Transepithelial urate transport by avian renal proximal tubule epithelium in primary culture
1 Department of Physiology and Neurobiology, University of Connecticut,
Storrs, Connecticut 06269, USA
2 Department of Internal Medicine, Yale University School of Medicine, New
Haven, CT 06520, USA
3 Department of Physiology, University of Arizona, College of Medicine,
Tucson, AZ 85724, USA
* Author for correspondence (e-mail: larry.renfro{at}uconn.edu)
Accepted 9 September 2005
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Summary |
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Key words: renal secretion, organic anion, MK-571, para-aminohippuric acid, uric acid, chicken
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Introduction |
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In birds and the urate-secreting mammals an organic anion transport system
has been implicated in basolateral membrane (BLM) urate uptake, which is
Na+- and -ketoglutarate (
-KG)-dependent
(Brokl et al., 1994
;
Dantzler, 1969
;
Werner and Roch-Ramel, 1991
).
The mammalian PAH/
-KG exchangers in the BLM have been identified as
OAT-type (SLC22A; major facilitator family of organic anion transporters;
Sweet et al., 1997
) with urate
a demonstrated substrate (Uwai et al.,
1998
). Human OAT3 has been strongly implicated in urate secretion
by expression studies in Xenopus oocytes
(Bakhiya et al., 2003
). Both
OAT1 and OAT3 transport urate, and OAT3 may have the higher affinity
(Bakhiya et al., 2003
). Some
discrimination in the two transporters has been achieved with shared
transported substrates for which affinities vary (see
Wright and Dantzler,
2004
).
Among the vertebrates, the modalities of urate transport across the apical
membranes of the proximal tubule epithelium include electroneutral anion
exchange (Enomoto et al.,
2002; Guggino et al.,
1983
), electrogenic urate transport
(Grassl, 2002a
;
Roch-Ramel et al., 1994
) and
primary active transport (Van Aubel et
al., 2005
). The last two processes have been associated with urate
secretion, and NPT1 (sodiumphosphate transporter I;
Uchino et al., 2000
),
Oatv1 (voltage-dependent organic anion transporter 1;
Jutabha et al., 2003
) and MRP4
(multidrug resistance peptide 4; Van Aubel
et al., 2005
) have been proposed as candidate transporters.
Chicken orthologs of NPT1 and Oatv1 have yet to be reported, and no
human ortholog of Oatv1 has been found
(Hediger et al., 2005
).
Overexpression of MRP4 in HEK293 cells revealed an ATP-dependent urate export
capability (Van Aubel et al.,
2005
). The relationship of these processes in mammals to urate
secretion is still unclear, and even less is known about the mechanisms of
apical urate efflux in avian species (see
Dantzler, 2002
).
A general model of the mechanism of urate secretion by avian proximal
tubule was recently reviewed by Dantzler
(2005). This model represents
the uric acid uptake at the BLM as two processes, one urate/
-KG
exchange and a process that apparently exchanges urate for an unknown
counteranion. The apical cell-to-lumen step is down an electrochemical
gradient; however, neither apical nor BLM urate transporters have been fully
defined. The initial step in urate secretion as determined from basolateral
membrane vesicles (BLMV) of turkey kidney is consistent with the `classical'
organic anion (OA) uptake system of transporters
(Grassl, 2002b
).
In the present study, chicken proximal tubule epithelial cell primary monolayer cultures (PTCs) were used to investigate net active transepithelial transport of urate together with passive `leak' fluxes under short-circuited conditions. The PTCs were shown to express mRNA for orthologs of OAT1, OAT3, MRP2 and MRP4. The data are consistent with OAT-like-mediated urate transport across the BLM. Active net urate secretion was not sensitive to apical membrane OH, Cl-, or K+ concentration gradients but was greatly reduced by inhibitors of MRP4.
