Facilitated diffusion of urate in avian brush-border 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 mediating transcellular secretion of urate across the proximal tubule were investigated in brush-border membrane vesicles (BBMV) isolated from avian kidney. An inside-positive K diffusion potential induced a conductive uptake of urate to levels exceeding equilibrium. Protonophore-induced dissipation of membrane potential significantly reduced voltage-driven urate uptake. Conductive uptake of urate was inhibitor sensitive, substrate specific, and a saturable function of urate concentration. Urate uptake was trans-stimulated by urate and cis-inhibited by p-aminohippurate (PAH). Conductive uptake of PAH was cis-inhibited by urate. Urate uptake was unaffected by an outward alpha -ketoglutarate gradient. In the absence of a membrane potential, urate uptake was similar in the presence and absence of an imposed inside-alkaline pH gradient or an outward Cl gradient. These observations suggest a uniporter-mediated facilitated diffusion of urate as a pathway for passive efflux across the brush border membrane of urate-secreting proximal tubule cells.

organic anion secretion; renal proximal tubule; urate transporter


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TRANSCELLULAR URATE SECRETION across the avian proximal tubule arises from transporter-mediated urate uptake and efflux at the basolateral and apical membranes, respectively. In a companion paper (Ref. 5a; this issue), a basolateral membrane anion exchanger coupling fluxes of urate and alpha -ketoglutarate was identified by observing urate- and alpha -ketoglutarate-driven alpha -ketoglutarate and urate uptake in membrane vesicles. Urate transport was dependent on urate and alpha -ketoglutarate concentration, specific for alpha -ketoglutarate, Cl dependent, inhibited by probenicid, and membrane potential insensitive. These findings suggest a Cl-dependent, alpha -ketoglutarate-coupled anion exchanger as a transport pathway for active uptake of urate across the basolateral membrane of urate-secreting proximal tubule cells.

In urate-secreting species, urate transport pathways across the apical membrane have been investigated in membrane vesicles isolated from pig (12, 26, 27) and rabbit (11, 17) kidney. A facilitated diffusion of urate has been functionally identified and characterized in the brush-border membrane of these species. This is in contrast to an anion exchanger observed to mediate urate transport in the brush-border membrane of urate-reabsorbing species (8-10). On the basis of these observations, the presence of a brush-border membrane urate/anion exchanger has been proposed to distinguish functionally between urate-reabsorbing and urate-secreting species (8).

Because of its capacity for urate secretion, we have used the avian proximal tubule to study both brush-border and basolateral membrane urate transport. Here we report studies on the mechanism of urate transport across the avian brush-border membrane. The evidence obtained identifies a mechanism mediating facilitated diffusion or uniport of urate as an apical membrane transport pathway in urate-secreting proximal tubules.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Membrane preparation. Brush-border membrane vesicles were isolated from turkey kidney by divalent cation aggregation and differential centrifugation as previously described with minor modifications (4, 13). 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 in a SS-34 Sorvall rotor at 1,100 g for 10 min (brake off), and the resulting supernatant was diluted 2:1 for further centrifugation 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 in sucrose buffer without EDTA and resuspended by completing three strokes with a 250-ml Lurex Teflon-glass homogenizer. MgCl2 was added to the membrane suspension to a concentration of 12 mM and was incubated for 15 min on ice. The membrane suspension was centrifuged at 3,000 g for 15 min 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 the cycle of divalent cation aggregation and centrifugation was repeated. The brush-border membrane pellet was resuspended in sucrose buffer, aliquoted, and stored frozen (-70°C) for use within 4 weeks. The brush-border membrane marker enzyme activities, alkaline phosphatase and delta -glutamyltranspeptidase, were enriched 10- to 20-fold compared with homogenate and represented 3-5% of the homogenate enzyme activity. The specific activity of Na-glucose cotransport, a brush-border membrane marker transport activity, was comparable to levels measured in nonavian species. Membrane protein was determined from the sodium dodecyl sulfate Lowry assay with bovine serum albumin as standard (19).

