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Overlapping in vitro and in vivo specificities of the organic anion transporters OAT1 and OAT3 for loop and thiazide diuretics

Division of Nephrology and Hypertension, Departments of ¹Medicine, ²Pharmacology, ³Pediatrics, and ⁴Cellular and Molecular Medicine, University of California, and ⁵Department of Medicine, San Diego Veterans Affairs Healthcare System, La Jolla, California

Submitted 8 November 2007 ; accepted in final form 17 January 2008

	ABSTRACT

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Organic anion transporter (OAT) genes have been implicated in renal secretion of organic anions, but the individual in vivo contributions of OAT1 (first identified as NKT) and OAT3 remain unclear. Potential substrates include loop diuretics (e.g., furosemide) and thiazide diuretics (e.g., bendroflumethiazide), which reach their tubular sites of action mainly by proximal tubular secretion. Previous experiments in Oat1 knockout (–/–) mice revealed an almost complete loss of renal secretion of the prototypic organic anion p-aminohippurate (PAH) and a role of OAT1 in tubular secretion of furosemide (Eraly SA, Vallon V, Vaughn D, Gangoiti JA, Richter K, Nagle M, Monte JC, Rieg T, Truong DM, Long JM, Barshop BA, Kaler G, Nigam SK. J Biol Chem 281: 5072–5083, 2006). In this study we found that both furosemide and bendroflumethiazide inhibited mOat1- and mOat3-mediated uptake of a labeled tracer in Xenopus oocytes injected with cRNA, consistent with their being substrates for mouse OAT1 and OAT3. Experiments in Oat3^–/– mice revealed intact renal secretion of PAH, but the dose-natriuresis curves for furosemide and bendroflumethiazide were shifted to the right and urinary furosemide excretion was impaired similar to the defect in Oat1^–/– mice. Thus, whereas OAT1 (in contrast to OAT3) is the classic basolateral PAH transporter of the proximal tubule, both OAT1 and OAT3 contribute similarly to normal renal secretion of furosemide and bendroflumethiazide, and a lack of either one is not fully compensated by the other. Although microarray expression analysis in the kidneys of Oat1^–/– and Oat3^–/– mice revealed somewhat altered expression of a small number of transport-related genes, none were common to both knockout models. When searching for polymorphisms involved in human diuretic responsiveness, it may be necessary to consider both OAT1 and OAT3, among other genes.

organic anion transport; proximal tubule; tubular secretion

The basolateral uptake of OA into proximal tubules is believed to operate by coupling the entry of OA to the exit of dicarboxylates along their concentration gradient. Dicarboxylates like -ketoglutarate are maintained at high intracellular concentrations through intracellular synthesis and Na⁺-dicarboxylate cotransport, the latter being driven by the Na⁺ gradient established by the Na⁺-K⁺-ATPase. The classic basolateral OAT system, long referred to as the p-aminohippurate (PAH) transporter, was originally identified as novel kidney transporter (NKT) (24), which was proposed to function in renal organic ion secretion and was later directly shown to function in this capacity and named OAT1 (7, 34). The other primary candidate for basolateral OA uptake is OAT3, originally identified as Roct [reduced in osteosclerosis (oc) transporter] (4), and several immunohistochemical studies have localized both OAT1 and OAT3 to the basolateral membrane of the proximal tubule of human, rat, and mouse (1, 8, 15, 16, 20, 23, 26, 35). When expressed in Xenopus oocytes or epithelial cell lines, OAT1 couples OA entry to dicarboxylate exit and was trans-stimulated by preloading with PAH (34). Similarly, OAT3 operates as an organic anion/-ketoglutarate exchanger (2), and OAT3-mediated uptake of PAH was trans-stimulated by glutarate (32). Thus both transporters are candidates for basolateral uptake of OA in the proximal tubule.

