Urate/alpha -ketoglutarate exchange in avian basolateral membrane vesicles

Steven M. Grassl

Department of Pharmacology, State University of New York, Upstate Medical University, Syracuse, New York 13210


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Membrane transport pathways for transcellular secretion of urate across the proximal tubule were investigated in avian kidney. The presence of coupled urate/alpha -ketoglutarate exchange was investigated in basolateral membrane vesicles (BLMV) by [14C]urate and [alpha -3H]ketoglutarate flux measurements. An inward Na gradient induced accumulation of alpha -ketoglutarate of sufficient magnitude to suggest a Na-dicarboxylate cotransporter. An inward Na gradient also induced concentrative accumulation of urate in the presence of alpha -ketoglutarate but not in its absence, suggesting urate/alpha -ketoglutarate exchange. alpha -Ketoglutarate-dependent stimulation of urate uptake was not observed in brush-border membrane vesicles. An outward urate gradient induced concentrative accumulation of alpha -ketoglutarate. alpha -Ketoglutarate-coupled urate uptake was specific for alpha -ketoglutarate, Cl dependent, and insensitive to membrane potential. alpha -Ketoglutarate-coupled urate uptake was inhibited by increasing p-aminohippurate (PAH) concentrations, and alpha -ketoglutarate-coupled PAH uptake was observed. alpha -Ketoglutarate-coupled PAH uptake was inhibited by increasing urate concentrations, and an outward urate gradient induced concentrative accumulation of PAH. These results suggest a Cl-dependent, alpha -ketoglutarate-coupled anion exchange mechanism as a pathway for active urate uptake across the basolateral membrane of urate-secreting proximal tubule cells.

organic anion secretion; renal proximal tubule; urate transporter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN HUMANS, the catabolic degradation of dietary and endogenously synthesized purine bases ends in the formation of uric acid, a metabolite eliminated primarily by renal excretion. The renal handling of uric acid by the human kidney is confined to the proximal nephron, where a four-part process is postulated: 1) glomerular filtration, 2) presecretory reabsorption, 3) secretion, and 4) postsecretory reabsorption (7, 26). The renal handling of uric acid is species specific, reflecting the differences in net reabsorption and net secretion occurring in S1, S2, and S3 segments of the proximal tubule (1, 8). Net reabsorption is dominant in humans, dogs, and rats, which 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.

In species demonstrating net secretion of urate, pathways for transcellular transport of urate across the proximal tubule have been investigated with membrane vesicles isolated from pig (17, 36, 37) and rabbit (16, 19) kidney. The transcellular secretion of urate across the proximal tubule originates from the concentrative accumulation of intracellular urate from the blood across the basolateral membrane. The urate transport pathway(s) mediating active uptake of urate across the proximal tubular basolateral membrane remains to be identified and characterized. Previous membrane vesicle studies identified alpha -ketoglutarate/p-aminohippurate (PAH) exchange in the basolateral membranes of both urate-reabsorbing and urate-secreting species (18, 22, 29, 33, 37). However, when assayed in the same experiment with the same rat basolateral membrane vesicles, alpha -ketoglutarate-driven PAH uptake but not urate uptake was observed, suggesting that urate is not a substrate for the alpha -ketoglutarate/PAH exchange mechanism (21). In pig, a urate-secreting species, a single-membrane vesicle study suggests an anion exchange mechanism coupling efflux of intracellular alpha -ketoglutarate to influx of extracellular urate across the basolateral membrane (37). These observations suggest that two independent alpha -ketoglutarate-coupled anion exchange mechanisms, distinguished by a difference in substrate specificity, may mediate uptake of PAH and urate across the basolateral membrane.

Among urate-secreting species, the largest net secretion may occur in the avian kidney, which clears urate from the blood at a rate up to 10-fold greater than the rate of glomerular filtration (32). This urate secretion results from the large transcellular secretory flux of urate across the entire length of the bird proximal tubule and the absence of reabsorption (2). Despite these features of avian urate excretion, the transport mechanism(s) mediating active uptake at the basolateral membrane has not been identified.

