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【摘要】 We compared the characteristics of several cloned rabbit organic electrolyte (OE) transporters expressed in cultured cells with their behavior in intact rabbit renal proximal tubules (RPT) to determine the contribution of each to basolateral uptake of the weak acid ochratoxin A (OTA) and the weak base cimetidine (CIM). The activity of organic anion transporters OAT1 and OAT3 proved to be distinguishable because OAT1 had a high affinity for PAH ( K t of 20 µM) and did not support estrone sulfate (ES) transport, whereas OAT3 had a high affinity for ES ( K t of 4.5 µM) and a weak interaction with PAH (IC 50 1 mM). In contrast, both transporters robustly accumulated OTA. Intact RPT also accumulated OTA, with OAT1 and OAT3 each responsible for 50%: ES and PAH each reduced uptake by 50%, and the combination of the two eliminated mediated OTA uptake. The weak base CIM was transported by OAT3 ( K t of 80 µM) and OCT2 ( K t of 2 µM); OCT1 had a comparatively low affinity for CIM, and CIM uptake by OAT1 was equivocal. Intact RPT accumulated CIM, with TEA and ES reducing CIM uptake by 20 and 75%, respectively, suggesting that OAT3 plays a quantitatively more significant role in CIM uptake in the early proximal tubule than OCT1/2. In single S2 segments of RPT, ES and TEA each blocked 50% of CIM uptake. Thus the fractional contribution of different OE transporters to renal secretion is influenced by their affinity for substrate and relative expression level in RPT.
【关键词】 Relative contribution transporters electrolyte transport proximal
kidney
ONE OF THE CRITICALLY IMPORTANT functions of the kidney is modulation of the plasma concentrations of an extraordinary array of organic compounds, some of endogenous (i.e., physiological) origin and many more of exogenous (i.e., xenobiotic) origin. The latter group of compounds includes a wide range of substances of both pharmacological and toxicological significance. To deal with this broad array of molecules, evolution has responded by producing an equally diverse collection of transport processes organized in ways that result in the net secretion of some compounds and the net reabsorption of others. The landmark studies of Ullrich and colleagues (see Ref. 51 ) defined what are frequently referred to as the "classic" pathways associated with the secretion by the kidney of organic electrolytes, i.e., compounds that, at physiological pH, carry either a net negative charge [i.e., organic anions (OAs)] or a net positive charge [organic cations (OCs)]. In recent years, it has become increasingly apparent that renal secretion of OAs and OCs involves the integrated activity of a number of distinct transport proteins (see Ref. 56 ), many of which belong to a single family of solute carriers (SLC22A).
Functionally, members of the SLC22A family can be subdivided into organic cation transporters, to include the OCTs and OCTNs, and organic anion transporters, to include the OATs and RST/URATs ( 56 ). Of the OATs, four homologs have been functionally characterized, and endogenous substrates include hippurates, folates, cyclic nucleotides, products of neurotransmitter metabolism, prostaglandins, and selected hormone conjugates ( 56 ). Xenobiotics secreted by various OATs include both clinically important pharmaceuticals [e.g., nonsteroidal anti-inflammatory drugs (NSAIDS), -lactam antibiotics, loop and thiazide diuretics, and antiviral agents] and anionic toxins (e.g., ochratoxin, and 2,4-D) ( 56 ). The principal site of secretion of all these compounds in the kidney is the proximal tubule.
Proximal tubule OA secretion is a two-step process: OAs 1 ) are transported from the peritubular plasma across the basolateral membrane and 2 ) subsequently move into the tubular lumen by means of apical transporters. Although the mechanism(s) associated with luminal OA transport remain unclear, there is strong physiological evidence that the uphill basolateral uptake of OAs into proximal cells is a tertiary active process that involves indirect coupling of three parallel transport processes ( 39, 45 ): 1 ) OA/ -ketoglutarate ( -KG) exchange that uses an outwardly directed -KG gradient to support the uphill accumulation of exogenous OAs; 2 ) Na-dicarboxylate cotransport that assists in the maintenance of the outwardly directed -KG gradient; and 3 ) the Na-K-ATPase that maintains the inwardly directed Na gradient required to sustain activity of the aforementioned cotransporter.
Two of the OATs, i.e., OAT1 and OAT3, have been implicated as being key contributors to the OA/ -KG exchange element of this energetically linked set of processes. Both OAT1 and OAT3 support OA/ -KG exchange ( 4, 46, 48 ) [OAT2 does not ( 43 )] and are expressed in the basolateral membrane of proximal tubules in all species studied ( 29, 37 ) [OAT2 and OAT4 are not ( 2, 29 )]. Furthermore, levels of OAT1 and OAT3 mRNA expression in the human and the rat are sufficiently high [compared with the other OATs ( 6, 37 )] to support the hypothesis that they both contribute significantly to renal OA secretion. Importantly, the selectivity of these two processes displays marked similarities for some substrates and clear differences for others. For example, whereas PAH, the prototypical substrate for the classic OA secretory process in the kidney, is a substrate for both rodent ( 30, 44, 48 ) and human ( 10, 25, 32 ) orthologs of OAT1 and OAT3, estrone sulfate (ES) is transported by OAT3 but not by OAT1 ( 47 ). Consistent with this profile is the observation that ES uptake is eliminated, and PAH uptake reduced by 60%, in renal slices from the OAT3-null mouse.
The relative role of the several transporters involved in the basolateral uptake of organic anions (and other organic electrolytes) is influenced by both their differential selectivity for substrates and their relative expression level. Moreover, despite qualitative evidence showing coexpression of different transporter mRNAs or proteins in the kidney, there is virtually no quantitative information about the relative contribution different transporters have in the secretion of organic electrolytes in the kidney. The rabbit offers one of the few working models of intact renal tubule function and offers the opportunity to examine the relative physiological roles of organic electrolyte transporters in the uptake of test substrates into the proximal tubule. We recently reported that PAH and ES are transported across the basolateral membrane of isolated single S2 segments of rabbit (rb) renal proximal tubule (RPT) by kinetically distinct processes and suggested that PAH uptake reflected activity of OAT1 and that ES uptake reflected activity of OAT3 ( 33 ). In the present report, we compare the characteristics of rbOAT1 and rbOAT3, confirming that PAH and ES are "homolog-selective" substrates capable of distinguishing between the activities of these two transporters. We use these tools to assess the relative contribution of several organic electrolyte transporters, including OAT1 and OAT3, to the uptake of the weak acid ochratoxin A (OTA) and the weak base cimetidine (CIM), both of which have been shown to interact with multiple secretory pathways in the kidney (e.g., 21, 31, 52). The results indicate that both OAT1 and OAT3 play a quantitatively significant role in the renal transport of the anionic OTA and that OAT3 plays a significant role in the transport of the cationic CIM in rabbit kidney.
METHODS
Materials. [ 3 H]ES (44 or 46 Ci/mmol), [ 3 H]PAH (4 Ci/mmol), [ 3 H]CIM (9.8 Ci/mmol), [ 3 H]OTA (6 Ci/mmol), and [ 14 C]uric acid (49 mCi/mmol) were acquired from American Radiolabeled Chemicals (St. Louis, MO) or Sigma (St. Louis, MO). 6-Carboxyfluorescein (6CF) was supplied from Molecular Probes (Eugene, OR; Leiden, The Netherlands). The Flp-In expression system and the mammalian expression vector pcDNA3.1 were purchased from Invitrogen (Carlsbad, CA). All other chemicals were purchased from Sigma or from other standard sources. Cell culture media and all other molecular biology reagents were purchased from Life Technologies (Gaithersburg, MD), Invitrogen, or from GIBCO-BRL (Karlsruhe, Germany).
Isolation of mRNA. Total RNA was prepared from rabbit kidney tissues or isolated rabbit kidney tubules following the method of Sambrook et al. ( 42 ). Organs were removed from anesthetized animals and extensively washed or perfused with saline buffer to remove most of the remaining blood or further dissected to prepare renal tubules. Poly(A) + RNA was selected on oligo(dT) cellulose columns from the total RNA preparation and analyzed by agarose gel electrophoresis.