The loop diuretic furosemide and the thiazide diuretic bendroflumethiazide are both OA that have very high plasma protein binding and thus gain access to their tubular sites of action mainly by proximal tubular secretion. Previous expression studies in Xenopus oocytes with rat OAT1 (36) or in mouse S2 cell lines with human OAT1 and human OAT3 (13) had indicated that, in vitro, both OAT1 and OAT3 can transport thiazides as well as loop diuretics, but the in vivo relevance of these transport mechanisms remained unclear. Because of the tremendous clinical importance of diuretic handling by the kidney, it is important to examine transport in vivo. Whereas for PAH a number of transporters have been suggested by in vitro studies [including OAT3 (8, 32, 33)], our previous experiments in mice lacking OAT1 (Oat1^–/–) revealed an almost complete loss of renal secretion of PAH, indicating that OAT1 is the key transporter in vivo (11). The importance of in vivo studies is further confirmed in the present experiments showing that renal PAH secretion is intact in mice lacking OAT3. In this study we have further examined the interactions of furosemide and bendroflumethiazide with mOat1 and mOat3 in vitro and assessed the in vivo relevance. Unlike PAH, which is almost exclusively transported through Oat1, loop diuretics and thiazide diuretics are each bound by the Oat1 and Oat3 transporters when expressed in Xenopus oocytes, and there are defects in the action of both diuretics in Oat1 and Oat3 knockout mice. The results suggest how the two Oat transporters work in parallel during the elimination of common organic anionic drugs and have clinical implications.

	MATERIALS AND METHODS

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Xenopus oocyte uptake assay. Xenopus oocyte assays were performed as described previously (11, 18). Capped RNA was synthesized from linearized plasmid DNA (mOat1 Image clone ID 4163278; mOat3 Image clone ID 4239544) using mMessage mMachine in vitro transcription kit (Ambion, Austin, TX) and was injected (1 µg/µl, 23 nl/oocyte) into the oocytes using the Nanoliter 2000 nanoinjector (World Precision Instruments, Sarasota, FL). Two days after injection, groups of 16–20 oocytes were placed in a 24-well plate with 1 ml of Barth's buffer containing a fluorescent anion tracer transported by mOat1 or mOat3 [30 µM 6-carboxyfluorescein (6-CF) for mOat1-injected oocytes and 50 µM 5-carboxyfluorescein (5-CF) for mOat3-injected oocytes (Kaler G, Troung D, and Nigam S, unpublished observations)] and increasing concentrations of furosemide or bendroflumethiazide, the known Oat1 substrate PAH, or the Oat3 substrate estrone 3-sulfate (ES), with no inhibitor in the control group. After a 1-h incubation at 25°C, the oocytes were washed five times with ice-cold Barth's buffer, and each group was then divided into four samples of four to five oocytes. The oocytes were lysed before measurements, the lysate was centrifuged, and the fluorescence of the supernatant was measured using a fluorometer (PolarStar plate reader; BMG Labtechnologies, Durham, NC). Considering the amount of the diuretic in the incubation fluid and the volume used to lyse the oocytes, the maximum concentration of the diuretic in the fluorescence assay was 20 µM. Adding this concentration of furosemide or bendroflumethiazide to serial concentrations of 6-CF or 5-CF, respectively, did not reveal any quenching of the fluorescence signal of the tracers. The Oat-mediated clearance (difference in clearance between cRNA-injected and noninjected oocytes) of the fluorescent tracer in the presence of an inhibitor was calculated as the mean ± SE of quadruplicate or triplicate samples. The median inhibition concentrations (IC₅₀) were calculated using Prism 4.0 software (GraphPad, San Diego, CA). All tested compounds were purchased from Sigma-Aldrich (St. Louis, MO).

Generation of OAT3 knockout mice. Oat3^–/– mice (33) were backcrossed to C57BL/6J mice for eight generations. Heterozygous mice from the final backcross were bred to each other to generate knockout and wild-type (WT) mice, from which all the animals used in the experiments described were descended. Only male mice were used in the present experiments. Mice were genotyped by PCR. All experimental protocols were in accordance with the Guide for the Care and Use of Laboratory Animals (National Institutes of Health, Bethesda, MD) and were approved by the Institutional Animal Care and Use Committee.