Basolateral membrane transport pathways mediating proximal tubule urate secretion were investigated with membrane vesicles prepared from avian kidney. The evidence suggests that transcellular urate secretion across the proximal tubule is initiated by an alpha -ketoglutarate-coupled anion exchange mechanism in the basolateral membrane.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Membrane preparation. Basolateral membrane vesicles were isolated from turkey kidney by differential and density gradient centrifugation as described previously with minor modifications (11). Kidneys from four to six turkeys obtained at slaughter at the Plainville Turkey Farm (Plainville, NY) were immediately placed in ice-cold 250 mM sucrose, 2 mM EDTA, and 10 mM HEPES-tetramethylammonium (TMA), pH 7.6. All subsequent preparation occurred on ice or in refrigerated centrifuges at 4°C. Kidneys were finely minced and homogenized in 4 ml/g buffer containing 0.1 mM phenylmethylsulfonyl fluoride by completing 30 strokes at 2,000 rpm with a 250-ml Lurex Teflon-glass homogenizer (0.006-in. clearance). The homogenate was centrifuged at 1,100 g for 10 min (brake off), and the resulting supernatant was further centrifuged at 43,500 g for 30 min. The white, "fluffy" upper layer of the pellet was collected and recentrifuged at 43,500 g for 30 min. The crude plasma membrane fraction was again collected and resuspended in 16% Percoll by completing two strokes with a 110-ml Lurex Teflon-glass homogenizer. Percoll density gradient centrifugation was performed in a Sorvall SS-34 rotor at 48,000 g for 60 min (brake off). Membrane fractions were collected by aspiration from above with a Haake-Buchler Auto-Densi-Flow apparatus pumping to fraction collector. Membrane fractionation studies were performed to assess the spatial density-dependent distribution of brush-border membrane marker transport activity (Na-glucose cotransport) and basolateral membrane marker transport activity (alpha -ketoglutarate/urate exchange). Basolateral membranes were collected from membrane fractions where Na-glucose cotransport was minimal or absent and where alpha -ketoglutarate/urate exchange was maximal. Percoll was removed from pooled fractions by centrifugation at 200,000 g for 60 min. The pelleted membrane was gently separated from the solid underlying Percoll and resuspended in sucrose buffer to a concentration of 8-12 mg membrane protein/ml. Membrane protein was determined from the sodium dodecyl sulfate Lowry assay with bovine serum albumin as standard (20). The basolateral membrane preparation was aliquoted and stored frozen at -70°C for use within 4 wk.

Brush-border membrane vesicles were collected from the remaining Percoll gradient at a density enriched in Na-glucose cotransporter activity and were further purified by divalent cation aggregation (10). The collected membrane fractions were centrifuged at 200,000 g for 60 min, and the pelleted membrane was gently removed from the solid underlying Percoll pellet. The membranes were resuspended in sucrose buffer by completing three strokes with a Lurex Teflon-glass homogenizer. The membrane suspension was incubated in 12 mM MgCl2 for 15 min and centrifuged for 15 min at 3,000 g to pellet the Mg-induced membrane aggregates. The resulting supernatant was decanted and centrifuged at 31,000 g for 30 min. The resulting pellet was resuspended and subjected to a second and third cycle of divalent cation aggregation and centrifugation. The final brush-border membrane vesicle pellet was resuspended in sucrose buffer, aliquoted, and stored frozen at -70°C for use within 4 wk. Brush-border membrane marker enzyme activity (alkaline phosphatase) was typically enriched 10- to 16-fold compared with homogenate, and the specific activity of Na-glucose cotransport was comparable to levels observed in nonavian species.

Isotopic flux measurements. Thawed (20-25°C) aliquots of membrane vesicles were diluted in 125 mM KCl, 10 mM HEPES-TMA, pH 7.5, and centrifuged 36,600 g for 30 min. Membrane pellets were resuspended and recentrifuged. The second pellet was resuspended, and isosmotic solutions of appropriate ionic composition were added to obtain the desired intravesicular solution described for each experiment in Tables 1-3 and Figs. 1-11. The membrane suspension was preincubated for 120 min at room temperature to attain transmembrane equilibration of the added solutions. The extravesicular solutions were prepared similarly, and the extravesicular ionic composition for each experiment is given in Tables 1-3 and Figs. 1-11. The intravesicular uptake of [14C]urate, [alpha -3H]ketoglutarate, and [3H]PAH was assayed at least in triplicate for each time point determination by a rapid filtration technique described previously (12). Briefly, 10 µl of membrane vesicles were rapidly mixed by vortex with 40 µl of isotope buffer and incubated for a designated time interval measured by stopwatch. Isotope uptake was quenched by rapid dilution with 125 mM NaCl or isosmotic K gluconate, 2 mM TMA probenicid, and 10 mM HEPES-TMA, pH 7.5, kept at 4°C. The diluted membrane suspension was passed through a 0.65-µm Millipore filter (DAWP) and washed with additional quench buffer. The process of quenching, filtration, and washing occurred routinely within a 15-s interval. The filters were dissolved in 3 ml of Ready Safe (Beckman) and counted by scintillation spectroscopy. The timed uptake values obtained were corrected by the nonspecific retention of isotope by the filters. Although the absolute uptake values expressed per milligram of membrane protein were noted in some instances to vary from membrane preparation to membrane preparation, suggesting differences in transporter activity, the relative changes in uptake occurring within each experiment were highly reproducible among different membrane preparations. All experiments were performed at least in triplicate, using a different membrane vesicle preparation in each instance. Where appropriate, statistical significance was calculated with an unpaired t-test for two means, where P < 0.05 is taken as the limit denoting statistical significance. Km and Vmax values were quantified from Hanes-Woolf replots of the kinetic data with linear regression.