Isotope flux measurements. Frozen aliquots of membrane vesicles were thawed at room temperature and diluted for centrifugation at 12,000 g for 30 min in buffers designated for each experiment: 125 mM TMA gluconate, 25 mM HEPES-TMA, pH 7.5; 100 mM TMA gluconate, 86 mM HEPES-TMA, pH 7.5; and 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. 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 and 2 and Figs. 1-10. The membrane suspension was preincubated at room temperature for 120 min 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 and 2 and Figs. 1-10. The intravesicular uptake of [14C]urate, [alpha -3H]ketoglutarate, and 3H-labeled p-aminohippurate (PAH) was assayed at least in triplicate for each time point determination by a rapid filtration technique described previously (6). Briefly, 10 µl of membrane vesicles were rapidly mixed by vortexing with 40 µl of isotope buffer and incubated for a designated time interval measured by stopwatch. Isotope uptake was quenched by rapid dilution with 3 ml of ice-cold 2 mM probenicid, 25 mM HEPES-TMA, pH 7.5, and 125 mM K gluconate, 125 mM NaCl, or 205 mM TMA gluconate. The diluted membrane suspension was passed through a 0.65-µm Millipore filter (DAWP) and washed twice with additional volumes of 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) scintillation cocktail and counted by spectroscopy. The timed uptake values obtained were corrected for 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 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 chemical analogs on conductive PAH and urate influx


                              
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Table 2.   Effect of drugs and transport inhibitors on conductive PAH and urate influx



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Fig. 1.   Effect of membrane potential on urate influx. Membrane vesicles were equilibrated with 25 mM HEPES-tetramethylammonium (TMA), pH 7.5, and 125 mM TMA gluconate or 62.5 mM TMA gluconate-62.5 mM K gluconate. Uptake of urate (300 µM) occurred from extravesicular solutions containing 25 mM HEPES-TMA, pH 7.5, and 100 mM K gluconate-25 mM TMA gluconate or 62.5 mM TMA gluconate-62.5 mM K gluconate. Membranes were preincubated with or without 0.25 mg/ml valinomycin (Val) for a minimum of 30 min. A representative experiment of 3 independent observations, each performed with a different membrane preparation, is shown. Subscript o indicates extravesicular concentration; subscript i indicates intravesicular concentration.



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Fig. 2.   Effect of FCCP on conductive urate influx. Membrane vesicles were equilibrated with solutions described in Fig. 1. The 10-s uptake of urate (300 µM) occurred from extravesicular solutions described in Fig. 1 and containing 5 µM FCCP or an equivalent volume of ethanol (0.5%). Membranes were preincubated with or without 0.25 mg/ml valinomycin for a minimum of 30 min. Urate influx is shown as % of urate uptake measured in the absence of membrane potential (Ko = Ki + valinomycin). Means + SE of 4 independent observations are shown.



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Fig. 3.   Effect of urate concentration gradient on urate influx. Membrane vesicles were equilibrated with (2 mM) and without urate and with 125 mM KCl and 10 mM HEPES-TMA, pH 7.5. Uptake of urate (50 µM) occurred from extravesicular solutions containing 125 mM KCl and 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. 4.   Kinetics of conductive urate influx. Membrane vesicles were equilibrated with solutions described in Fig. 1. The 10-s uptake of urate occurred from extravesicular solutions described in Fig. 1 and containing the urate concentrations indicated. Membrane vesicles were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. At each urate concentration, conductive urate influx was determined from the difference in urate uptake measured in the presence (Ko > Ki) and absence (Ko = Ki) of membrane potential. Means ± SE of 3 experiments, each performed with a different membrane preparation, are shown.



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Fig. 5.   Effect of membrane potential on p-aminohippurate (PAH) index. Membrane vesicles were equilibrated with solutions described in Fig. 1. Uptake of PAH (50 µM) occurred from extravesicular solutions described in Fig. 1. Membrane vesicles were preincubated with or without 0.25 mg/ml valinomycin for a minimum of 30 min. A representative experiment of 4 independent observations is shown.