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Materials and methods |
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Solutions and chemicals
Hanks' balanced salt solution (HBSS) was purchased from Mediatech (Herndon,
VA, USA). Krebs-Henseleit buffer was purchased from Sigma Chemicals (St Louis,
MO, USA). This medium was supplemented with 4 mmol l1
NaHCO3 (pH 7.4). The final plating medium and maintenance medium
consisted of Dulbecco's modified Eagle's mediumHank's balanced salt
solution (DMEMHam's) F12 supplemented with insulin/transferrin/selenium
pre-mix (ITS; 5 µg ml1 insulin, 5 µg
ml1 transferrin, 5 ng ml1 selenite), 20
µmol l1 ethanolamine, 300 µmol l1
L-glutamine, and 10% fetal bovine serum (FBS). The saline solution
used for Ussing chamber experiments contained (in mmol l1)
1.1 CaCl2, 4.2 KCl, 0.3 MgCl2, 0.4 MgSO4, 120
NaCl, 0.4 NaH2PO4, 0.5 Na2HPO4,
1.0 glycine, 25 NaHCO3 (pH 7.4 with 5% CO295%
O2, 290 mosmol kg1 H2O). Additionally,
330 µmol l1 urate and 5.5 mmol l1
glucose were added to the saline solution in both lumen and interstitium at
the start of each experiment (t=0).
PAH, probenecid, Bromcresol Green, ouabain, cimetidine, methotrexate (MTX), estrone sulfate (ES), ethanolamine, L-glutamine, oxonic acid, pyrazine, adenosine, nicotinic acid, nocodozole, cytochalasin D, and all components of the saline solution were purchased from Sigma (St Louis, MO, USA). DMEMHam's F-12 was from Mediatech (Herndon, VA, USA). FBS and lithium chloride were purchased from Fisher (Pittsburgh, PA, USA). ITS was purchased from Collaborative Biomedical Products (Bedford, MA, USA). Glutarate was purchased from Aldrich (Sheboygan, WI, USA). MK-571 was purchased from Biomol (Plymouth Meeting, PA, USA). Percoll was purchased from Amersham Pharmacia Biotech (Piscataway, NJ, USA).
Preparation of chicken PTCs
Chicken renal tubule segments were isolated and dispersed
(Sutterlin and Laverty, 1998)
and modified (Dudas and Renfro,
2001
) as previously described. Briefly, kidneys were removed,
rinsed in HBSS, cleaned of blood vessels, ducts and connective tissue, and
minced. The tissue fragments were incubated in an enzyme solution containing
collagenase A (0.13 U ml1) and dispase II (0.54 U
ml1) at 37°C for 10 min. Nephron segments were further
dissociated by trituration and filtration through a stainless steel sieve (380
µm). The dissociated tissue was rinsed three times with HBSS, with the last
rinse containing DNase I (2161 U ml1), and resuspended in a
1:1 mixture of Percoll and 2x KrebsHenseleit buffer. The
suspension was centrifuged at 17,500 g and the high-density
band consisting of small proximal tubule segments (probably a mixture from
cortical, transitional and long-looped medullary nephrons) was removed, rinsed
with HBSS, suspended in culture medium with 10% serum, and plated on native
rat-tail collagen as previously described
(Dickman and Renfro, 1993
).
After 6 days in culture, the collagen gels were detached from the culture
dishes, and after
14 days, these floating collagen gels had been
contracted by the epithelial monolayers to
40% (17 mm to 10 mm
diameter).
Ussing chamber studies
During days 1529, transepithelial electrical characteristics and
urate transport were measured. The tissues were supported by 150 µm nylon
mesh and mounted in Ussing chambers as previously described
(Gupta and Renfro, 1989). The
saline bathing the luminal and interstitial sides of the tissue was maintained
at 39°C, and continuously gassed (95% O2, 5% CO2)
and stirred throughout the experiment.
Transepithelial electrical potentials (VT) were determined with a pair of reference electrodes connected to the luminal and interstitial compartments via 3 M KCl2% agar bridges. Electrode asymmetry was corrected at the beginning and end of each experiment with compensation for fluid resistance. Current was passed through AgAgCl electrodes connected to each compartment with 3 M KCl2% agar bridges. Electrical properties were measured with a pair of computer-controlled, high-impedance automatic dual voltage clamps (DVC 1000; World Precision Instruments, Sarasota, Fl, USA). Transepithelial electrical resistance (TER) was determined from the change in VT produced by a 10 µA current pulse. The sensitivity of the transepithelial current to 104 M phloridzin in the lumen side, a specific inhibitor of Na+-dependent glucose transport, was determined at the end of every experiment in all tissues. This current (Iglu; sodium-dependent glucose current) served as a check on proximal tubule-like function and tissue vitality.