                              
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Table 1.   Effect of increasing PAH concentration on urate influx


                              
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Table 2.   Effect of analogs on PAH and urate influx


                              
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Table 3.   Effect of drugs and transport inhibitors on PAH and urate influx



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Fig. 1.   Effect of Na concentration gradient on alpha -ketoglutarate influx. Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-tetramethylammonium (TMA), pH 7.5. Uptake of alpha -ketoglutarate (5 µM) occurred from extravesicular solutions containing 100 mM NaCl or TMA Cl, 25 mM KCl, 10 mM HEPES-TMA, pH 7.5. Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. A representative experiment of 3 independent observations is shown. Subscript o indicates extravesicular concentration; subscript i indicates intravesicular concentration.



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Fig. 2.   Effect of Na concentration gradient and alpha -ketoglutarate on urate influx. Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. Uptake of urate (300 µM) occurred in the presence (5 µM) and absence of alpha -ketoglutarate and from extravesicular solutions containing 100 mM NaCl or TMA Cl, 25 mM KCl, 10 mM HEPES-TMA, pH 7.5. Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. A representative experiment of 3 independent observations is shown.



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Fig. 3.   alpha -Ketoglutarate concentration dependence of urate influx. Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. The 10-s uptake of urate (300 µM) occurred from extravesicular solutions containing 100 mM NaCl, 25 mM KCl, 10 mM HEPES-TMA, pH 7.5, and the alpha -ketoglutarate concentrations shown. Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. Urate influx is shown as % of urate uptake measured in the absence of alpha -ketoglutarate (291 ± 9 pmol/mg). Means + SE for 4 experiments, each performed with a different membrane preparation, are shown.



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Fig. 4.   Effect of urate concentration gradient on alpha -ketoglutarate influx. Basolateral (A) and brush-border (B) membrane vesicles were equilibrated with (1 mM) and without urate and with 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. Uptake of alpha -ketoglutarate (12.5 µM) occurred from extravesicular solutions containing 12.5 µM urate, 125 mM KCl, 10 mM HEPES-TMA, pH 7.5 Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. Representative experiments of 3 independent observations are shown.



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Fig. 5.   Kinetics of alpha -ketoglutarate-dependent urate influx. Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. The 10-s uptake of urate occurred in the presence (5 µM) and absence of alpha -ketoglutarate and from extravesicular solutions containing 100 mM NaCl, 25 mM KCl, 10 mM HEPES-TMA, pH 7.5, and the urate concentrations shown. Membrane vesicles were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. Mean ± SE alpha -ketoglutarate-dependent urate influx of 3 experiments, each performed with a different membrane preparation, is shown.



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Fig. 6.   Carboxylate specificity of Na gradient-induced urate influx. Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. The 30-s uptake of urate (300 µM) occurred from extravesicular solutions containing 100 mM NaCl, 25 mM KCl, 10 mM HEPES-TMA, pH 7.5, and the anions shown (5 µM). Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. Means + SE of 4 experiments, each performed with a different membrane preparation, are shown.



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Fig. 7.   Anion dependence of urate/alpha -ketoglutarate exchange. Membrane vesicles were equilibrated with (2 mM) and without ketoglutarate and with 125 mM K gluconate, 10 mM HEPES-TMA, pH 7.5. The 20-s uptake of urate (300 µM) occurred from extravesicular solutions containing 25 µM alpha -ketoglutarate, 10 mM HEPES-TMA, pH 7.5, 1.6 mM K gluconate, and the K anions shown (123.4 mM). Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. The alpha -ketoglutarate gradient-dependent difference in urate uptake (pmol/mg) measured in vesicles preloaded with and without alpha -ketoglutarate is shown as the mean + SE of 3 experiments, each performed with a different membrane preparation.