Cloning of rbOAT3. To clone rbOAT3, specific sense and antisense oligonucleotide primers were designed from consensus sequences of human and rat OAT3 (sense: 5'-GAAGAATTTGGAGTCAAC-3'; antisense: 5'-TCACCCGTGATTTTCACCAG-3'). For first-strand synthesis, 0.5 µg of rabbit kidney poly(A) + RNA was reverse transcribed using Moloney murine leukemia virus reverse transcriptase (RT) H - at 37°C for 20 min. After incubation at 70°C for 15 min, ribonuclease (RNase) H was added, and the reactions were again kept at 37°C for 20 min. The RT reaction (2 µl) was used directly for amplification. The PCR solution was assembled and heated at 94°C for 3 min before Pfu DNA polymerase was added. Subsequently, PCR was performed using the following profile: 94°C for 1 min, 54°C for 1 min, and 72°C for 2 min for 35 cycles. The last cycle was terminated after an elongation time of 7 min. A 246-bp RT-PCR product was gel purified and sequenced. To obtain the remaining 5'- and 3'-portion of the rabbit kidney OAT3 sequence, the PCR-based 5'- and 3'-rapid amplification of cDNA ends (RACE) systems (GIBCO-BRL) were utilized. Briefly, the gene-specific primers 5'-TCCCAGCCAAGTTCATCAC-3' (for 3'-end RACE) and 5'-TAGAGGAAGAGGCAGCTGAAG-3' (for 5'-end RACE) were designed from the partial rbOAT3 sequence. The 5'- and 3'-RACE reactions were primed with the internal gene-specific primers and adapter primers. The PCRs were performed according to the manufacturers protocols. The RACE products were gel purified and subcloned into the mammalian expression vector pcDNA3.1. The 5'-RACE product and the 3'-RACE product were sequenced and showed an overlapping region. Since there was no restriction site that could be used to subclone the two fragments of the RACE product into a mammalian expression vector, we designed two sequence-specific primers covering the full open reading frame of rbOAT3 to amplify rbOAT3 cDNA. The resulting rbOAT3 cDNA was then inserted into the TOPO cloning vector of pcDNA 3.1, and the sequence was confirmed in the sense and antisense strands by an Applied Biosystems model 373A sequencing unit at the University of Arizona sequencing facility.
Expression of rbOAT1 and rbOAT3 activity in cultured cells. Clonal lines of the nonpolarized Chinese hamster ovary (CHO) cell line that stably expressed either rbOAT1 or rbOAT3 were established by using the Flp-In expression system (Invitrogen) according to the manufacturers protocol. Briefly, a cDNA fragment containing the open reading frame of cloned rbOAT1 or rbOAT3 was digested by Hin dIII and Eco R V and then subcloned into the Flp-In expression vector pcDNA5/FRT, containing a Flp recombination target (FRT) site linked to the hygromycin resistance gene. The new vector, pcDNA5/FRT-rbOAT1 or pcDNA5/FRT-rbOAT3, was then cotransfected with the Flp recombinase expression vector pOG44 into Flp-In CHO cells. Cells stably expressing rbOAT1 or rbOAT3 were then selected and maintained in hygromycin (200 µg/ml) according to the manufacturers protocol. Stable expression of transport activity was verified by visual inspection of mediated accumulation of 6CF. In some experiments, transport activity was measured after transient transfection of rbOAT3 (in pcDNA3.1) into COS-7 cells, or of rbOAT1 into CHO cells, as described previously ( 3, 22 ).
Cell culture. Flip-In CHO cells that stably expressed rbOAT1 or rbOAT3 were grown at 37°C in a humidified atmosphere (5% CO 2 ) in plastic culture flasks. The medium was Kaighns modification medium supplemented with 10% fetal calf serum and hygromycin (200 µg/ml). The medium was changed every 3 days, and the culture was split every 3 days. Uptake studies were performed with 100% confluent cells. Separate studies made use of the nonpolarized cell line established from green monkey kidney (COS-7). COS-7 cells were cultivated in plastic flasks or petri dishes in DMEM with 580 mg/l glutamine, 110 mg/l Na-pyruvate and with 10% heat-inactivated FCS in 8.5% CO 2 at 37°C. Five micrograms of rbOAT3-pcDNA3.1 construct were transiently transfected into COS-7 cells by electroporation (GenePulser II; Bio-Rad, Munich, Germany) at 250 V and 300 µF. Twenty-four hours after transfection, the cells were plated in 24-well plastic dishes at a density of 5 x 10 5 cells/well.
Transport assays. Uptake was measured at 25°C. For the measurement of radiolabeled substrate transport, CHO cells were incubated in Waymouths buffer [WB; (in mM) 135 NaCl, 13 HEPES-NaOH, pH 7.4, 28 D -glucose, 5 KCl, 1.2 MgCl 2, 2.5 CaCl 2, and 0.8 MgSO 4 ] to which labeled substrate plus appropriate test agents were added. Incubation was stopped by rinsing the cells three times with 2 ml of ice-cold WB. The cells were then solubilized using 400 µl of 0.5 N NaOH with 1% (vol/vol) SDS (the extract was then neutralized with 200 µl of 1 N HCl). Accumulated radioactivity was determined by liquid scintillation spectrometry. Uptakes are expressed as moles per square centimeter of nominal cell surface of the confluent monolayer. Uptake of fluorescent substrates involved incubation in transport buffer to which the optically active substrate plus appropriate test agents were added. The incubation was stopped, and the extracellular tracer was removed by washing the monolayer two to three times with ice-cold PBS. Cells were dissolved in 0.5-1 ml 0.5 N NaOH. To assess accumulation of fluorescein (FL) and of 6CF, fluorescence was measured in a fluorescence spectrophotometer (Hitachi, Tokyo, Japan) at 492/512 nm (excitation/emission), with the amount of probe calculated from standard curves for each fluorophore.
Preparation of rabbit RPT suspension. Rabbit RPT were isolated and purified from New Zealand White rabbits (1.3-1.5 kg) as described previously ( 23, 40 ). The final tubule pellet was resuspended at a protein concentration of 2 mg/ml in the medium used for dissection of single S2 segments. Tubular protein was measured using a Bio-Rad protein assay (Hercules, CA) with a -globulin standard.
Measurement of transport in suspensions of rabbit RPT. Tubule suspensions (2 mg/ml) were preincubated in Erlenmeyer flasks for 15 min at 37°C and gassed with 95% O 2 -5% CO 2. An aliquot of tubule suspension (0.5 ml) was transferred to a 15-ml tube containing 0.5 ml of incubation medium containing radiolabeled substrate and varying concentrations of unlabeled PAH or ES. After 30 s, 5 ml of ice-cold DMEM/F-12 (Sigma) were added to stop the uptake, and the tubules were pelleted ( 30 s at 1,480 g). The rinse was repeated, the final pellet was dissolved in 0.5 N NaOH/1% SDS, and aliquots were taken for counting radioactivity.
Preparation of isolated tubules. New Zealand White rabbits (1.5 kg, Harlan, Indianapolis, IN) were killed by an intravenous injection of pentobarbital sodium. All protocols employing rabbits were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. The kidneys were flushed via the renal artery with an ice-chilled HEPES-sucrose buffer containing 250 mM sucrose and 10 mM HEPES, adjusted to pH 7.4 with Tris base, and bubbled with 100% O 2 before use. They were then gently removed and sliced transversely using a single-edge razor. A kidney slice was transferred to the lid of a plastic petri dish on ice, which contained the standard solution used for dissecting and bathing tubules (in mM: 110 NaCl, 25 NaHCO 3, 5 KCl, 2 NaH 2 PO 4, 1 MgSO 4, 1.8 CaCl 2, 10 Na-acetate, 8.3 D -glucose, 5 L -alanine, 0.9 glycine, 1.5 lactate, 1 malate, and 1 sodium citrate). This standard solution was aerated continuously with 95% O 2 -5% CO 2 to maintain the pH at 7.4. The osmolality of the solutions averaged 290 mosmol/kgH 2 O. S2 segments of proximal tubules were individually dissected from the cortical zone without the aid of enzymatic agents as described by others ( 9 ).
Measurement of transport in nonperfused single, isolated RPT. These experiments were performed in a manner similar to that used previously ( 12, 13, 20 ). Briefly, tubules were maintained in oxygenated (95% O 2 -5% CO 2 ) buffer transferred to oil-covered wells in a temperature-controlled chamber containing bicarbonate-buffer solution at 4°C to prevent evaporation until the start of each experiment, and photographed through a dissecting microscope equipped with a digital image-capture system (Snappy, Play). Five minutes before the experiment, the bathing medium was warmed to 37°C. The tubules were then individually transferred to the oil-covered incubation medium at 37°C containing labeled substrate and appropriate test agents. After a 15-s to 5-min period, uptake was stopped by transferring each tubule into 1 N NaOH for extraction. Accumulated labeled substrate was determined by liquid scintillation counting. Control and experimental uptakes were determined alternately and sequentially in tubules from the same kidney.