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Fig. 6.   Effect of PAH on urate influx. Membrane vesicles were equilibrated with solutions described in Fig. 3. Uptake of urate (50 µM) occurred from extravesicular solutions described in Fig. 3 and in the presence (5 mM) or absence (gluconate) of PAH. Membranes were preincubated with 0.25 mg/ml valinomycin for a minimum of 30 min. A representative experiment of 4 independent observations is shown.



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Fig. 7.   Effect of Na gradient on alpha -ketoglutarate influx. Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. Uptake of alpha -ketoglurate (5 µM) occurred from extravesicular solutions containing 100 mM NaCl or TMA Cl, 25 mM KCl, and 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. 8.   Effect of alpha -ketoglutarate on urate influx. Membrane vesicles were equilibrated as described in Fig. 7. Uptake of urate (300 µM) occurred in the presence (5 µM) and absence of alpha -ketoglutarate from extravesicular solutions containing 100 mM NaCl, 25 mM KCl, and 10 mM HEPES-TMA, pH 7.5. Membranes were preequilibrated 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. 9.   Effect of pH gradient on urate influx. Membrane vesicles were equilibrated with 110 mM TMA gluconate, 57.3 mM K gluconate, 45.3 mM HEPES, 25 mM TMA (OH) and at pH 6 with 52 mM 2-(N-morpholino)ethanesulfonic acid (MES) or at pH 7.5 with 52 mM mannitol. Uptake of urate (30 µM) occurred from extravesicular solutions containing 110 mM TMA gluconate and 57.3 mM K gluconate with 52 mM MES, 45.3 mM HEPES, 25 mM TMA (OH) (pHo 6/pHi 6), 47 mM MES, 42 mM mannitol, 9 mM HEPES, 25 mM TMA (OH) (pHo 6/pHi 7.5), or 52 mM mannitol, 45.3 HEPES, 25 mM TMA (OH) (pHo 7.5/pHi 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. 10.   Effect of Cl gradient on urate influx. Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-TMA, pH 7.5. Uptake of urate (300 µM) occurred from extravesicular solutions containing 125 mM KCl or 112.5 mM K gluconate-12.5 mM KCl, 10 mM HEPES-TMA, pH 7.5. Membranes were preincubated with and without 0.25 mg/ml valinomycin for a minimum of 30 min. A representative experiment from 3 independent observations is shown.

Chemicals. [8-14C]urate (53 mCi/mmol) and [alpha -3H(G)]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). FCCP, alpha -ketoglutarate, probenicid, TMA, gluconate, and other compounds shown in Tables 1 and 2 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 and FCCP dissolved in ethanol (95%) were added to membrane suspensions in 1:100 and 1:200 dilutions, respectively. 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

Facilitated diffusion of urate. Pathways for urate transport across the apical membrane of avian proximal tubules were investigated by testing for facilitated diffusion. If a facilitated diffusion mechanism mediates conductive uptake of urate, an inside-positive voltage difference will drive urate accumulation. An inwardly directed K gradient accelerated urate uptake in vesicles treated with and without valinomycin but to levels above equilibrium only in valinomycin-treated vesicles (Fig. 1). Therefore, an inside-positive voltage difference promotes urate uptake via a conductive uptake pathway in brush-border membrane. The nature of this conductive pathway was investigated by testing the effect of the protonophore FCCP on urate uptake. The increase in urate uptake induced by an inside-positive potential may have been secondary to the formation of a pH gradient, inside alkaline, driving urate accumulation in exchange for OH. These transport mechanisms may be distinguished by testing the effect of FCCP on voltage-driven urate uptake. Charge-compensating movements of protons across FCCP-absorbed membranes will enhance formation of an inside-alkaline pH gradient while dissipating transmembrane voltage. As shown in Fig. 2, a marked reduction in urate uptake was measured in vesicles with FCCP, suggesting that urate uptake does not result from a pH gradient-driven anion exchanger. Furthermore, an inside-alkaline pH gradient did not stimulate urate uptake (see Fig. 9). These observations indicate that voltage-driven urate uptake occurs by a conductive mechanism possibly mediated by facilitated diffusion.

