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
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ABSTRACT |
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
-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
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INTRODUCTION |
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
-ketoglutarate was identified by observing urate- and
-ketoglutarate-driven
-ketoglutarate and urate uptake in membrane vesicles. Urate transport was dependent on
urate and
-ketoglutarate concentration, specific for
-ketoglutarate, Cl dependent, inhibited by probenicid, and membrane
potential insensitive. These findings suggest a Cl-dependent,
-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.
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METHODS |
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
-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,
[
-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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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 -ketoglutarate influx.
Membrane vesicles were equilibrated with 125 mM KCl, 10 mM HEPES-TMA,
pH 7.5. Uptake of -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 -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
-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.
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Chemicals.
[8-14C]urate (53 mCi/mmol) and
[
-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,
-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.
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RESULTS |
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
-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
-ketoglutarate gradient at low
extravesicular
-ketoglutarate. Imposition of an inward Na gradient
induced concentrative accumulation of
-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/
-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/
-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
-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.
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DISCUSSION |
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/
-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
-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
-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
-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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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.
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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.
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