Quantitative real-time PCR for Oat1, Oat3, and Urat1. RNA prepared from WT and Oat3 and Oat1 (11) knockout kidneys (n = 3 per group) was purified on RNeasy columns (Qiagen, Valencia, CA) and then reverse transcribed using SuperScript III (Invitrogen, Carlsbad, CA). Each cDNA sample was subjected to duplicate real-time PCR reactions using the Amplifluor detection system (Serologicals, Clarkston, GA) in an Applied Biosystems ABI Prism 7700 sequence detector. [Reactions were performed at the University of California, San Diego/Veterans Affairs Medical Center's (UCSD/VAMC) Center for AIDS Research Genomics Core laboratory (http://cfar.ucsd.edu/genomics).] Gene expression values were normalized to that of GAPDH in the corresponding cDNA samples. Gene-specific primer sequences (5' to 3') were as follows [please note that the first 18 bases (ACT GAA CCT GAC CGT ACA) on each forward primer correspond to the "Z sequence" that is complementary to the "uniprimer" used in the Amplifluor system]: mOat1 (mSlc22a6; GenBank accession no. NM_008766), ACT GAA CCT GAC CGT ACA GCA TGA CTG CCG AGT TCT ACC (forward) and CAG CGC CGA AGA TGA AGA G (reverse); mOat3 (mSlc22a8; GenBank accession no. NM_031194) ACT GAA CCT GAC CGT ACA GCA GCC CTT CAT CCC TAA TG (forward) and CCT CCC AGT AGA GTC ATG GTC AC (reverse); and mUrat1 (mSlc22a12; GenBank accession no. NM_009203), ACT GAA CCT GAC CGT ACA CCA TGC TAG GGC CTT TGG TA (forward) and GCA TCC AGG AGC CAT AGA CAC (reverse).

Microarray analyses of OATs. Microarray analysis was performed by the UCSD/VAMC GeneChip Core laboratory (http://www.vmrf.org/research-websites/gcf). Briefly, RNA prepared as described above was linearly amplified (reverse transcribed and then in vitro transcribed) with labeling by incorporation of biotinylated nucleotides during in vitro transcription and was then hybridized to Affymetrix microarrays, washed, and scanned per the standard Affymetrix protocol (n = 3 per group).

Renal clearance experiments in anesthetized Oat3^–/– mice. Mice were anesthetized for terminal clearance experiments with 100 mg/kg thiobutabarbital intraperitoneally and 100 mg/kg ketamine intramuscularly (17, 38). The femoral artery was cannulated for blood pressure measurement and for blood sample withdrawal. The jugular vein was cannulated for continuous maintenance infusion of 2.25 g/dl BSA in 0.85% NaCl at a rate of 0.5 ml·h^–1·30 g body wt^–1. For assessment of glomerular filtration rate (GFR), [³H]inulin was added to deliver 20 µCi·h^–1·30 g body wt^–1. In six pairs of Oat3^+/+ and Oat3^–/– mice, PAH was infused at doses ranging from 70 to 2,520 ng·min^–1·g body wt^–1 (the same doses were given to pairs of mice of different genotype) to determine renal PAH clearance. A timed urine collection was performed for 30 min using a bladder catheter. Blood was withdrawn at the beginning and end of the collection period to determine [³H]inulin and PAH levels. [³H]inulin level was determined using liquid scintillation counting, and PAH level was determined with a colorimetric assay as previously described (37).

