann J, Vallon V: Adenosine A(1) receptors determine effects of caffeine on total fluid intake but not caffeine appetite. Eur J Pharmacol 555 : 174 –177, 2007
    OpenUrlCrossRefPubMed
View Abstract
Back to top

In this issue

View Selected Citations (0)
Print
Download PDF
Email Article
Citation Tools
Request Permissions
Organic Anion Transporter 3 Contributes to the Regulation of Blood Pressure
Volker Vallon, Satish A. Eraly, William R. Wikoff, Timo Rieg, Gregory Kaler, David M. Truong, Sun-Young Ahn, Nitish R. Mahapatra, Sushil K. Mahata, Jon A. Gangoiti, Wei Wu, Bruce A. Barshop, Gary Siuzdak, Sanjay K. Nigam
JASN Sep 2008, 19 (9) 1732-1740; DOI: 10.1681/ASN.2008020180
del.icio.us logo Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo

Jump to section

More in this TOC Section

Cited By...

Similar Articles

Related Articles