Data analysis. Uptake values are presented as means ± SE. In each experiment, a minimum of three wells was used to generate each data point, and experiments were typically repeated at least three times. Statistical comparison of observed differences used one-way ANOVA followed by the the Student-Newman-Keuls post hoc test (StatMost, Dataxion, Los Angeles, CA). Differences at the 0.05 level were considered indicative of significance.
Amino acid sequences and pairwise sequence alignments were analyzed with default parameters using the ClustalW algorithm available on the internet from Network Protein Sequence Analysis ( http://npsa-pbil.ibcp.fr/cgi-bin/npsa_automat.pl?page=/NPSA/npsa_clustalw.html ).
RESULTS
Molecular properties of rbOAT3. A 246-bp RT-PCR product of rbOAT3 was generated by PCR amplification using oligonucleotide primers designed from consensus sequences of human and rat OAT3. PCR-based 5'- and 3'-RACE systems were used to obtain the remaining 5'- and 3-'portions of the rbOAT3 sequence. The full-length cDNA is 2,104 bp and contains a 4-bp 5'-untranslated region, a 1,629-bp open reading frame, and a 471-bp 3'-untranslated region. It encodes a protein of 542 amino acids with a predicted mass of 60 kDa (GenBank accession nos. AF533644 and AJ489526 ). BLAST searches of the protein and gene databases indicated that the rbOAT3 protein belongs to the SLC22 family of solute carriers (also the OCT family of the major facilitator superfamily; 2.A.1.19) ( 41 ). Assessment of possible secondary structure (TMpred) was consistent with the presence of two large hydrophilic loops and 12 membrane-spanning domains typical of the major facilitator superfamily ( 38 ). The protein sequence contains four potential N-linked glycosylation sites (N-X-T/S) at positions 54, 81, 86, and 102. In addition, excluding domains within the postulated long extracellular loop, were one consensus PKA phosphorylation site ( position 324 ), four consensus PKC phosphorylation sites ( positions 259, 269, 514, and 527 ), and four consensus casein kinase II phosphorylation sites ( positions 2, 293, 313, and 503 ). rbOAT3 has high sequence homology with its orthologs cloned from humans, mice, and rats (82-84% identity) and strong homology with the related OAT1 paralog (45-49% for humans, rats, and mice and 48% for rabbits).
Substrate specificity of rbOAT3. Expression of rbOAT3 in CHO cells resulted in a 20-fold increase in the 10-min uptake of 20 nM [ 3 H]ES (compared with wild-type cells), and this uptake was eliminated by the presence of 1 mM unlabeled ES ( Fig. 1 A ). Similarly, uptake of 5 µM 6CF was increased 17-fold by expression of rbOAT3, and this was blocked by 1 mM probenecid ( Fig. 1 B ). rbOAT3 also supported uptake of [ 3 H]CIM, [ 14 C]urate, and [ 3 H]OTA ( Fig. 1, C - E ).
Fig. 1. Rabbit organic anion transporter (rbOAT3)-mediated transport of selected substrates. Ten-minute uptakes of [ 3 H]estrone sulfate (ES; 20 nM; A ); 6-carboxyfluorescein (6CF; 5 µM; B ); [ 3 H]cimetidine (CIM; 96 nM; C ); [ 14 C]urate (20.8 µM; D ); [ 3 H]ochratoxin A (OTA; 150 nM; E ); and [ 3 H]PAH (253 nM; F ) were measured in wild-type and rbOAT3-expressing Chinese hamster ovary (CHO) cells. Unlabeled ES (1 mM), probenecid (1 mM), CIM (1 mM), or PAH (2.5 mM) was added, as indicated, to test for inhibition of rbOAT-mediated transport. Each point represents the means ± SE of substrate accumulation in 3 individual experiments with 3 wells of cells for each experiment.
Interestingly, and in contrast to the human and rodent orthologs of OAT3 ( 10, 30 ), rbOAT3 supported comparatively little mediated PAH uptake at either 10 ( Fig. 1 F ) or 30 min (data not shown). Figure 2 compares (to control uptake in wild-type CHO cells) uptake of ES and PAH as mediated by the rabbit orthologs of OAT1 and OAT3. Although rbOAT1 did support ES uptake ( P < 0.05), the level of uptake was dwarfed by that mediated by rbOAT3, in general agreement with previous work on selectivity of murine (m)OAT1 ( 47 ). These data are consistent with recent work comparing PAH and ES uptake in isolated, single rabbit RPT that suggested the presence of mutually exclusive transport pathways for the two substrates in rabbit tubules ( 33 ). We built on that observation in subsequent experiments that used PAH and ES as homolog-selective inhibitors of OAT1 and OAT3 in intact rabbit RPT.
Fig. 2. Transport of ES and PAH mediated by rbOAT1 or rbOAT3. Ten-minute uptakes of 20 nM [ 3 H]ES or 250 nM [ 3 H]PAH were measured in triplicate wells of cells stably expressing either rbOAT1 or rbOAT3, with uptake expressed relative to uptake measured in wild-type CHO cells. All uptakes mediated by rbOAT1 and rbOAT3 were significantly greater than that measured in the control CHO cells ( P < 0.05).
Kinetics of ES and 6CF transport by rbOAT3. We examined the concentration dependence of [ 3 H]ES uptake by rbOAT3. ES uptake into CHO cells was time dependent and reasonably linear for 60 s; 30-s uptakes were used for all kinetic analyses of ES transport. The effect of increasing substrate concentration on the rate of ES uptake was adequately described by Michaelis-Menten kinetics
where J is the rate of transport of a labeled OA (in this case, ES) from a substrate concentration equal to ; J max is the maximum rate of mediated transport; K t is the OA concentration that resulted in half-maximal transport (Michaelis constant); and D is a constant that represents the "first-order" component of total OA uptake (i.e., nonsaturable over the range of substrate concentrations tested) and presumably reflects the combined influence of diffusive flux, nonspecific binding, and/or incomplete rinsing of the cell layer. Figure 3 A shows the kinetics of rbOAT3-mediated ES transport in a representative experiment. In three separate experiments in cells expressing rbOAT3, J max was 24.8 ± 5.9 pmol·cm -2 ·min -1 with a K t of 4.5 ± 1.2 µM, the latter in close agreement with the value of 3.4 µM measured in isolated single S2 segments of rabbit RPT ( 33 ).
Fig. 3. Kinetics of ES ( A ) or 6CF ( B ) transport in Flp-in CHO cells stably expressing rbOAT3. Thirty-second uptakes of [ 3 H]ES were measured in the presence of increasing concentrations of unlabeled ES. Five-minute uptakes of 6CF were measured in the presence of increasing concentrations of 6CF. The kinetics of total substrate accumulation were adequately described as involving a single mediated uptake process and a parallel first-order process (see text). Each point is the means ± SE of uptakes measured in 3 wells in a single representative experiment. The lines were calculated using a least-squares fit of Eq. 1 to the individual data points (SigmaPlot 3.0). Solid line, total uptake; dashed line, Michaelis-Menten component of total uptake. For the representative example of ES transport, the Michaelis constant ( K t ) for ES transport mediated by rbOAT3 was 3.8 µM, with a maximum velocity ( J max ) of 20.0 pmol·cm -2 ·min -1. For the representative example of 6CF transport, the Michaelis constant ( K t ) was 10.7 µM, with a J max of 15.1 pmol·cm -2 ·min -1.
Transport of 6CF into rbOAT3-expressing CHO cells was linear for at least 10 min (data not shown), and 5-min uptakes were used in subsequent studies of the kinetics of 6CF transport. Figure 3 B shows the kinetics of 6CF uptake into CHO cells stably expressing rbOAT3. As with ES, 6CF accumulation was adequately described by Michaelis-Menten kinetics ( Eq. 1 ). In four separate experiments in cells expressing rbOAT3, J max was 13.3 ± 3.8 pmol·cm -2 ·min -1 with a K t of 10.2 ± 2.8 µM. The fluorescent monocarboxylate FL was also transported by rbOAT3-expressing CHO cells. In three experiments (at pH 7), the FL J max was 11.8 ± 2.4 pmol·cm -2 ·min -1 with a K t of 9.8 ± 2.8 µM.