Acute natriuretic responses to furosemide or bendroflumethiazide. Acute natriuretic responses to furosemide or bendroflumethiazide were assessed in anesthetized Oat3^–/– mice as previously described for furosemide in Oat1^–/– mice (11). Briefly, 30 min after clearance experiments were finished and the infusion of PAH was discontinued (see above), slow bolus application of vehicle (0.85% NaCl for furosemide and 2.25 g/dl BSA and 1% DMSO in 0.85% NaCl for bendroflumethiazide, 30 µl/25 g body wt over 1 min) was followed by application of increasing doses of furosemide (0.1, 0.3, 1, 3, and 10 mg/kg) or bendroflumethiazide (0.003, 0.01, 0.03, 0.1, 0.3, and 1 mg/kg). After each bolus, allowing 2 min for drug distribution, urine was collected via a bladder catheter for 5–10 min depending on urinary flow rate to determine Na⁺ excretion. As previously described in detail (11), furosemide concentrations in urine were determined by modification of the PAH assay, which can detect sulfonamide therapeutic agents (28). Sufficient urine for colorimetric detection of furosemide in both genotypes was obtained after doses of 0.3 or 1 mg/kg and higher. At these time points, plasma PAH (and therefore likely urinary PAH, as well, was estimated to be <10^–5 of initial levels). Consistent with primary detection of furosemide in the urine, absorbance readings showed a nearly linear increase in urinary excretion with diuretic dose in both genotypes (see Fig. 2A). In comparison, the assay was not applicable to measure bendroflumethiazide. In additional experiments, the natriuretic effect of bendroflumethiazide was tested in Oat1^–/– mice, the generation of which had previously been described (11). WT and knockout mice used in these experiments were selected to have similar basal blood pressure to avoid the confounding effect of any blood pressure changes on diuretic responsiveness.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Concentration-dependent inhibition of fluorescent anion tracer uptake by furosemide and bendroflumethiazide in mouse organic anion transporter 1 (mOat1)- (A) and mOat3-injected oocytes (B). Values are means ± SE of IC50 values for furosemide and bendroflumethiazide. p-Aminohippurate (PAH) and estrone 3-sulfate (ES), well-known substrates of Oat1 and Oat3, respectively, were included for comparison. IC50, median inhibition concentrations.

View larger version (33K): [in this window] [in a new window]   Fig. 2. Expression of renal transporters in Oat1–/– and Oat3–/– mice. A: renal expression of Oat1, Oat3, and the organic anion transporter renal-specific transporter (Rst or Urat1) in Oat1–/– and Oat3–/– mice was determined by quantitative real-time PCR. Expression values in each sample were normalized to that of GAPDH in the same sample and are presented relative to the level of normalized expression in wild-type (WT) kidney. Data are means ± SE in 3 each of WT and knockout mice (n = 3/group). *P < 0.05 vs. WT. B: renal expression of the indicated transporters implicated in organic anion transport was determined in Oat1–/– or Oat3–/– mice by microarray hybridization (n = 3/group). Other than Oat1 and Oat3, none of these genes were significantly and consistently altered by >50% in expression in OAT knockout compared with WT mice. See text for details with regard to findings on Slco4c1.

	RESULTS AND DISCUSSION

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The individual contributions of OAT1 and OAT3 in loop and thiazide diuretic handling in vivo remain unclear. Therefore, we performed a systematic analysis at the level of both the transporter and the whole animal. All experiments were performed in male animals.