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Fig. 8.   Cl concentration dependence of urate/alpha -ketoglutarate exchange. Membrane vesicles were equilibrated with 125 mM K gluconate, 100 mM N-methyl-D-glucamine (NMDG) gluconate, and 10 mM HEPES-TMA, pH 7.5. The 30-s uptake of urate (300 µM) was measured in the presence (5 µM) and absence of alpha -ketoglutarate and the Cl concentrations shown. Uptake occurred from extravesicular solutions containing 125 mM K gluconate, 80 mM Na gluconate, 20 mM NMDG gluconate, and 10 mM HEPES-TMA, pH 7.5. Where indicated (0 Clo), Cl was substituted with gluconate. Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. The alpha -ketoglutarate-dependent difference in urate uptake measured in the presence and absence of alpha -ketoglutarate is shown as % of uptake (567 ± 51 pmol/mg) measured at 100 mM Cl. Means ± SE for 8 experiments, each performed with a different membrane preparation, are shown.



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Fig. 9.   Effect of Cl and Cl concentration gradient on urate influx. Membrane vesicles were equilibrated with 100 mM NMDG gluconate or Cl, 100 mM K gluconate, 10 mM HEPES-TMA, pH 7.5. Uptake of urate (300 µM) occurred in the presence (5 µM; A) and absence (B) of alpha -ketoglutarate and from extravesicular solutions containing 100 mM K gluconate, 10 mM HEPES-TMA, pH 7.5, and 80 mM Na gluconate, 20 mM NMDG gluconate (0 Cl), 80 mM NaCl, 20 mM NMDG Cl (Clo = Cli), or 80 mM NaCl, 20 µM NMDG gluconate (Clo > Cli). Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. Representative experiments of 3 independent observations are shown.



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Fig. 10.   Effect of Na concentration gradient and alpha -ketoglutarate on p-aminohippurate (PAH) influx. Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-TMA (OH), pH 7.5. Uptake of PAH (300 µM) occurred in the presence (5 µM) or absence of alpha -ketoglutarate and from extravesicular solutions containing 100 mM NaCl or TMA Cl, 25 mM KCl, 10 mM HEPES-TMA, pH 7.5. Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. A representative experiment of 3 independent observations is shown.



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Fig. 11.   Effect of PAH concentration gradients on urate influx. Membrane vesicles were equilibrated with 5 mM TMA-PAH or TMA gluconate, 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. Uptake of urate (62.5 µM) occurred from an extravesicular solution containing 62.5 µM PAH, 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. A representative experiment of 3 independent observations is shown.

Chemicals. [8-14C]urate (53 mCi/mmol) and [3H(G)]alpha -ketoglutarate (10.3 Ci/mmol) were obtained from Moravek Biochemicals (Brea, CA). Glycyl-2-3H-PAH (1.3 Ci/mmol) was obtained from New England Nuclear (Boston, MA). Percoll, alpha -ketoglutarate, probenicid, TMA, gluconate, nitrate, and other compounds shown in Tables 2 and 3 were obtained from Sigma (St. Louis, MO) and the Sigma-Aldrich library of rare chemicals. 4-Nitrohippurate and pyrazinoate were purchased from Aldrich (Milwaukee, WI). Losartan was a gift from Merck (Rahway, NJ). Valinomycin was dissolved in ethanol (95%) and was added to membrane suspensions in a 1:100 dilution. Where indicated, equivalent volumes of ethanol were added to control aliquots of membrane. All solutions were prepared with distilled-deionized water and passed through a 0.22-µm Millipore filter.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Functional identification of urate/alpha -ketoglutarate exchange. Transport pathways mediating active accumulation of urate across the basolateral membrane of avian proximal tubules were investigated by testing for urate/alpha -ketoglutarate exchange in membrane vesicles. Evidence for a Na-dicarboxylate cotransporter and the generation of an outwardly directed alpha -ketoglutarate gradient was obtained from observing the effect of Na on alpha -ketoglutarate uptake. Figure 1 shows such evidence: a Na gradient induced a transient alpha -ketoglutarate accumulation 30-fold above equilibrium. In the absence of Na, alpha -ketoglutarate accumulation was well below equilibrium. An indirect electrostatic coupling of alpha -ketoglutarate and Na uptake via parallel conductive pathways is unlikely because concentrative accumulation of alpha -ketoglutarate was observed in the presence of conditions [extravesicular K (Ko) < intravesicular K (Ki) + valinomycin (Val)] expected to induce an inside-negative voltage difference. Na-dicarboxylate cotransport in avian basolateral membranes is consistent with previous observations of Na-dependent alpha -ketoglutarate uptake in basolateral membrane vesicles isolated from rat (4) and rabbit (39) kidney.