Inhibitory interaction of rbOAT3 with selected organic electrolytes. Figure 4 shows the effect of a structurally diverse array of organic electrolytes on OAT3-mediated transport activity. Having observed previously ( 27 ) that the kinetic parameters of cloned transporters can differ between expression systems (see also Ref. 56 ), we assessed inhibitory interactions for selected substrates in two different expression systems, CHO cells and COS-7 cells. The qualitative pattern of inhibition was similar in both cell types. Consistent with the observations noted above, PAH exhibited a very weak (IC 50 1 mM) inhibition of OAT3 when expressed in either cell type. Table 1 presents an extended list of the compounds tested as inhibitors of rbOAT3 in CHO or COS-7 cells and IC 50 values determined for selected inhibitors. Of the dicarboxylates tested, -KG and glutarate were comparatively effective inhibitors of rbOAT3 activity, whereas succinate, malate, and fumarate were not, a profile similar to that observed for rbOAT1 ( 3 ). Urate and CIM both blocked rbOAT3-mediated ES transport, but TEA, which has a fixed cationic charge, had no effect on rbOAT3 transport activity.
Fig. 4. Selectivity of rbOAT3-mediated transport in CHO (light gray bars) or COS-7 cells (dark gray bars). -KG, -ketoglutarate. In CHO cells, rbOAT3 was stably expressed and uptake was determined by accumulation of 20 nM [ 3 H]ES. In COS-7 cells, rbOAT3 was transiently expressed and uptake was determined by accumulation of 1 µM 6CF. The length of each bar represents the average uptake (±SE) measured in triplicate in at least 3 separate experiments. Uptakes were measured in the absence (control) or presence of the indicated inhibitors. Inhibitor concentration was 1 mM (except for 10 mM TEA in CHO cells).
Table 1. Selectivity of rbOAT3-mediated transport
rbOAT3 serves as an anion/dicarboxylate exchanger and urate transporter. Although the initial studies of human and rat OAT3 concluded that it does not behave as a secondary active exchanger of ES for other substrates (including -KG) ( 30 ), more recent studies examined this issue directly and concluded that rOAT3 as well as hOAT3 support ES/dicarboxylate exchange (analogous to the PAH/dicarboxylate exchange mediated by OAT1) ( 4, 46 ). To confirm and extend this observation, we examined the effect of preloading rbOAT3-expressing CHO cells with 0.5 mM glutarate or 1 mM urate on the transport of [ 3 H]ES ( Fig. 5 A ). Both compounds served to trans -stimulate [ 3 H]ES uptake by more than twofold, compared with the nonpreloaded control condition. In addition, preloading cells with glutarate was sufficient to trans -stimulate uptake of [ 14 C]urate ( Fig. 5 B ). Thus, in addition to confirming for the rabbit that OAT3 supports OA/DC exchange, these data suggest that OAT3 can support the secondary active transport of urate via mediated exchange with dicarboxylates and other substrates, and, therefore, play a role in the active basolateral uptake of OAs that is the first step in transepithelial OA secretion. The fact that orotate and nicotinate did not alter 6CF uptake via rbOAT3 significantly (cf. Table 1 ) indicates different driving forces for the basolateral and luminal side of the cell, where urate reabsorption by URAT1 was specifically inhibited with orotate or trans -stimulated by nicotinate ( 17 ).
Fig. 5. Trans -stimulation of rbOAT3-mediated ES ( A ) and urate ( B ) uptake. A : 30-s uptakes of 20 nM [ 3 H]ES into rbOAT3-expressing CHO cells preloaded for 15 min with Waymouths buffer (WB; control) or WB containing 500 µM glutarate or 1 mM urate. The height of each bar represents means ± SE uptake of those measured in triplicate from a representative experiment. B : 5-min uptakes of 21 µM [ 14 C]urate into rbOAT3-expressing CHO cells preloaded for 15 min with WB (control) or WB containing 500 µM glutarate. Urate uptake in untransfected CHO cells was 0.1 fmol·cm -2 ·min -1. The height of each bar represents the means ±SE of uptakes measured in triplicate from a representative experiment.
Influence of OAT1 and OAT3 on basolateral OTA transport in intact rabbit proximal tubules. In a previous study of OTA uptake into rabbit cortical RPT, we concluded that there are at least two mediated pathways capable of supporting basolateral transport of OTA ( 21 ). This conclusion was based on the observation that, although 1 mM probenecid eliminated mediated OTA uptake, 2.5 mM PAH (i.e., a concentration sufficient to eliminate mediated PAH uptake in intact tubules) ( 15 ) only reduced OTA uptake by 50%. OTA was subsequently shown to be a substrate for both OAT1 ( 50 ) and OAT3 ( 30 ), suggesting that the parallel activity of these processes could account for the presence of (at least) two pathways for the basolateral uptake of OTA in rabbit RPT. In light of the present results showing that PAH and ES can be used to discriminate between rbOAT1 and rbOAT3, respectively, we examined the effect on tubular OTA uptake of selectively blocking each process (using PAH and ES, respectively). We first confirmed that OTA is a substrate for both rbOAT1 and rbOAT3 ( Fig. 6 A ) and then determined the effect of probenecid, PAH, ES, and the combination of PAH and ES on the accumulation of OTA into a suspension of intact, isolated cortical renal RPT ( Fig. 6 B ). A 1 mM concentration of probenecid, which is sufficient to block both OAT1 and OAT3, reduced OTA uptake into rabbit RPT by 95%. In contrast, concentrations of PAH and ES that should have reduced the activity of OAT1 and 90% failed to produce a complete block of OTA uptake. However, the combination of PAH and ES did effectively eliminate mediated OTA uptake into rabbit RPT. These data support the conclusions that 1 ) basolateral uptake of OTA into a mixed population of cortical proximal tubules (i.e., including both S1 and S2 segments) can be accounted for by the interaction of OTA with OAT1 and OAT3; and 2 ) both transporters play quantitatively similar roles in mediating this uptake
Fig. 6. A : uptake of OTA by rbOAT1 and rbOAT3. Ten-minute uptakes of 0.15 µM [ 3 H]OTA were measured in triplicate wells of cells stably expressing either rbOAT1 or rbOAT3, with uptake expressed as a percentage of uptake measured in wild-type CHO cells, in the absence or presence of unlabeled PAH or ES (as indicated). The height of each bar represents the means ± SE uptake measured in 3 wells in a single experiment. B : effect of ES and PAH on uptake of OTA in isolated, suspended rabbit renal proximal tubules. Thirty-second uptakes of 40 nM [ 3 H]OTA were measured in the absence (control) of presence of unlabeled PAH, ES, and/or probenecid (Prob; as indicated). The height of each bar represents the means ± SE of uptakes measured in triplicate.
Influence of OAT3 and OCT1/2 on basolateral CIM transport in intact rabbit proximal tubules. CIM is a weak base (p K a of 6.9) and long known to be a substrate of the classic organic cation secretory pathway ( 54 ). However, evidence suggests that some fraction of CIM clearance may involve interaction with one or more elements of the organic anion secretory process, including observations that probenecid blocks a substantial fraction of renal CIM secretion in the rat ( 31 ), rabbit ( 35 ), and human ( 19 ). CIM has been shown to be transported by human and flounder orthologs of OAT1. However, the apparent affinity of OAT1 for CIM appears to be rather low (IC 50 for inhibition 1 mM) ( 53 ), placing in question the quantitative significance of OAT1 in the renal handling of CIM. OAT3 also transports CIM [human ( 10 ); rat ( 18, 30 ); rabbit, Fig. 1 of the present study], and the apparent affinity of OAT3 for CIM is sufficiently high ( K t /IC 50 of 40-60 µM) (10, 18; Table 1 ) to make credible the hypothesis that OAT3 may play a quantitatively significant role in the basolateral uptake of cimetidine. Figure 7 compares the kinetics of CIM uptake mediated by the rabbit orthologs of OAT1 and OAT3. Consistent with the inhibition of OAT3-mediated ES reported in Table 1, in three separate experiments the K t for OAT3-mediated CIM uptake was 89.0 ± 0.80 µM, with a J max of 2.8 ± 0.55 pmol·cm -2 ·min -1. rbOAT1 also supported CIM uptake. Interestingly, the apparent affinity of rbOAT1 for CIM was comparatively high (25.4 ± 10.6 µM; n = 3), comparable to the K t for PAH (27.8 ± 3.9 µM; n = 2). However, the maximal rate of rbOAT1-mediated CIM transport was very low (0.15 pmol·cm -2 ·min -1 ) compared with that for PAH (9.0 ± 2.0 pmol·cm -2 ·min -1; n = 2), or to the maximal rate of CIM transported mediated by rbOAT3. The suggestion of high apparent affinity of rbOAT1 for CIM, combined with a very low apparent rate of transport, prompted us to examine in more detail the interaction of CIM with rbOAT1. Figure 8 shows the effect of 1 mM concentrations of unlabeled PAH and CIM on the uptake of radiolabeled PAH and CIM into CHO cells that stably expressed rbOAT1. PAH (1 mM), as expected, reduced accumulation of 0.3 µM [ 3 85% ( Fig. 8 A ). If, as implied by the results in Fig. 7 A, CIM is transported by rbOAT1 with a K t of 20 µM, then a 1 mM concentration of CIM should also have effectively eliminated [ 3 H]PAH uptake. Instead, 1 mM CIM reduced PAH uptake by only 50%, consistent with the weak interaction of CIM with flOAT1 observed by Burckhardt et al. ( 7 ). As shown in Fig. 8 B, accumulation of [ 3 H]CIM into rbOAT1-expressing cells, although significant ( P < 0.05), was only 10% of that noted for an equal concentration of [ 3 H]PAH. The "PAH-inhibitable" transport of [ 3 H]CIM (i.e., that fraction that may have been transported by rbOAT1) was only 2.5% of that noted for [ 3 H]PAH (there was no displaceable uptake of [ 3 H]CIM into wild-type CHO cells). In the light of these results, we suggest that, although both OAT1 and OAT3 can support transport of CIM, only the interaction with OAT3 is likely to play a role in renal clearance of CIM.