Inhibition of tracer uptake in mOat1- and mOat3-injected oocytes by furosemide and bendroflumethiazide. We tested the interaction of mouse Oat1 and mouse Oat3 with furosemide and bendroflumethiazide, together with PAH and ES (controls for inhibition of mOat1 and mOat3, respectively). Tracer uptake in oocytes expressing mOat1 was inhibited by PAH with an IC₅₀ value of 8 ± 2 µM (Fig. 1A). This is consistent with previous studies showing that coincubation of mOat1-transfected oocytes with unlabeled PAH resulted in dose-dependent inhibition of uptake of labeled PAH with an estimated K_i of 13 µM (11), which is similar to previously reported values for the affinity of PAH for mOat1 (22). In comparison, ES inhibited tracer uptake with an IC₅₀ value for mOat3 of 8 ± 2 µM (Fig. 1B). This is consistent with previous studies reporting affinities of ES for mouse, rat, and human OAT3 of 3 µM (8, 21, 33). Furosemide and bendroflumethiazide inhibited tracer uptake with IC₅₀ values for mOat1 of 8 ± 1 and 8 ± 3 µM, respectively (Fig. 1A), which were comparable with previously reported K_i values for rat and human OAT1 (13, 36). The IC₅₀ values for furosemide and bendroflumethiazide in mOat3 were 2.8 ± 0.1 and 21 ± 3 µM, respectively (Fig. 1B), also comparable with values previously obtained for human OAT3 in one prior study (13). Thus both furosemide and bendroflumethiazide inhibited tracer uptake by mOat1 and mOat3 in a concentration-dependent manner, consistent with their being substrates for these transporters.

Quantitative PCR and microarray analyses of transporter gene expression. As expected, there was no significant expression of Oat3 and Oat1 in the corresponding knockout mice, as determined by quantitative PCR (Fig. 2A). Possibly as a consequence of the Oat1 and Oat3 genes being localized adjacent to each other and the application of gene-targeting strategies to delete one of the genes, the expression of Oat1 was reduced to 40% in Oat3^–/– mice and the expression of Oat3 to 40% in Oat1^–/– mice compared with WT mice. A previous study in Oat3^–/– mice had reported a similar reduction of renal mRNA expression for Oat1 (39). In comparison, the expression of another proximal tubular OAT, renal-specific transporter (Rst or Urat1) (25), was affected in neither OAT3^–/– nor Oat1^–/– mice compared with WT mice (Fig. 2A). However, it should be noted that the renal transport of the Oat3 substrate ES is unaffected in Oat1^–/– mice (11), and renal transport of the Oat1 substrate PAH is unaffected in Oat3^–/– mice (Fig. 3). Thus the fact that Oat3 expression is decreased approximately by one-half in Oat1^–/– mice, and vice versa, does not appear to significantly impact function at the level of the intact organism.

View larger version (15K): [in this window] [in a new window]   Fig. 3. Inulin and PAH clearance in OAT3–/– mice. The line of identity indicates similar values for knockout and WT mice. Renal clearance of inulin (as a measure of glomerular filtration rate; n = 11) and PAH (n = 6) was not different between Oat3–/– and WT mice, indicating preserved glomerular filtration and normal renal secretion of PAH in the absence of OAT3. Data are means ± SE. Published data for Oat1–/– mice (11) are shown for comparison: whereas inulin clearance was normal, the clearance of PAH was significantly reduced in Oat1–/– mice.

Intact renal PAH clearance in Oat3^–/– mice. No significant differences (NS) were observed between WT and Oat3^–/– in body weight (28 ± 1 vs. 26 ± 1 g, NS, n = 11 per group) and kidney weight (166 ± 9 vs. 176 ± 13 mg, NS) or in plasma concentrations of Na⁺ or K⁺ (153 ± 3 vs. 156 ± 4 and 4.4 ± 0.2 vs. 4.4 ± 0.2 mM, respectively, both NS). No significant differences were observed between the genotypes in renal [³H]inulin clearance as a measure of GFR as well as in the renal clearance of PAH (Fig. 3), which amounted to approximately five times the clearances of [³H]inulin, indicating intact renal PAH secretion in the absence of Oat3. The applied infusion protocol induced steady-state plasma concentrations of PAH of 10–250 µM. The concentration range thus included the values for PAH affinities of 60–90 µM previously reported for rat and human OAT3 in oocyte expression studies (8, 21). The present renal clearance experiments significantly refined the results from a previous study, which showed an unaltered whole body clearance of both inulin and PAH in Oat3^–/– mice using low-dose PAH and maximum plasma concentrations of 20 nM, i.e., significantly lower than the above-mentioned PAH affinities (39). Together with previous studies showing that renal PAH secretion is almost completely absent in Oat1^–/– mice (11) (shown in Fig. 3), the present data are consistent with OAT1 being the classic basolateral PAH transporter in the kidney. Assuming that protein expression is affected similarly to mRNA expression (unfortunately, no commercially available antibody is available that recognizes mouse Oat1), a reduced Oat1 mRNA expression in Oat3^–/– mice (see above) suggested that normal renal PAH secretion is maintained despite a significant reduction of Oat1 expression. In accordance, previous studies provided evidence that significantly lower renal Oat1 mRNA levels in female versus male WT mice are sufficient to maintain similar PAH elimination (5, 39). In contrast to PAH, recent studies in Oat3^–/– mice indicated an important role of this transporter in the clearance of both penicillin G and methotrexate (39, 40).