The observed stimulation of urate uptake by an inside-positive voltage is not evidence for a mediated transport process. A simple conductive pathway could account for voltage-driven urate uptake. To characterize further the nature of urate transport, we tested the effect of trans-urate on urate uptake. Membrane vesicles were preloaded with and without 2 mM urate and diluted into a buffer containing [14C]urate. As shown in Fig. 3, both the rate and magnitude of urate uptake were increased in vesicles with a large outward urate gradient compared with vesicles initially devoid of urate. The trans-stimulation of urate uptake by intravesicular urate, or counterflow (24), is a functional property of carrier-mediated transport (5) and is consistent with facilitated diffusion of urate. Basolateral membrane contamination could not account for the facilitated diffusion of urate because conductive urate uptake was absent when assayed in basolateral membrane vesicles (unpublished observations).

Functional properties of facilitated diffusion mechanism. The kinetic properties of conductive urate uptake were estimated from initial rate determinations of urate uptake measured at 10 s and at the urate concentrations shown in Fig. 4. Consistent with the presence of a mediated transport process, the rate of conductive urate uptake increased as a hyperbolic function of urate concentration. A Hanes-Woolf replot of these data (Fig. 4) is linear, consistent with interaction of urate at a single site with an apparent Km of 300 µM.

PAH was tested as an alternate substrate for the urate transporter. PAH is the prototype substrate of multispecific organic anion transport mechanisms in the apical and basolateral membranes of proximal tubules. PAH influx was accelerated in valinomycin-treated membranes by an inwardly directed K gradient compared with uptake with no K gradient, suggesting a conductive uptake pathway for PAH (Fig. 5). Extravesicular urate inhibited conductive PAH uptake (IC50 ~300 µM), consistent with a common uptake pathway (unpublished observations). As shown in Fig. 6, urate uptake stimulated by an outward urate gradient was also inhibited by extravesicular PAH. The inhibition by PAH was measured in valinomycin-treated membranes with equal transmembrane K. Therefore, inhibition by PAH is not by dissipation of membrane potential and the presence of parallel conductive uptake pathways. These results further support the mediated conductive uptake of urate and PAH by a common mechanism.

If urate and PAH are substrates for the same facilitated diffusion mechanism, a quantitatively similar profile of urate and PAH transport inhibition would be expected for the same analogs, transport inhibitors, and drugs. The effect of urate and hippurate analogs on conductive urate and PAH uptake is shown in Table 1. In most instances, conductive urate and PAH transport were reduced to the same level in the presence of the same analogs. The substrate specificity was investigated further with the anionic drugs and transport inhibitors shown in Table 2. Once again, in most instances, conductive urate and PAH uptake were reduced to similar levels by the same test compounds, suggesting a common transport mechanism.

We have identified an alpha -ketoglutarate-coupled anion exchange mechanism as a pathway for urate transport across the basolateral membrane of avian proximal tubules (5a). If urate secretion arises from an active accumulation across the basolateral membrane and a passive efflux across the brush-border membrane, the anion exchange mechanism would not be expected in the brush-border membrane. First, we tested for a Na-dicarboxylate cotransporter for the purpose of generating a large outwardly directed alpha -ketoglutarate gradient at low extravesicular alpha -ketoglutarate. Imposition of an inward Na gradient induced concentrative accumulation of alpha -ketoglutarate to levels 16-fold above equilibrium (Fig. 7). This suggests a Na-dicarboxylate cotransport mechanism in avian renal brush-border membrane, consistent with previous observations of its presence in the brush-border membrane of all species examined (18). Next we tested for urate/alpha -ketoglutarate exchange by determining the dependence of urate uptake on the Na gradient and ketoglutarate. In contrast to basolateral membrane vesicles (5a), Fig. 8 shows no such dependence in brush-border membrane vesicles. This evidence suggests the absence of urate/alpha -ketoglutarate exchange in the avian brush-border membrane.