Fig. 7. Kinetics of CIM transport mediated by the rabbit orthologs of OAT1 ( A ), OAT3 ( B ), OCT1 ( C ), and OCT2 ( D ). The transporters were stably expressed in CHO cells. Each point is the means ± SE of triplicate 5-min uptakes of 35-40 nM [ 3 H]CIM measured in 3 separate experiments ( n = 3). The larger plots show the reduction in accumulation of labeled CIM arising from exposure to increasing concentrations of unlabeled CIM. The line fit to these data was based on the kinetics of competitive interaction of labeled and unlabeled substrate from which were calculated K t and J max values and constants of first-order uptake ( 34 ). Insets : Michaelis-Menten transformation of these data (i.e., total CIM uptake vs. total CIM concentration) in which the data points and solid lines show the kinetics of total CIM transport (mediated plus first order); the dashed lines are the apparent Michaelis-Menten components of total CIM uptake.
Fig. 8. Inhibition of rbOAT1-mediated transport of 0.3 µM concentrations of [ 3 H]PAH ( A ) and [ 3 H]CIM ( B ) produced by unlabeled CIM (1 mM) or PAH (1 mM). The height of each bar represents the means ± SE of 5-min uptakes measured in triplicate in a representative experiment.
Figure 7 also compares the kinetics of CIM uptake as mediated by the principal organic cation transporters of rabbit RPT, OCT1 and OCT2. rbOCT2 had a much higher affinity for CIM that did rbOCT1, with K t values of 1.3 ± 0.12 and 142 ± 59.7 µM, respectively ( n = 3). Interestingly, J max for rbOCT1-mediated CIM uptake, 3.7 ± 1.8 pmol·cm -2 ·min -1, was some 60-fold larger than that measured for rbOCT2 (56.4 ± 14.2 fmol·cm -2 ·min -1 ). Although that could reflect different levels of protein expression in the two clonal cell lines used to make the measurements, the maximal rate of TEA transport in these cell lines was virtually identical (7 vs. 12 pmol·cm -2 ·min -1 for rbOCT1 and rbOCT2, respectively; data not shown), lending credence to the suggestion that the higher affinity for CIM is associated with a lower turnover number for OCT2-mediated CIM transport (compared with OCT1). Taken together, these data suggest that the basolateral element of CIM secretion can involve interactions with OAT3, as well as with OCT1 and -2.
Although the characteristics of cloned OATs and OCTs expressed in heterologous systems implicated these distinct transporters as potential contributors to renal CIM secretion, these comparisons provided no information concerning the fractional contribution each transporter provides to CIM uptake in native RPT. We assessed the relative roles of these OA and OC transport processes play in basolateral CIM uptake by measuring accumulation of [ 3 H]CIM uptake into isolated cortical RPT in the absence and presence of increasing concentrations of either ES, as an inhibitor of OAT3, or TEA, as an inhibitor of OCT1 and OCT2 ( Fig. 9 ). ES proved to be a very effective inhibitor of [ 3 H]CIM uptake into cortical RPT, blocking 85% of total [ 3 H]CIM accumulation with an IC 50 of 4.1 ± 0.12 µM ( n = 2). This ES IC 50 agreed closely with the 4.5 µM K t for OAT3-mediated ES uptake determined in the present study ( Fig. 3 ), and with the K t of 7 µM for ES uptake in isolated, suspended rabbit RPT determined previously ( 33 ). Moreover, we found that ES exerts only a weak inhibition of OCT activity (IC 50 of OCT1-mediated 1 mM; IC 50 of OCT2-mediated TEA transport of 250 µM; data not shown). TEA (2 mM) only blocked 25% of total CIM uptake (with an IC 50 of 281 ± 181 µM, n = 2). Figure 10 A shows the effect on CIM uptake into cortical RPT of adding a single concentration of ES sufficient to block 90% of OAT3-mediated transporter activity; it blocked 70% of 90% of OCT1/2-mediated activity failed to significantly reduce CIM uptake. The combined influence of ES plus TEA did appear to decrease CIM uptake slightly more than ES alone. We also examined the effect of PAH on CIM uptake into cortical RPT. The concentration employed, 0.6 mM, was selected to target the contribution of OAT1 to total CIM uptake. As shown in Fig. 10 A, 0.6 mM PAH reduced CIM uptake by 35%. Collectively, these data suggested that [ 3 H]CIM uptake into cortical rabbit RPT is dominated by interactions with OATs, rather than with OCTs.
Fig. 9. Kinetics of inhibition of [ 3 H]CIM (30 nM) uptake into rabbit cortical renal proximal tubules (RPT) produced by increasing concentrations of ES ( A ) or TEA ( B ). Each point is the means ± SE of triplicate 30-s uptakes measured in a representative experiment. The lines were calculated assuming the observed inhibitions were competitive ( 20 ).
Fig. 10. Inhibition of [ 3 H]CIM uptake into cortical RPT ( left ) or isolated single S2 segments of RPT ( right ). In experiments with cortical RPT, tubules were exposed to 30 nM [ 3 H]CIM for 30 s in the absence (control) or presence of 1 mM CIM, 50 µM ES, 1 mM TEA, 50 µM ES+1 mM TEA, or 0.6 mM PAH. The height of each bar represents the means ± SE of triplicate uptakes measured in a representative experiment. In experiments with isolated single S2 segments, tubules were exposed to 1.5 µM [ 3 H]CIM for 30 s in the absence (control) or presence of 200 µM CIM, 50 µM ES, 0.5 mM TEA, 50 µM ES+0.5 mM TEA, or 0.5 mM PAH. The height of each bar represents the means ± SE of triplicate uptakes measured in experiments with 3-4 rabbits.
The comparatively modest contribution of OC transporters to CIM uptake was unexpected. However, as shown in Fig. 7, rbOCT1 and rbOCT2 interact differentially with CIM, with the latter having a 100-fold higher affinity for CIM than the former. With that fact in mind, it is potentially significant that OCT1 and OCT2 are distributed differentially along the length of the proximal tubule in rabbit [and rat ( 28 )] kidney, with functional expression of OCT1 dominating TEA transport in the S1 segment, whereas OCT2 dominates TEA transport in the S2 and S3 segments ( 56a ). Owing to the method of isolation, the cortical tubule suspension used in the experiments presented in Fig. 10 A was likely to consist of both S1 and S2 segments of RPT. Consequently, we elected to examine the pathways associated with CIM transport in a "defined" tubule segment, i.e., the single isolated S2 segment of rabbit RPT, in which OCT2 is the dominant OCT homolog ( 27, 57 ). As shown in Fig. 10 B, unlabeled ES (50 µM) and TEA (500 µM) both reduced total basolateral CIM uptake by 40-50%, and the combination of the two appeared to have an even greater effect. In contrast to the observations made with cortical RPT, PAH had no effect on CIM uptake into isolated single S2 segments. These data suggest that CIM uptake in the S2 segment of RPT involves approximately equal contributions from CIMs interaction with OAT3 and OCT2.