Using the Na-dicarboxylate cotransport mechanism to generate a large outwardly directed alpha -ketoglutarate concentration gradient, we assessed the presence of a basolateral membrane urate/alpha -ketoglutarate exchange mechanism by observing the dependence of urate uptake on the Na concentration gradient and on alpha -ketoglutarate. Figure 2 shows that urate uptake was low in the absence of a Na gradient and the presence of alpha -ketoglutarate and slowly approached equilibrium. In contrast, there was a marked stimulation of urate uptake in the presence of both alpha -ketoglutarate and a Na gradient, with levels reaching more than twofold above equilibrium. The level of urate uptake at equilibrium indicates a volume of distribution approximating 1-2 µl/mg vesicle protein, which suggests that nonspecific urate binding to the vesicle membrane does not contribute significantly to urate uptake. The concentrative accumulation of urate observed in the presence of a Na gradient and alpha -ketoglutarate suggests the presence of an anion exchange mechanism mediating direct coupling of alpha -ketoglutarate efflux to urate influx. An indirect electrostatic coupling of alpha -ketoglutarate efflux to urate influx via parallel conductive pathways is unlikely because concentrative accumulation of urate was observed in the presence of conditions (Ko < Ki + Val) in which an inside-negative voltage difference is expected. The localization of the anion exchange activity to the basolateral membrane is proven by the absence of Na gradient- and alpha -ketoglutarate-dependent urate uptake in brush border membrane vesicles despite a comparable magnitude of Na-dicarboxylate cotransport (unpublished observations).

The urate/alpha -ketoglutarate exchange mechanism was investigated further by assessing the alpha -ketoglutarate concentration dependence of urate uptake (Fig. 3). A biphasic relation between increasing extravesicular alpha -ketoglutarate concentration and urate uptake is observed where cis-inhibition is indicated at higher alpha -ketoglutarate concentrations. The presence of urate/alpha -ketoglutarate exchange was further suggested by the concentration-dependent inhibition (IC50 ~250 µM) of Na gradient- and alpha -ketoglutarate-dependent urate uptake induced by probenicid, an organic anion transport inhibitor (unpublished observations).

Urate/alpha -ketoglutarate exchange should also mediate accumulation of alpha -ketoglutarate driven by a urate gradient. The uptake of alpha -ketoglutarate was measured in vesicles in the presence and absence of an outwardly directed urate gradient (Fig. 4). Imposition of a urate gradient induced alpha -ketoglutarate uptake to levels threefold above equilibrium. An indirect coupling of conductive alpha -ketoglutarate uptake to an inside-positive urate gradient-induced diffusion potential is excluded because of a charge-compensating flux of K across valinomycin-treated membranes. In brush border membrane vesicles, an outwardly directed urate gradient had no effect on alpha -ketoglutarate uptake, consistent with a basolateral membrane localization of urate/alpha -ketoglutarate exchange.

Functional properties of urate/alpha -ketoglutarate exchange mechanism. Figure 5 shows that the initial rate of urate influx increased with increasing extravesicular urate concentration, approaching a maximum at ~1 mM urate. The Hanes-Woolf replot of the data is linear, suggesting that urate interacts with the anion exchange mechanism at a single saturable site with an apparent Km value of 350 µM and a maximal transport rate of 8 nmol · mg-1 · min-1. The apparent Km value is similar to the urate concentrations measured in the avian circulation (9).

We next determined which di- and tricarboxylic citric acid cycle intermediates may serve as intracellular driving forces for concentrative urate accumulation across the basolateral membrane. In addition to alpha -ketoglutarate, the dicarboxylates glutarate, succinate, fumarate, malate, and oxolacetate and the tricarboxylate citrate are reported to be substrates transported by the basolateral membrane Na-dicarboxylate cotransporter (4, 39). Figure 6 shows the effects of these anions on urate uptake. Only alpha -ketoglutarate and its nonphysiological analog glutarate stimulated urate uptake to levels exceeding the control.

