Urate/
-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
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ABSTRACT |
Membrane transport pathways for
transcellular secretion of urate across the proximal tubule were
investigated in avian kidney. The presence of coupled
urate/
-ketoglutarate exchange was investigated in basolateral
membrane vesicles (BLMV) by [14C]urate and
[
-3H]ketoglutarate flux measurements. An inward Na
gradient induced accumulation of
-ketoglutarate of sufficient
magnitude to suggest a Na-dicarboxylate cotransporter. An inward Na
gradient also induced concentrative accumulation of urate in the
presence of
-ketoglutarate but not in its absence, suggesting
urate/
-ketoglutarate exchange.
-Ketoglutarate-dependent
stimulation of urate uptake was not observed in brush-border membrane
vesicles. An outward urate gradient induced concentrative accumulation
of
-ketoglutarate.
-Ketoglutarate-coupled urate uptake was
specific for
-ketoglutarate, Cl dependent, and insensitive to
membrane potential.
-Ketoglutarate-coupled urate uptake was
inhibited by increasing p-aminohippurate (PAH)
concentrations, and
-ketoglutarate-coupled PAH uptake was observed.
-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,
-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
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INTRODUCTION |
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
-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,
-ketoglutarate-driven PAH uptake but not urate uptake was observed, suggesting that urate is not a substrate for the
-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
-ketoglutarate to influx of extracellular urate across the
basolateral membrane (37). These observations suggest that
two independent
-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
-ketoglutarate-coupled anion
exchange mechanism in the basolateral membrane.
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METHODS |
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 (
-ketoglutarate/urate exchange). Basolateral membranes were
collected from membrane fractions where Na-glucose cotransport was
minimal or absent and where
-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,
[
-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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Fig. 1.
Effect of Na concentration gradient on -ketoglutarate influx.
Membrane vesicles were equilibrated with 125 mM KCl, 10 mM
HEPES-tetramethylammonium (TMA), pH 7.5. Uptake of -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 -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 -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.
-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 -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 -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 -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 -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 -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 -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
-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/ -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 -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
-ketoglutarate gradient-dependent difference in urate uptake
(pmol/mg) measured in vesicles preloaded with and without
-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/ -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 -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
-ketoglutarate-dependent difference in urate uptake measured in the
presence and absence of -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 -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 -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
-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.
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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). Percoll,
-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.
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RESULTS |
Functional identification of urate/
-ketoglutarate exchange.
Transport pathways mediating active accumulation of urate across the
basolateral membrane of avian proximal tubules were investigated by
testing for urate/
-ketoglutarate exchange in membrane vesicles. Evidence for a Na-dicarboxylate cotransporter and the generation of an
outwardly directed
-ketoglutarate gradient was obtained from
observing the effect of Na on
-ketoglutarate uptake. Figure 1 shows such evidence: a Na gradient
induced a transient
-ketoglutarate accumulation 30-fold above
equilibrium. In the absence of Na,
-ketoglutarate accumulation was
well below equilibrium. An indirect electrostatic coupling of
-ketoglutarate and Na uptake via parallel conductive pathways is
unlikely because concentrative accumulation of
-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
-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
-ketoglutarate concentration gradient, we
assessed the presence of a basolateral membrane urate/
-ketoglutarate exchange mechanism by observing the dependence of urate uptake on the
Na concentration gradient and on
-ketoglutarate. Figure 2 shows that urate uptake was low in the
absence of a Na gradient and the presence of
-ketoglutarate and
slowly approached equilibrium. In contrast, there was a marked
stimulation of urate uptake in the presence of both
-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
-ketoglutarate suggests the presence of an anion exchange mechanism
mediating direct coupling of
-ketoglutarate efflux to urate influx.
An indirect electrostatic coupling of
-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
-ketoglutarate-dependent urate
uptake in brush border membrane vesicles despite a comparable magnitude
of Na-dicarboxylate cotransport (unpublished observations).
The urate/
-ketoglutarate exchange mechanism was investigated further
by assessing the
-ketoglutarate concentration dependence of urate
uptake (Fig. 3). A biphasic relation
between increasing extravesicular
-ketoglutarate concentration and
urate uptake is observed where cis-inhibition is
indicated at higher
-ketoglutarate concentrations. The presence of
urate/
-ketoglutarate exchange was further suggested by the
concentration-dependent inhibition (IC50 ~250 µM) of Na
gradient- and
-ketoglutarate-dependent urate uptake induced by
probenicid, an organic anion transport inhibitor (unpublished observations).
Urate/
-ketoglutarate exchange should also mediate accumulation of
-ketoglutarate driven by a urate gradient. The uptake of
-ketoglutarate was measured in vesicles in the presence and absence
of an outwardly directed urate gradient (Fig.
4). Imposition of a urate gradient
induced
-ketoglutarate uptake to levels threefold above equilibrium.
An indirect coupling of conductive
-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
-ketoglutarate uptake, consistent
with a basolateral membrane localization of urate/
-ketoglutarate exchange.
Functional properties of urate/
-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
-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
-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
-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
-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/
-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/
-ketoglutarate exchange to membrane potential
indicates an electroneutral exchange process. This may be either the
coupled exchange of a monovalent cation and
-ketoglutarate for urate or the exchange of a monovalent anion and urate for
-ketoglutarate. The kinetics of urate uptake is not consistent with the exchange of two
urate molecules for
-ketoglutarate (Fig. 5). When
urate/
-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
-ketoglutarate for urate.
Previous studies of a related transport mechanism,
PAH/
-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/
-ketoglutarate exchange may arise from coupling with Cl. A
coupling to Cl was tested by determining the effect of Cl substitution
on urate/
-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/
-ketoglutarate exchange. The Cl dependence of
-ketoglutarate-driven urate uptake suggests a mechanism mediating
electroneutral exchange of
-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
-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
-ketoglutarate gradient-driven 36Cl transport also suggest that Cl transport is not
mediated by urate/
-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/
-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
-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/
-ketoglutarate exchange.
Is
-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/
-ketoglutarate exchange and
PAH/
-ketoglutarate exchange may occur by a common mechanism. An
outward
-ketoglutarate gradient had no effect on
-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
-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
-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
-ketoglutarate-coupled organic anion exchange mechanism. A
kinetic study of
-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 |
These membrane vesicle studies of urate transport
demonstrate a novel, Cl-dependent,
-ketoglutarate-coupled anion
exchanger as a pathway for urate transport across the basolateral
membrane of urate-secreting proximal tubules. The evidence includes
1)
-ketoglutarate-driven concentrative urate
accumulation, 2) urate-driven concentrative
-ketoglutarate accumulation, 3) cis-inhibition of urate transport by
-ketoglutarate, and 4) inhibition
of urate transport by probenicid. The basolateral membrane location of the anion exchanger is proven by the absence of both
-ketoglutarate-driven urate transport and urate-driven
-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-
-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/
-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/
-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/
-ketoglutarate exchange and a coupled 36Cl flux
was not observed (unpublished observations). Although the evidence
shows that urate/
-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
-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
-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
-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)
-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
-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,
-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.
 |
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