Determination of transepithelial urate fluxes
Tissues were continuously short-circuited during flux determinations (with
the exceptions noted), i.e. there were no transepithelial electrical or
chemical gradients. Short-circuit current was 45 µA
cm2 in these low-resistance tissues prior to glucose
addition. Unidirectional tracer fluxes were initiated by the addition of 0.4
µCi [8-14C]urate (ARC, St Louis, MO, USA) to the appropriate
hemichamber. Duplicate 50 µl samples were taken from the unlabeled side
every 30 min over a period of 1.5 h and replaced with equal volumes of
unlabeled saline (see Fig. 1).
The specific activity of the labeled solution was determined at the beginning
and end of each experiment based on the urate concentration of 330 µM and
radioactivity of the radioactive-side bathing medium.
|
Intracellular recordings
The cultured proximal tubule cell monolayers on collagen gels were affixed
to 18 mm circular coverslips (Fisher, Pittsburgh, PA, USA) with chicken
plasma/thrombin (1:1) clots and immersed in warmed (37°C) saline (same
composition as used for Ussing chamber experiments; pH 7.4 with 5%
CO2, 95% O2, 290 mosmol kg1
H2O). The coverslips with the attached monolayers were mounted on
the stage of a Nikon Eclipse E-600 FN microscope (Nikon, Melville, NY, USA)
and continuously perfused with saline. Cultures were visualized using infrared
differential interference contrast (IR-DIC) microscopy. Electrodes were pulled
from capillary tubing (Garner Glass N51A; Garner Glass Co., Claremont, CA,
USA) using a Narishige multi-step electrode puller (Model PP-830) (Tritech
Research, Los Angeles, CA, USA) and had resistances of 30 M
. The
electrode solution was 3 M KCl. Current-clamp recordings were made using an
Axon Instruments 200B amplifier (Axon Instruments Inc., Foster City, CA, USA),
and low pass filtered at 1 kHz. Currents were digitally sampled at 10 kHz and
monitored with pCLAMP 8.0 software (Axon Instruments Inc.) running on a PC
pentium computer.
To examine depolarization resulting from elevated luminal K+ or
the presence of luminal glucose we used a picospritzer (Picospritzer II,
General Valve Corporation, Fairfield, NJ, USA) to locally apply either 30 mmol
l1 K+, or glucose-free solution. Following a
24 s baseline recording, a brief pulse of 30 mmol l1
K+ (200 ms), or glucose-free solution (100 ms) was applied within
100 µm of the punctured cell and the response of the cell was
monitored for durations of
2535 s. Currents were identified using
Clampfit 8 software (Axon Instruments).
Preparation of total mRNA from PTCs
Four PTCs, each containing 107 cells were used for
isolation of total RNA using the Qiagen RNeasy Midi Kit according to the
manufacturer's instructions (Valencia, CA, USA). PTC mRNA was isolated from
total RNA (
150 ng µl1) using the Qiagen Oligotex
mRNA kit (Valencia, CA, USA). Final concentration of chicken PTC mRNA was 25
ng µl1.
RT-PCR for detecting known organic anion transporters in chick PTCs
Chick PTC mRNA was used for RT-PCR as described in the Qiagen one-step
RT-PCR kit. The initial reverse transcription and subsequent PCR were carried
out in a single step with primers (Table
1) against known sequences for OAT1-like (BBSRC Chick EST ID
603807902F1), OAT3-like (BBSRC Chick EST ID 603812145F1), MRP2 (GenBank
accession XM_421698) and MRP4 (GenBank accession XM_416986) transporters from
the domestic chicken. Since the OAT1 and OAT3 sequences in the database are
only partial sequences and correspond to different regions of the mammalian
OAT1 and OAT3 open reading frame, there is no certainty that these sequences
correspond to unique OATs or whether they are from the same OAT-like
transporter. They are termed OAT1- and OAT3-like since BLAST searches of the
individual sequences found them to be most similar (50% each) to human
OAT1 and OAT3, respectively. In contrast, full sequences for chicken MRP2 and
MRP4 transporters are available and amino acid alignments indicate that they
are 60% and 86% similar to their human orthologs, respectively. The primers
(0.6 µmol l1) and 25 ng of template mRNA were used in
each RT-PCR reaction. The reverse transcription reaction was conducted at
50°C for 30 min followed by a 15-min incubation at 95°C to denature
the reverse transcriptase. The PCR reaction was run for 38 cycles with a
denaturing temperature of 94°C, an annealing temperature of 58.7°C
(OAT1), 61.6°C (OAT3), 58.7°C (MRP2) or 56°C (MRP4), and an
elongating temperature of 72°C. PCR products were separated on a 2%
agarose gel and stained with Gel-Star (Fisher, Pittsburgh, PA, USA).