Assuming a stoichiometric coupling ratio of 1, the exchange of the divalent anion alpha -ketoglutarate for the monovalent anion urate requires net transfer of negative charge across the membrane and a voltage sensitivity of at least one step in the anion exchange process. To test this, the effect of an inside-negative voltage difference on alpha -ketoglutarate-driven urate uptake was determined. The exchanger should transport negative charge from the vesicle interior, and an inside-negative potential should serve as an additional driving force accelerating urate/alpha -ketoglutarate exchange. However, when measured in the presence of an inside-negative voltage (Ko < Ki + Val), urate uptake (650 ± 105 pmol/mg; n = 3) did not exceed urate uptake (652 ± 82 pmol/mg; n = 3) in the absence of a membrane potential (Ko = Ki + Val). The apparent insensitivity of urate/alpha -ketoglutarate exchange to membrane potential indicates an electroneutral exchange process. This may be either the coupled exchange of a monovalent cation and alpha -ketoglutarate for urate or the exchange of a monovalent anion and urate for alpha -ketoglutarate. The kinetics of urate uptake is not consistent with the exchange of two urate molecules for alpha -ketoglutarate (Fig. 5). When urate/alpha -ketoglutarate exchange was measured with an outward K gradient (Ko < Ki - Val), urate uptake (607 ± 126 pmol/mg; n = 3) was no greater than in the absence of a K gradient (652 ± 82 pmol/mg; n = 3). This is evidence against coupled exchange of K and alpha -ketoglutarate for urate.

Previous studies of a related transport mechanism, PAH/alpha -ketoglutarate exchange, demonstrated a voltage sensitivity when assayed in the absence of Cl (3, 18). This observation suggests the possibility that the voltage insensitivity of urate/alpha -ketoglutarate exchange may arise from coupling with Cl. A coupling to Cl was tested by determining the effect of Cl substitution on urate/alpha -ketoglutarate exchange. To control for possible diffusion potentials due to gradients of Cl or other ions, experiments were conducted in vesicles with no membrane potential (Ko = Ki + Val). Urate uptake was reduced by 75% by Cl substitution with gluconate, isothionate, or chlorate and by 50% by substitution with nitrate (Fig. 7). Figure 8 shows the Cl concentration dependence (EC50 ~20 mM) of increased urate/alpha -ketoglutarate exchange. The Cl dependence of alpha -ketoglutarate-driven urate uptake suggests a mechanism mediating electroneutral exchange of alpha -ketoglutarate for urate and Cl.

A coupling of Cl to the anion exchanger was investigated further by assessing the effect of a Cl gradient on urate uptake. If Cl and urate influx are coupled, then Cl concentration contributes to the driving force on the exchanger and imposition of an inwardly directed Cl gradient should stimulate urate uptake. Figure 9A shows a marked stimulation of urate uptake by Cl to a level above equilibrium. In the absence of alpha -ketoglutarate, Cl did not stimulate urate uptake (Fig. 9B). The Cl-dependent increase in urate uptake was similar in the presence and absence of a Cl gradient (Fig. 9A). This observation is inconsistent with the cotransport of Cl and urate. Studies of urate- and alpha -ketoglutarate gradient-driven 36Cl transport also suggest that Cl transport is not mediated by urate/alpha -ketoglutarate exchange (unpublished observations). Further study is necessary to determine the nature of Cl interaction with this novel anion exchanger and to account for the observed electroneutral exchange.

PAH was assessed as a substrate transported by the urate/alpha -ketoglutarate exchanger. PAH is the prototype substrate for a multispecific organic anion transporter (OAT) mediating active accumulation across the basolateral membrane of the proximal tubule. Table 1 shows a PAH concentration-dependent decrease in urate uptake (IC50 ~1 mM), suggesting a competition between urate and PAH for interaction with the anion exchanger. A stimulation of PAH uptake to levels well above equilibrium was observed when measured in the presence of both alpha -ketoglutarate and a Na gradient but not either alone (Fig. 10). The concentrative accumulation of PAH strongly suggests the presence of a transporter mediating PAH/alpha -ketoglutarate exchange.

Is alpha -ketoglutarate-coupled urate and PAH exchange mediated by the same anion exchange mechanism? This possibility was investigated by assessing the anion exchange coupling of urate and PAH fluxes. An outwardly directed PAH gradient induced a stimulation of urate uptake to levels well above those measured in the absence of a PAH gradient (Fig. 11). The PAH-driven urate uptake suggests a direct coupling of PAH efflux to urate influx, consistent with mediated urate/PAH exchange. This functional mode of anion exchange suggests that urate/alpha -ketoglutarate exchange and PAH/alpha -ketoglutarate exchange may occur by a common mechanism. An outward alpha -ketoglutarate gradient had no effect on alpha -ketoglutarate uptake, indicating an asymmetry of the exchanger and no homoexchange of dicarboxylates (unpublished observations).

If urate and PAH are both substrates for the same alpha -ketoglutarate-coupled anion exchanger, a similar pattern of transport inhibition would be expected for a series of urate and hippurate analogs. Table 2 shows that a similar level of urate and PAH transport inhibition was observed for most of the same chemical analogs. These results are consistent with transport of urate and PAH by a common anion exchanger.