In humans and other urate-reabsorbing species, brush-border membrane vesicle studies suggest that the active accumulation of urate across the proximal tubule apical membrane is mediated by a probenicid-sensitive anion exchange mechanism (2, 7-10, 22, 23). The active uptake of urate appears to be coupled to efflux of hydroxyl ions, bicarbonate, lactate, hippurate, and nicotinate but not alpha -ketoglutarate or other divalent organic anions. If the avian proximal tubule mediates transcellular urate secretion, but not reabsorption, a similar anion exchanger would not be expected in the brush-border membrane. As shown in Fig. 9, no increase in urate uptake was induced by an inside-alkaline pH gradient, indicating the absence of a hydroxyl-coupled anion exchange mechanism in avian brush-border membranes.

A previous membrane vesicle study of urate transport mechanisms in the avian proximal tubule suggested the presence of a Cl-coupled anion exchange mechanism as a pathway for efflux of intracellular urate across the brush-border membrane (13). We tested for Cl gradient-induced urate uptake in the avian brush-border membrane. An outward Cl gradient stimulated urate uptake to levels exceeding equilibrium in vesicles not treated with valinomycin (Fig. 10). There was no stimulation of urate uptake in valinomycin-treated vesicles. These observations suggest that the nature of coupling of urate uptake to the Cl gradient is indirect, a consequence of an inside-positive, Cl gradient-induced diffusion potential driving urate uptake via the conductive uptake pathway described above.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

These membrane vesicle studies of urate transport demonstrate the presence of a facilitated diffusion mechanism or uniporter as a pathway for urate transport across the apical membrane of urate-secreting proximal tubules. The evidence includes 1) voltage-driven urate accumulation and 2) urate trans-stimulation of urate transport, demonstrating counterflow. The brush-border membrane location of the urate uniporter is verified by the absence of conductive urate transport in basolateral membrane vesicles. Facilitated diffusion of urate in the avian brush border is consistent with previous demonstrations of mediated, conductive urate transport in other urate-secreting species (11, 12, 17, 26, 27).

The facilitated diffusion mechanism was characterized with regard to 1) transport kinetics, 2) monocarboxylate substrate specificity, 3) drug and transport inhibitor sensitivity, and 4) PAH transport. The kinetic evidence suggests that urate associates with the uniporter at a single saturable site, with an apparent affinity similar to its association with the basolateral membrane urate/alpha -ketoglutarate exchange mechanism (see Ref. 5a). A single Km for the uniporter further suggests uniporter-mediated transfer of one urate molecule across the membrane. A suggestion of a similar apical and basolateral urate transport capacity is also indicated by comparable maximal rates of urate transport mediated by the uniporter and anion exchanger, respectively.

The evidence suggests that urate and PAH are substrates for the same uniporter. 1) Extravesicular urate inhibited PAH transport. 2) PAH inhibited urate counterflow. 3) A series of analogs caused similar inhibition of urate and PAH transport. 4) A series of drugs and transport inhibitors caused similar inhibition of urate and PAH transport. Interestingly, conductive urate transport in brush-border membranes and alpha -ketoglutarate-driven urate transport in basolateral membranes were reduced to the lowest levels in the presence of the same test compounds (see Ref. 5a). This suggests that a common substrate specificity may link the uniporter and anion exchanger in mediating transcellular substrate transport. The observed similarity in substrate specificity also includes an apparent pyrazinoate insensitivity of both the uniporter and the anion exchanger, which further suggests that pyrazinoate suppression of urate excretion is unlikely to result from inhibition of either transporter.

We have identified and functionally characterized an alpha -ketoglutarate-coupled anion exchanger as a pathway for urate transport across the basolateral membrane of avian proximal tubules (see Ref. 5a). Transcellular urate secretion is a consequence of urate transporters mediating active accumulation across the basolateral membrane and passive efflux across the brush-border membrane. The anion exchanger would not be expected in the brush-border membrane because it would mediate cellular influx of urate. Indeed, there was no alpha -ketoglutarate-driven urate transport in brush-border membranes. This observation also verifies an absence of significant basolateral membrane contamination in the brush-border membrane preparation.