Impaired natriuretic responses to furosemide and bendroflumethiazide in Oat3^–/– mice. The dose-natriuresis curves for both furosemide (Fig. 4A) and bendroflumethiazide (Fig. 4B) were shifted to the right in Oat3^–/– mice in the absence of significant differences in maximum responses. Figure 4D summarizes the half-maximal effective dose values of these experiments. Moreover, impaired urinary excretion of furosemide was directly demonstrated in these mice (Fig. 4A). Furthermore, furosemide doses of 1 mg/kg in WT and 3 mg/kg in Oat3^–/– mice resulted in similar levels of renal excretion of the diuretic as well as of natriuresis, indicating that the pharmacodynamic response, once furosemide reached the tubular lumen, was preserved in the absence of OAT3. These findings demonstrate that an impaired pharmacokinetic response, i.e., impaired renal secretion, caused the observed rightward shift in the natriuretic response to furosemide, and likely to bendroflumethiazide, in Oat3^–/– mice.

View larger version (19K): [in this window] [in a new window]   Fig. 4. Acute natriuretic responses to furosemide in Oat3–/– mice and to bendroflumethiazide in Oat3–/– and Oat1–/– mice. A: Oat3–/– mice showed a significant rightward shift in the natriuretic response to furosemide and lower urinary excretion of furosemide vs. WT, whereas maximum natriuresis was unaffected. Furosemide doses of 1 mg/kg in WT and 3 mg/kg in Oat3–/– mice resulted in similar levels of both renal furosemide excretion and natriuresis (n = 6 per group). B: Oat3–/– mice showed a similar maximum natriuresis but a rightward shift in the natriuretic response to bendroflumethiazide compared with WT (n = 5 per group). C: as observed in Oat3–/– mice, Oat1–/– mice showed a similar maximum natriuresis but a rightward shift in the natriuretic response to bendroflumethiazide compared with WT (n = 5 per group). D: summary of half-maximal effective dose (ED50) values. Data for Oat1–/– mice for furosemide were taken from the original publication (11). UNaV, absolute Na+ excretion rates. Data are means ± SE. *P < 0.05 vs. WT.

	GRANTS

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This work was supported by National Institutes of Health Grants DK-56248 and DK-28602 (to V. Vallon), DK-064839 and DK-075486 (to S. A. Eraly), and AI-057695 and HL-35018 (to S. K. Nigam), the Department of Veterans Affairs (to V. Vallon), and Deutsche Forschungsgemeinschaft RI 1535/3-1 and 3-2 (to T. Rieg).

	ACKNOWLEDGMENTS

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

Some of these data were presented in abstract form at the Annual Meeting of the American Society of Nephrology, Philadelphia, PA, 2005.

	FOOTNOTES

 

Address for reprint requests and other correspondence: V. Vallon, Depts. of Medicine and Pharmacology, Univ. of California, San Diego and VASDHCS, 3350 La Jolla Village Drive (9151), San Diego, CA 92161 (e-mail: vvallon{at}ucsd.edu) or S. K. Nigam, Dept. of Medicine, Univ. of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093 (e-mail: snigam{at}ucsd.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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