DISCUSSION
The renal proximal tubule is the principal site of secretion of a wide array of organic electrolytes. The cloning of multiple homologous transport proteins that can interact, to one extent or another, with selected organic anions and/or organic cations has made it apparent that renal organic electrolyte secretion involves the concerted activity of multiple transporters. Despite the evidence showing the potential of these processes to influence renal organic electrolyte secretion, the physiological role each plays is generally unclear. The present study determined the relative role of selected members of the SLC22 family of organic electrolyte transporters in mediating basolateral uptake of two organic electrolytes (the weak acid OTA and the weak base CIM) shown previously to interact with multiple transporters in the renal proximal tubule.
Our studies made use of the rabbit RPT, thereby taking advantage of previous studies that characterized the transport of OTA and CIM in physiologically intact tubules ( 21, 35, 36, 55 ). Although the rabbit orthologs of OCT1, OCT2, and OAT1 have been cloned ( 3, 49, 57 ), rbOAT3 had to be cloned and was found to share the general transport properties noted previously for the human ( 10 ), rat ( 30, 46 ), and mouse ( 47 ) orthologs of OAT3, most notably a broad substrate specificity ( Fig. 1 ) and the capacity to mediate OA/dicarboxylate exchange ( Fig. 5 ). These characteristics support the contention that OAT3 could play a quantitatively significant role in the active secretion of a broad range of organic electrolytes. Although rbOAT3 transported many of the substrates accepted by other OAT3 orthologs (including urate, CIM, and OTA), it had a comparatively weak interaction with PAH, and this proved useful in subsequent examination of the relative role played by OAT3 in the renal transport of OTA and CIM.
PAH is generally viewed as the "prototypic" substrate of the classic organic anion secretory pathway. After the initial observation that OAT1 supports PAH/ -KG exchange ( 44, 48 ), it was generally assumed that OAT1 was the molecular identity of the basolateral element of the classic OA secretory process (e.g., Ref. 16 ). It was subsequently found that PAH is also transported by the homologous proteins OAT2, OAT3, and OAT4 ( 11, 30, 43 ). Although there is broad consensus that neither OAT2 nor OAT4 plays a significant role in the basolateral step of active OA secretion ( 56 ), several observations implicate OAT3 as a quantitatively significant element in renal OA secretion. First, although it was initially suggested that OAT3 does not support OA/dicarboxylate exchange ( 30 ), more recent results clearly showed that OAT3 mediates exchange of ES for dicarboxylates ( 4, 46 ). The present results confirmed this and, in addition, showed that OAT3 can mediate ES/urate exchange as well ( Fig. 5 ). Second, OAT3 is expressed at comparatively high levels in the basolateral membrane of proximal tubule cells ( 37 ) and is coexpressed with OAT1 in at least some segments of RPT ( 33, 37 ). Finally, studies with an OAT3-null mouse showed that elimination of this transporter eliminates mediated basolateral uptake of ES and taurocholate into renal cortical slices and, interestingly, reduces PAH uptake by 50% ( 47 ).
A contribution by OAT3 in renal uptake of PAH in the mouse was not unexpected (although the amount may have been). The human and rat OAT3 orthologs transport PAH with relatively high affinity ( K t values of 87 and 65 µM, respectively) ( 10, 30 ). In the rabbit, however, the IC 50 for PAH inhibition of rbOAT3 activity 1 mM ( Fig. 4 ), similar to the inhibition by PAH of basolateral ES uptake in isolated single S2 segments of rabbit RPT ( 33 ). Although rbOAT3 did support PAH transport, the ratio of J PAH / was only 1.5% that of J ES /. OAT1 showed a reverse selectivity, i.e., transporting PAH but not ES ( Fig. 2 ), in accordance with the selectivity profile of mOAT1 ( 46 ). ES can inhibit rbOAT1 activity, but the inhibition is not competitive ( 33 ), and the IC 50 for ES inhibition of rbOAT1 is high ( 100 µM) ( 33 ) compared with the K t for OAT3-mediated ES transport (4 µM; Fig. 3 ). Taken together, these data suggest that, in the rabbit, PAH and ES can be used as homolog-selective inhibitors of OAT1 and OAT3, respectively.
We used PAH and ES to determine the relative contribution of OAT1 and OAT3 to the uptake of an organic acid, OTA, and an organic base, CIM, both of which are known to interact with multiple secretory processes in renal RPT. We previously reported that OTA (p K a 3.3) is accumulated across the basolateral membrane of rabbit proximal tubules by an organic anion secretory process that includes at least two distinct pathways that, collectively, display a high affinity for this mycotoxin (half-saturated at 1 µM) ( 21 ). The evidence for the involvement of (at least) two transporters included the observation that 2.5 mM unlabeled PAH, a concentration sufficient to saturate mediated PAH transport, only blocked 50% of total OTA accumulation, whereas probenecid (0.6 mM) effectively eliminated mediated OTA uptake ( 21 ). OTA has subsequently been shown to be a substrate for both OAT1 (human, K t of 2 µM) ( 50 ) and OAT3 (rat, K t of 0.7 µM) ( 30 ). Here, we confirmed that the rabbit orthologs of OAT1 and OAT3 transport OTA ( Fig. 6 A ) and that OTA is accumulated into rabbit cortical RPT ( Fig. 6 B ). The combined influence of OAT1 and OAT3 in this uptake was evident in the observation that PAH and ES, applied singly (each at a concentration 10 times higher than their K t values for uptake by OAT1 and OAT3, respectively), blocked only 50% of total mediated OAT uptake, whereas the combination of the two effectively eliminated mediated uptake ( Fig. 6 B ). The present results, therefore, support the contention that OTA uptake into cortical RPT involves the parallel activity of OAT1 and OAT3, with each responsible for approximately half of total OTA uptake.
Renal transport of the organic base, CIM, is particularly intriguing in that it appears to involve interactions with secretory pathways for both organic cations and organic anions ( 19, 31, 35, 54 ). The present results confirmed that CIM is an effective substrate for transporters normally considered selective for either organic cations or organic anions. The interaction of this weak base with OCTs is not surprising and supports the demonstrated influence of the organic cation secretory pathway with renal secretion of CIM as observed both in renal clearance studies ( 54 ) and in experiments with isolated single, perfused rabbit proximal tubules ( 35 ). Although OCT2 clearly displays a higher affinity for CIM than does OCT1, the transport capacity of the latter for CIM is sufficiently large ( Fig. 7 ) to support the conclusion that the OCT-mediated flux of CIM is likely to reflect the sum of expression of both OCT homologs. The comparatively weak inhibition of CIM uptake into cortical RPT produced by TEA, and the large inhibition of CIM uptake produced by ES, suggests that the functional expression of OATs (particularly OAT3) in the early proximal tubule may exceed that of OCTs.
The interaction of CIM with elements of the organic anion secretory pathway, particularly OAT3, is consistent with the observation that probenecid reduces CIM clearance in rats ( 31 ) and humans ( 19 ) and in isolated, single perfused rabbit RPT ( 35 ). Although CIM was transported by both rbOAT1 and rbOAT3 ( Fig. 7 ), OAT3-mediated transport was much more robust. The comparatively weak interaction of rbOAT1 was consistent with the failure of PAH to inhibit CIM uptake into isolated single S2 segments. Although there was a modest effect of PAH on uptake of CIM into the cortical tubule suspension ( Fig. 9 ), the poor interaction of CIM with the cloned rbOAT1 and the failure of PAH to influence CIM transport in single tubules suggest that the presence of PAH may have exerted an indirect inhibition on the CIM-OAT3 interaction (perhaps via accelerated, OAT1-mediated efflux of -KG and subsequent competition between -KG and CIM at the level of OAT3).
The surprising observation in the present report is that OAT3 can play as large or larger a role in basolateral CIM uptake into proximal tubules than the OCT pathways. In the mixed tubule suspension isolated from the outer cortex, OAT3 appeared to account for at least 80% of total CIM uptake, whereas TEA-inhibitable pathways (i.e., OCT1 plus OCT2) account for no more than 15-20% (or less) of CIM uptake ( Figs. 8 and 9 ). Owing to the method of isolation, our tubule suspension will be limited to S1 and S2 segments and may well be dominated by S1 segments. TEA uptake in the S1 segment is dominated by the activity of OCT1 ( 56a ), which has a much lower affinity for CIM than does OCT2 ( 27, 57 ). In contrast, the S2 segment is dominated by the activity of OCT2 ( 27, 57 ), so it was expected that the contribution of OC transport pathways would be more evident in isolated single S2 segments of rabbit RPT, and this was the case. However, even in the S2 segment, interaction with OCTs appeared to account for only 50% of total CIM uptake, with the remaining 50% occurring through a process inhibited by ES, i.e., probably OAT3.