|
Statistics
Experimental results are expressed as means ± S.E.M.
Sample means were compared with paired one-tailed Student's t-tests.
Differences were judged significant if P<0.05.
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Results |
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Pharmacological characterization
A general pharmacological characterization of the effects of a battery of
inhibitors and (or) competitive substrates on active urate secretion is shown
in Tables 2,
3 and
4. In the proximal tubule,
alteration of the TER is indicative of a change in conductance through the
paracellular shunts, and a change in Iglu can reflect a
change in plasma membrane Na+ electrochemical potential. Thus, a
`nonspecific' effect in this circumstance is defined as a change due to
interaction of a compound with ATP production, Na/K-ATPase, membrane
integrity, etc., rather than, or in addition to, specific interaction with a
urate transporter. If a particular compound caused significant changes in TER,
Iglu, or VT, the direction of change
is shown.
|
|
Interstitial-side effects
Others have provided evidence that urate shares an OA/PAH transport system
in the avian kidney (Dantzler,
2002). High concentrations (210 mmol l1)
of PAH in the bathing medium inhibit BLM urate uptake by both chicken isolated
proximal tubules (Brokl et al.,
1994
) and turkey BLMV (Grassl,
2002b
). As shown in Table
2, addition of PAH (10 mmol l1) to the
interstitial side of PTCs in Ussing chambers completely abolished net
transepithelial urate secretion (negative sign indicates net reabsorption)
with no nonspecific effects. It should be noted that this high concentration
of PAH in the interstitial side significantly stimulated unidirectional
reabsorptive urate flux, an effect consistent with PAH entering the cell in
exchange for intracellular urate.
Bromocresol Green, generally considered to be a high affinity substrate for proximal tubule OA secretion, inhibited net transepithelial urate secretion; however, its effect was also non-specific as it caused a significant increase in unidirectional reabsorptive (leak) flux together with a significant decrease in TER (Table 2).
The cationic OAT substrate, cimetidine, increased leak in both secretory and reabsorptive directions but, because of extremely high variance, had no effect on net urate transport (Table 2). Consistent with the increased leakiness, both TER and Iglu decreased, indicative of toxicity.
At the basolateral membrane, interaction of urate with at least OAT1- or
OAT3-like transporters should be dependent upon exchange for intracellular
-KG. Thus, lithium (5 mmol l1), an inhibitor of
Na+dicarboxylate cotransport should inhibit re-uptake of
-KG, depleting intracellular dicarboxylate concentration, and slowing
urate secretion. Table 3 shows
that lithium, added to the interstitial side only of PTCs in Ussing chambers
decreased net urate secretion by
40%, mainly through decreasing the
secretory flux. Lithium affected only urate secretion and had no effect on
reabsorption or electrical properties. Consistent with the lithium effect,
ouabain, an inhibitor of the Na+/K+ ATPase, also
inhibited net transepithelial urate secretion by 40% and, as expected,
significantly inhibited Iglu.