Table 3 shows the effect of anionic drugs and transport inhibitors on alpha -ketoglutarate-driven urate and PAH uptake. Similar magnitudes of urate and PAH transport inhibition were observed for most of the compounds tested. The evidence presented in Tables 2 and 3 strongly suggests that urate and PAH are substrates of the same alpha -ketoglutarate-coupled organic anion exchange mechanism. A kinetic study of alpha -ketoglutarate-driven PAH uptake indicates that PAH interacts with the anion exchanger at a single, saturable site with an apparent Km of 450 µM (unpublished observations). Thus the anion exchanger would appear to have greater affinity for urate (Km ~350 µM) than PAH.

The suppression of urate excretion by pyrazinoate has been attributed to inhibition of urate secretion (25), although more recent evidence suggests that a trans-stimulation of urate reabsorption by pyrazinoate may also account for the reduced excretion of urate (13, 28). If pyrazinoate inhibits proximal tubular urate secretion by decreasing urate uptake across the basolateral membrane, then inhibition of the anion exchanger by pyrazinoate may be anticipated. Table 3 shows that an excess of pyrazinoate had no significant inhibitory effect on urate uptake.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These membrane vesicle studies of urate transport demonstrate a novel, Cl-dependent, alpha -ketoglutarate-coupled anion exchanger as a pathway for urate transport across the basolateral membrane of urate-secreting proximal tubules. The evidence includes 1) alpha -ketoglutarate-driven concentrative urate accumulation, 2) urate-driven concentrative alpha -ketoglutarate accumulation, 3) cis-inhibition of urate transport by alpha -ketoglutarate, and 4) inhibition of urate transport by probenicid. The basolateral membrane location of the anion exchanger is proven by the absence of both alpha -ketoglutarate-driven urate transport and urate-driven alpha -ketoglutarate transport in brush-border membrane vesicles.

The functional properties of the identified anion exchanger were characterized with regard to 1) the kinetics of urate transport, 2) electrogenicity, 3) Cl dependence, 4) drug and transport inhibitor sensitivity, 5) mono- and dicarboxylate substrate specificity, and 6) an assessment of PAH as a transported substrate. The kinetic evidence shows that urate interacts with the anion exchange mechanism at a single saturable site with an apparent Km value of 350 µM, which is similar to urate concentrations in the avian circulation (9). The linearity of the kinetic replot and the single Km value obtained are consistent with transport of one urate molecule.

Assuming a urate-to-alpha -ketoglutarate coupling ratio of 1, a negative charge transfer would accompany substrate exchange and it would be electrogenic. A recent electrophysiological study of a closely related OAT from flounder kidney (fROAT) expressed in frog oocytes demonstrates a PAH-induced depolarization and inward current consistent with mediated charge transfer (3). In contrast, our observations of an apparent voltage insensitivity of urate/alpha -ketoglutarate exchange suggest an electroneutral anion exchange. Two possibilities were considered to account for this observation: the coupled exchange of K and ketoglutarate for urate and the coupled exchange of Cl and urate for ketoglutarate. Coupling of K to the anion exchanger was excluded because a K gradient did not stimulate urate/alpha -ketoglutarate exchange. Coupling of Cl to the anion exchanger was suggested by the marked reduction in urate transport in the absence of Cl. However, a Cl gradient did not stimulate urate/alpha -ketoglutarate exchange and a coupled 36Cl flux was not observed (unpublished observations). Although the evidence shows that urate/alpha -ketoglutarate exchange is Cl dependent, the nature of Cl interaction and the observed electroneutrality are unexplained. An interaction of Cl with the coupled exchange of PAH for alpha -ketoglutarate has been described from rat (15) and bovine (30) basolateral membrane vesicle studies and more recently from heterologous functional expression of human (hOAT1; Refs. 14, 24) and flounder (fROAT; Ref. 3) kidney OAT. Cl transport was not assessed in these studies. The observed Cl dependence of the anion exchanger is a unique functional property, which may characterize a subset of OATs with related structure and function.

The substrate specificity of the anion exchanger was assessed to identify which organic anions are likely to drive anion exchange in vivo and to broadly characterize the chemical determinants necessary for substrate interaction with, and possible transport by, the anion exchanger. Of the di- and tricarboxylates tested, only alpha -ketoglutarate and its nonphysiological chemical analog glutarate were observed to drive anion exchange. Given the similar spatial configuration of charge and stereochemistry of the tested substrates, this evidence indicates a remarkable specificity for these two dicarboxylates. Perhaps only the high intracellular concentration of alpha -ketoglutarate in the proximal tubule serves to drive anion exchange in vivo (23). This specificity for dicarboxylates is also a functional property of related organic anion exchangers mediating PAH transport, and it may characterize a subset of OATs with related structure and function (5).