Burckhardt et al. ( 7 ) recently showed that the human and flounder orthologs of OAT1 support cimetidine transport, albeit at comparatively low rates and with low affinity. Importantly, their results were consistent with the conclusion that OAT1 interacts more effectively with unprotonated, i.e., electroneutral, CIM than with the charged species. We also found that the rabbit ortholog of OAT3 interacts with CIM with higher apparent affinity at elevated pH, consistent with the view that the neutral species binds more effectively to the transporter than does the charged species (unpublished observations). However, ES transport showed the same sensitivity to pH, despite the fact that over the tested pH range (pH 7-8) there is no significant change in the concentration of the protonated species of ES. Consequently, it remains equivocal if the charge status of CIM plays a role in its binding to rabbit OAT3. Nevertheless, the likelihood that OAT3 interacts preferentially with the neutral species of CIM (CIM 0 ) could explain an interesting discrepancy concerning the renal handling of CIM. Ullrich et al. ( 53 ), noting the interaction of CIM with both anion and cation pathways in rat proximal tubule, classified CIM as a "bisubstrate." Indeed, Ullrich suggested that neither anion nor cation transporters are sensitive to the charge status of their substrates, citing experiments with microperfused rat proximal tubules in which changes in ambient pH (from 6 to 8) had comparatively little effect on peritubular CIM uptake (despite its p K a of 6.9) ( 52 ). More recent evidence ( 5 ) showed that charge does play a role in binding of weak bases to hOCT2. In that study, binding to hOCT2 of compounds that have a fixed cationic charge (e.g., TEA and MPP) was shown to be insensitive to ambient pH, whereas interaction of weak bases (including CIM) shifted in direct proportion to their pH-sensitive charge status. If, as suggested by Burckhardt et al. ( 7 ), OATs interact preferentially with CIM 0, and OCTs interact with CIM +, then a change in ambient pH could simply increase the effective concentration of substrate for one set of transporters (e.g., OATs), while reducing the concentration of substrate for the other set (e.g., OCTs).
Uric acid was also found to be a substrate for OAT3 ( Fig. 1 ). Urate is produced as the major end product of purine metabolism by muscle, liver, and intestine. The renal handling of urate is species specific, reflecting the differences in net reabsorption and net secretion occurring in S 1, S 2, and S 3 segments of the proximal tubule ( 1, 14 ). Net reabsorption is dominant in humans, dogs, and rats, species that excrete less urate than is filtered at the glomerulus, and net secretion is dominant in rabbits, pigs, and birds, which excrete more urate than is filtered at the glomerulus ( 8 ). Urate transport across the basolateral membrane of proximal tubules has been proposed to involve exchange for anions through an OAT protein ( 24 ). Among the cloned OATs, rat OAT1 ( 44 ), human OAT3 ( 4 ), and human OAT1 ( 26 ) are known to transport urate. Our data indicated that rbOAT3 transports urate ( Fig. 1 D ). The trans -stimulatory effect of urate on rbOAT3-mediated ES uptake, as well as the trans -stimulation of urate transport produced by preloading cells with glutarate ( Fig. 5 ), indicates that rbOAT3 functions as an urate/anion exchanger. These data, along with previous observation that rbOAT1 interacts with urate ( 3 ), suggest that these processes may play a quantitatively significant role in basolateral urate flux in intact tubules. Further investigations will be required to determine whether differences in the affinities for urate of human and rabbit OAT1/3 are associated with the net reabsorption (in the human) vs. the net secretion (in the rabbit) of urate.
In summary, we cloned and functionally characterized rabbit OAT3. It transported a variety of anionic substrates, including ES, OTA, and urate. In addition, rabbit OAT3 transported the cationic substrate CIM. Rabbit OAT3 supported carrier-mediated exchange of extracellular ES and urate for intracellular dicarboxylate (glutarate). ES and PAH were shown to be "discriminatory" substrates, capable of distinguishing activity of OAT3 and OAT1 in intact rabbit RPT. These two distinct OATs can contribute significantly to renal cellular uptake and secretion of substrates, including nephrotoxicants, for which they share overlapping specificity, as well as for those substrates that are selectively handled by one or the other of these processes. Additionally, some compounds (e.g., CIM) can serve as substrates for selected elements of the secretory pathways for both organic anions and cations, serving to emphasize the functional redundancy of the complex array of renal organic electrolyte transporters.
GRANTS
This work was supported in part by National Institutes of Health Grants DK-56224, DK-58251, DK-062097, ES-04940, and ES-06694.
ACKNOWLEDGMENTS
We thank A. Hillemann for excellent technical assistance, A. Nolte (Dept. of Biochemistry, Univ. of Göttingen) for nucleotide sequencing, and L. Muñoz for contributions to the study of DMPS interactions with OAT3.
【参考文献】
Abramson RG and Lipkowitz MS. Evolution of the uric acid transport mechanisms in vertebrate kidney. In: Basic Principles in Transport, Comparative Physiology, edited by Kinne RKH. Basel: Karger, 1990, p. 115-153.
Babu E, Takeda M, Narikawa S, Kobayashi Y, Enomoto A, Tojo A, Cha SH, Sekine T, Sakthisekaran D, and Endou H. Role of human organic anion transporter 4 in the transport of ochratoxin A. Biochim Biophys Acta 1590: 64-75, 2002.
Bahn A, Knabe M, Hagos Y, Rodiger M, Godehardt S, Graber-Neufeld DS, Evans KK, Burckhardt G, and Wright SH. Interaction of the metal chelator 2,3-dimercapto-1-propanesulfonate with the rabbit multispecific organic anion transporter 1 (rbOAT1). Mol Pharmacol 62: 1128-1136, 2002.
Bakhiya A, Bahn A, Burckhardt G, and Wolff NA. Human organic anion transporter 3 (hOAT3) can operate as an exchanger and mediate secretory urate flux. Cell Physiol Biochem 13: 249-256, 2003.
Barendt WM and Wright SH. The human organic cation transporter (hOCT2) recognizes the degree of substrate ionization. J Biol Chem 277: 22491-22496, 2002.
Buist SC, Cherrington NJ, Choudhuri S, Hartley DP, and Klaassen CD. Gender-specific and developmental influences on the expression of rat organic anion transporters. J Pharmacol Exp Ther 301: 145-151, 2002.
Burckhardt BC, Brai S, Wallis S, Krick W, Wolff NA, and Burckhardt G. Transport of cimetidine by flounder and human renal organic anion transporter 1. Am J Physiol Renal Physiol 284: F503-F509, 2003.
Burckhardt G and Pritchard JB. Organic anion and cation antiporters. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW and Giebisch G. Philadelphia, PA: Lippincott Williams & Wilkins, 2000, p. 193-222.
Burg M, Grantham J, Abramov M, and Orloff J. Preparation and study of fragments of single rabbit nephrons. Am J Physiol 210: 1293-1298, 1966.
Cha SH, Sekine T, Fukushima JI, Kanai Y, Kobayashi Y, Goya T, and Endou H. Identification and characterization of human organic anion transporter 3 expressing predominantly in the kidney. Mol Pharmacol 59: 1277-1286, 2001.
Cha SH, Sekine T, Kusuhara H, Yu E, Kim JY, Kim DK, Sugiyama Y, Kanai Y, and Endou H. Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta. J Biol Chem 275: 4507-4512, 2000.
Chatsudthipong V and Dantzler WH. PAH/ -KG countertransport stimulates PAH uptake and net secretion in isolated rabbit renal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 263: F384-F391, 1992.
Chatsudthipong V and Jutabha P. Effect of steviol on para-aminohippurate transport by isolated perfused rabbit renal proximal tubule. J Pharmacol Exp Ther 298: 1120-1127, 2001.
Dantzler WH. Comparative aspects of renal organic anion transport. Cell Physiol Biochem 6: 28-38, 1996.
Dantzler WH, Evans KK, and Wright SH. Kinetics of interactions of para-aminohippurate, probenecid, cysteine conjugates, and N -acetyl cysteine conjugates with basolateral organic anion transporter in isolated rabbit proximal renal tubules. J Pharmacol Exp Ther 272: 663-672, 1995.
Dresser MJ, Leabman MK, and Giacomini KM. Transporters involved in the elimination of drugs in the kidney: organic anion transporters and organic cation transporters. J Pharm Sci 90: 397-421, 2001. <a href="/cgi/external_ref?access_num=10.1002/1520-6017(200104)90:4
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, and Endou H. Molecular identification of a renal urate anion exchanger that regulates blood urate levels. Nature 417: 447-452, 2002.