In humans and other urate-reabsorbing species, the active accumulation of urate across the brush-border membrane is accomplished by a probenicid-sensitive anion exchange mechanism coupling urate uptake to gradients of hydroxyl ions, bicarbonate, lactate, hippurate, and nicotinate (2, 7-10, 21-23). Because the avian proximal tubule mediates transcellular secretion, a similar monovalent anion exchanger mechanism is not expected in the brush-border membrane. Indeed, imposition of a hydroxyl ion gradient did not increase brush-border membrane urate transport. This observation is furthermore consistent with the absence of a reabsorptive urate flux across the avian proximal tubule (3). The antitubercular drug metabolite pyrazinoate is also a proven substrate transported by the brush-border membrane anion exchanger in urate-reabsorbing species (7). When assessed in avian brush-border membranes, a pyrazinoate gradient did not increase urate transport (unpublished observations), which further indicates the absence of an avian homolog of the anion exchanger identified in urate-reabsorbing species. This evidence supports the presence or absence of a brush-border membrane urate/anion exchanger as a criterion functionally distinguishing urate-reabsorbing from urate-secreting species (8).

A previous membrane vesicle study of urate transporters in the avian proximal tubule suggested that a Cl-coupled anion exchanger mediates urate efflux across the brush-border membrane (13). This is inconsistent with previous brush border membrane vesicle studies of urate transport in urate-secreting species, which suggest an absence of an anion exchanger (12, 17, 26). The possible indirect coupling of increased urate transport to a Cl gradient via generation of an inside-positive diffusion potential was not accounted for in the previous avian brush-border membrane studies. We reexamined the presence of a urate/Cl exchanger in the avian brush-border membrane by assessing the nature of Cl coupling to urate transport. We found no evidence for direct exchange coupling of urate and Cl (Fig. 10). Our finding of an indirect, electrostatic coupling of urate transport to an imposed Cl gradient further verifies the presence of conductive urate uniport across the avian brush-border membrane.

Recently, the rat (14, 15) and human (16) homologs of a genomic sequence encoding a putative urate transporter (UAT) have been identified based on function of the purified gene product in artificial lipid bilayers. The reconstituted protein was observed to mediate electrogenic urate transport, and the expressed gene product is localized to the plasma membrane with at least one extracellular domain (20). Remarkably, the deduced amino acid sequence of UAT is virtually identical to that of the galactin family of secreted galactoside-binding proteins (1). The difficulty in reconciling these two significantly different functions by essentially the same gene product is compounded by the apparent inability to heterologously express UAT transport function (20). Stable expression of a homologous protein, Gal9p, in HEK 293 cells conferred a potential-sensitive urate transport, but there was no trans-stimulation or substrate saturation (25). Thus there is insufficient evidence to suggest that the UAT-encoded protein is the urate uniporter identified in the avian brush-border membrane.

In summary, membrane vesicle studies were performed to identify and functionally characterize membrane transport pathways mediating efflux of intracellular urate across the apical membrane of urate-secreting avian proximal tubule cells. A conductive uptake pathway for urate mediated by facilitated diffusion was functionally identified and characterized. No evidence for the presence of a brush-border membrane anion exchange mechanism was obtained. Figure 11 summarizes the mechanisms proposed for urate secretion across the avian proximal tubule and their relation to Na-dicarboxylate cotransport. Thus proximal tubular urate secretion is a consequence of coordinate active accumulation of dicarboxylates across both apical and basolateral membrane, active accumulation of urate across the basolateral membrane, and passive efflux of urate across the apical membrane.


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Fig. 11.   Cellular model of transcellular urate secretion.


    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 (E-mail: 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.00380.2001

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


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 283(4):C1155-C1162
0363-6143/02 $5.00 Copyright © 2002 the American Physiological Society




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