In contrast, the anion exchanger had a much broader monocarboxylate substrate specificity. The general features of monocarboxylate substrate specificity characterizing this and related organic anion exchangers are consistent with the physical-chemical determinants identified by Ullrich (35): 1) one partial or full negative charge, 2) a hydrophobic moiety, and 3) the ability to form multiple hydrogen bonds.

PAH is the prototype substrate used to study multispecific OATs. Urate and PAH are apparently substrates for the same avian organic anion exchanger. The evidence includes 1) cis-inhibition of urate transport by PAH, 2) alpha -ketoglutarate-driven, concentrative accumulation of PAH, 3) PAH-driven, concentrative accumulation of urate, 4) cis-inhibition of urate and PAH transport by the same analogs, and 5) cis-inhibition of urate and PAH transport by the same drugs and transport inhibitors. The apparent Km values for PAH (450 µM) and urate (350 µM) indicate a modest preference for urate over PAH. Basolateral membrane vesicle studies of related OATs indicate a reversed order of affinity for these two substrates in urate-reabsorbing species (23).

Recently, the genomic sequences of a family of related OATs have been identified, including OAT1 from flounder, rat, and human (6). The functional properties of the OAT1-encoded protein have been characterized by heterologous expression and demonstrate a transport mechanism mediating the coupled exchange of PAH for alpha -ketoglutarate. The present evidence suggests that urate is a poor substrate for transport by OAT1 at physiological concentrations, and OAT1 is unlikely to be the transport pathway mediating active uptake of urate across the basolateral membrane of urate-secreting proximal tubules. Although PAH transport mediated by flounder OAT1 was modestly inhibited by urate at a high concentration (1 mM), transport of urate (50 µM) was not observed (38). Similarly, PAH transport (1 µM) mediated by human OAT1 was not decreased when measured with urate (100 µM), and transport of urate (45 µM) was not observed (24). However, 2 mM urate was observed to cis-inhibit human OAT1-mediated PAH transport (2 µM) (14). Whereas no decrease in PAH transport (50 µM) mediated by rat OAT1 was measured with 1 mM urate (34), a decrease in PAH transport (2 µM) was observed when measured in 2 mM urate (31). Urate transport by rat OAT1 was observed when measured at 100 µM urate, which is well above the levels (40 µM) of urate in the rat circulation (27). The increase in intracellular urate concentration (7 µM) estimated from urate uptake at 100 µM does not indicate active transport of urate by rat OAT1-expressing oocytes. (31).

The suppression of urate excretion induced by pyrazinoate is commonly attributed to inhibition of urate secretion (25). More recent membrane vesicle studies suggest that a pyrazinoate-induced trans-stimulation of urate reabsorption may also account for the reduced excretion of urate (13, 28). If pyrazinoate inhibits proximal tubule urate secretion by decreasing urate uptake across the basolateral membrane, pyrazinoate may inhibit urate transport mediated by the avian anion exchanger. Our results show that pyrazinoate does not inhibit urate transport by the avian anion exchanger. Assuming this transport pathway mediates the active step in transcellular urate secretion, this evidence would suggest that the increased excretion of urate induced by pyrazinoate does not result from a decrease in secretion.

In summary, membrane vesicle studies demonstrate the presence of a novel, Cl-dependent, alpha -ketoglutarate-coupled urate transport mechanism in the basolateral membrane of urate-secreting avian proximal tubules. This anion exchange mechanism can account for the active accumulation of urate from blood as the initial step in urate secretion across proximal tubule cells.


    ACKNOWLEDGEMENTS

The excellent technical assistance of Terri Novak is gratefully acknowledged. Dr. Philip Dunham is gratefully acknowledged for his many suggestions to improve the manuscript. The contributions of Francoise Roch-Ramel to the study of renal proximal urate transport are acknowledged.


    FOOTNOTES

This work was supported by the New York State Affiliate of the American Heart Association and the National Kidney Foundation of Central New York.

Address for reprint requests and other correspondence: S. M. Grassl, Dept. of Pharmacology, SUNY Upstate Medical Univ., 766 Irving Ave, Syracuse, NY 13210 (grassls{at}upstate.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.

June 13, 2002;10.1152/ajpcell.00379.2001

Received 7 August 2001; accepted in final form 6 June 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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