Feng B, Dresser MJ, Shu Y, Johns SJ, and Giacomini KM. Arginine 454 and lysine 370 are essential for the anion specificity of the organic anion transporter, rOAT3. Biochem 40: 5511-5520, 2001.
Gisclon LG, Boyd RA, Williams RL, and Giacomini KM. The effect of probenecid on the renal elimination of cimetidine. Clin Pharmacol Ther 45: 444-452, 1989.
Groves CE, Evans K, Dantzler WH, and Wright SH. Peritubular organic cation transport in isolated rabbit proximal tubules. Am J Physiol Renal Fluid Electrolyte Physiol 266: F450-F458, 1994.
Groves CE, Morales M, and Wright SH. Peritubular transport of ochratoxin A in rabbit renal proximal tubules. J Pharmacol Exp Ther 284: 943-948, 1998.
Groves CE, Munoz L, Bahn A, Burckhardt G, and Wright SH. Interaction of cysteine conjugates with human and rabbit organic anion transporter 1. J Pharmacol Exp Ther 304: 560-566, 2003.
Groves CE and Schnellmann RG. Suspensions of rabbit renal proximal tubules. In: Methods in Renal Toxicology, edited by Zalups RK and Lash LH. Boca Raton, FL: CRC, 1996, p. 147-162.
Hediger MA. Kidney function: gateway to a long life? Nature 417: 393-395, 2002.
Hosoyamada M, Sekine T, Kanai Y, and Endou H. Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney. Am J Physiol Renal Physiol 276: F122-F128, 1999.
Ichida K, Hosoyamada M, Kimura H, Takeda M, Utsunomiya Y, Hosoya T, and Endou H. Urate transport via human PAH transporter hOAT1 and its gene structure. Kidney Int 63: 143-155, 2003.
Kaewmokul S, Chatsudthipong V, Evans KK, Dantzler WH, and Wright SH. Functional mapping of rbOCT1 and rbOCT2 activity in the S2 segment of rabbit proximal tubule. Am J Physiol Renal Physiol 285: F1149-F1159, 2003.
Karbach U, Kricke J, Meyer-Wentrup F, Gorboulev V, Volk C, Loffing-Cueni D, Kaissling B, Bachmann S, and Koepsell H. Localization of organic cation transporters OCT1 and OCT2 in rat kidney. Am J Physiol Renal Physiol 279: F679-F687, 2000.
Kojima R, Sekine T, Kawachi M, Cha SH, Suzuki Y, and Endou H. Immunolocalization of multispecific organic anion transporters, OAT1, OAT2, and OAT3, in rat kidney. J Am Soc Nephrol 13: 848-857, 2002.
Kusuhara H, Sekine T, Utsunomiya-Tate N, Tsuda M, Kojima R, Cha SH, Sugiyama Y, Kanai Y, and Endou H. Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. J Biol Chem 274: 13675-13680, 1999.
Lin JH, Los LE, Ulm EH, and Duggan DE. Kinetic studies on the competition between famotidine and cimetidine in rats. Evidence of multiple renal secretory systems for organic cations. Drug Metab Dispos 16: 52-56, 1988.
Lu R, Chan BS, and Schuster VL. Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C. Am J Physiol Renal Physiol 276: F295-F303, 1999.
Lungkaphin A, Chatsudthipong V, Evans KK, Groves CE, Wright SH, and Dantzler WH. Interaction of the metal chelator, DMPS, with OAT1 and OAT3 in intact isolated rabbit renal proximal tubules. Am J Physiol Renal Physiol 286: F68-F76, 2004.
Malo C and Berteloot A. Analysis of kinetic data in transport studies: new insights from kinetic studies of Na + - D -glucose cotransport in human intestinal brush-border membrane vesicles using a fast sampling, rapid filtration apparatus. J Membr Biol 122: 127-141, 1991.
McKinney TD, Myers P, and Speeg KV Jr. Cimetidine secretion by rabbit renal tubules in vitro. Am J Physiol Renal Fluid Electrolyte Physiol 241: F69-F76, 1981.
McKinney TD and Speeg KV Jr. Cimetidine and procainamide secretion by proximal tubules in vitro. Am J Physiol Renal Fluid Electrolyte Physiol 242: F672-F680, 1982.
Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, Fukatsu A, Ogawa O, and Inui KI. Gene expression levels and immunolocalization of organic anion transporters in the human kidney. J Am Soc Nephrol 13: 866-874, 2002.
Pao SS, Paulsen IT, and Saier MH Jr. Major facilitator superfamily. Microbiol Mol Biol Rev 62: 1-34, 1998.
Pritchard JB. Luminal and peritubular steps in the renal transport of p -aminohippurate. Biochim Biophys Acta 906: 295-308, 1987.
Rodeheaver DP, Aleo MD, and Schnellmann RG. Differences in enzymatic and mechanical isolated rabbit renal proximal tubules: comparison in long-term incubation. In Vitro Cell Dev Biol 26: 898-904, 1990.
Saier MH Jr. A functional-phylogenetic classification system for transmembrane solute transporters. Microbiol Mol Biol Rev 64: 354-411, 2000.
Sambrook J, Fritsch EF, and Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor, 1989.
Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y, and Endou H. Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 429: 179-182, 1998.
Sekine T, Watanabe N, Hosoyamada M, Kanaim Y, and Endou H. Expression cloning and characterization of a novel multispecific organic anion transporter. J Biol Chem 272: 18526-18529, 1997.
Shimada H, Moewes B, and Burckhardt G. Indirect coupling to Na + of p -aminohippuric acid uptake into rat renal basolateral membrane vesicles. Am J Physiol Renal Fluid Electrolyte Physiol 253: F795-F801, 1987.
Sweet DH, Chan LM, Walden R, Yang XP, Miller DS, and 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.
Sweet DH, Miller DS, Pritchard JB, Fujiwara Y, Beier DR, and 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.
Sweet DH, Wolff NA, and Pritchard JB. Expression cloning and characterization of ROAT1: the renal basolateral organic anion transporter in rat kidney. J Biol Chem 272: 30088-30095, 1997.
Terashita S, Dresser MJ, Zhang L, Gray AT, Yost SC, and Giacomini KM. Molecular cloning and functional expression of a rabbit renal organic cation transporter. Biochim Biophys Acta 1369: 1-6, 1998.
Tsuda M, Sekine T, Takeda M, Cha SH, Kanai Y, Kimura M, and Endou H. Transport of ochratoxin A by renal multispecific organic anion transporter 1. J Pharmacol Exp Ther 289: 1301-1305, 1999.
Ullrich KJ. Affinity of drugs to the different renal transporters for organic anions and organic cations. Pharm Biotechnol 12: 159-179, 1999.
Ullrich KJ and Rumrich G. Renal contraluminal transport systems for organic anions (paraaminohippurate, PAH) and organic cations ( N 1 -methyl-nicotinanide, NMeN) do not see the degree of substrate ionization. Pflügers Arch 421: 286-288, 1992.
Ullrich KJ, Rumrich G, David C, and Fritzsch G. Bisubstrates: substances that interact with renal organic anion and organic cation transport systems. I. Amines, piperidines, piperazines, azepines, pyridines, quinolines, imadazoles, thiazoles, guanidines, and hydrazines. Pflügers Arch 425: 280-299, 1993.
Weiner IM and Roth L. Renal excretion of cimetidine. J Pharmacol Exp Ther 216: 516-520, 1981.
Welborn JR, Groves CE, and Wright SH. Peritubular transport of ochratoxin A by single rabbit renal proximal tubules. J Am Soc Nephrol 9: 1973-1982, 1998.
Wright SH and Dantzler WH. Molecular and cellular physiology of renal organic cation and anion transport. Physiol Rev 84: 987-1049, 2004.
Wright SH, Evans KK, Zhang X, Cherrington NJ, Sitar DS, and Dantzler WH. Functional map of TEA transport activity in isolated rabbit renal proximal tubules. Am J Physiol Renal Physiol 287: F442-F451, 2004.
Zhang X, Evans KK, and Wright SH. Molecular cloning of rabbit organic cation transporter rbOCT2 and functional comparisons with rbOCT1. Am J Physiol Renal Physiol 283: F124-F133, 2002.
作者单位：1 Department of Physiology, College of Medicine, University of Arizona, Tucson, Arizona 85724; 2 Zentrum für Physiologie und Pathophysiologie, Abteilung Vegetative Physiologie, Universität Göttingen, 37073 Göttingen, Germany; and 3 Department of Physiology, Mahidol University, Ba