The inhibition by lithium of unidirectional and net transepithelial urate
secretion in chick PTCs suggested that the well-characterized BLM tertiary
active OA uptake system (Wright and
Dantzler, 2004) was mediating urate uptake. However, acute
addition of 10 or 100 µmol l1 glutarate, the
non-metabolized form of
-KG, to the interstitial bath had no effect on
unidirectional secretory or reabsorptive urate fluxes in PTCs
(Table 3). Addition of 1 mmol
l1 glutarate to only the luminal bath had no effect on urate
fluxes (data not shown). To determine if 330 µmol l1
urate (physiological concentration) was preventing the stimulatory effect of
lower extracellular glutarate concentrations, the urate concentration in the
bathing medium was reduced to 5.8 µmol l1. However, no
stimulation of net transepithelial urate secretion resulted from the
combination of lower urate and 10 µmol l1 glutarate in
the interstitial side (data not shown). This apparent lack of effect was
probably due to production and efficient recycling of endogenous
-KG by
Na+/dicarboxylate cotransport since elevation of glutarate to 1
mmol l1 in the interstitial solution inhibited 80% of net
transepithelial urate secretion, as expected
(Table 3).
|
Chick PTCs avidly secreted PAH (Fig.
3) exhibiting a 5:1 flux ratio in short-circuited conditions. Net
PAH secretion was inhibited 34% by addition of 330 µmol
l1 urate, and the remaining net flux was totally blocked by
500 µM ES. The latter, alone, blocked both the urate-sensitive and
-insensitive net PAH secretion.
|
Lumen-side effects
Alterations in only the luminal fluid of several factors that are known to
change urate transport in vertebrate brush-border membrane vesicles (BBMV;
which facilitate both reabsorptive and secretory transport of urate) had no
effect on net urate secretion by chick PTCs
(Table 4). In all
ion-substitution experiments the tissues were open-circuited. Substituting
K+ for Na+ in the luminal bath (4.230 mmol
l1) caused only a small change in
Vapical (electrical potential across the apical
(brush-border) membrane) from approx. 79 to 72 mV,whereas
complete removal of luminal glucose shifted Vapical from
80.5 to 86.5 mV. In neither case were there effects on net
transepithelial urate transport. The increased lumen [K+] caused a
significant decrease in TER, perhaps reflecting the increase in the
transepithelial Na+ gradient caused by the ion substitution. There
was no effect on transepithelial urate transport of complete replacement of
luminal Cl- with gluconate
(Table 4). Apparently, urate
does not leave the cells in exchange for Cl-. Luminal pH of the
avian proximal tubule is slightly alkaline at 7.62
(Laverty and Alberici, 1987
),
and in dog BBMV OH/urate anion exchange can occur
(Kahn and Aronson, 1983
). To
assess whether a physiological luminal pH would alter urate transport, pH of
the physiological saline solution bathing the luminal sides of PTCs in Ussing
chambers was asymmetrically raised from 7.4 to 7.7 (a 50% decrease in
[H+]), again, with no effect on net transepithelial urate transport
(Table 4). The significant drop
in Iglu is probably due to increased apical
Na+/H+ exchange and depletion of the apical membrane
Na+ gradient.
Also shown in Table 4, a
series of substrates (oxonic acid, pyrazine, adenosine and nicotinic acid)
known to interfere with urate reabsorption or cause uricosuria
(Roch-Ramel et al., 1997), had
no effect on PTC urate transport or transepithelial electrophysiological
properties. These results coupled with the lack of effect of luminal
Cl- decrease the likelihood of a role for transporters such as
URAT1 (urate transporter 1), the electroneutal urate/Cl- exchanger
thought to be responsible for urate reabsorption in humans
(Enomoto et al., 2002
) and the
74%-similar mouse renal-specific transporter (RST) which may mediate
voltage-dependent urate transport (Imaoka
et al., 2004
).
Intracellular sequestration of OAs, subsequent bulk vesicular transport in
the cytosol, and release into the tubule lumen by exocytosis have been
suggested as part of the transepithelial OA transport process
(Miller and Pritchard, 1994;
Miller et al., 1993
). In the
chick PTCs concentrations of nocodozole (microtubule disrupter) or
cytochalasin D (microfilament disruptor) known to interfere with vesicular
trafficking were either ineffective or nonspecific
(Table 4).Nocodozole
significantly decreased Iglu and increased urate back leak
but, because of very high variance, had no significant effect on net urate
transport. Cytochalasin D significantly decreased TER and increased urate back
leak while totally blocking active urate secretion. The nonspecific effects of
these disrupters make conclusions about bulk vesicular transport of urate in
PTCs uncertain.
|
|
Detection of mRNA for known organic anion transporters from PTCs
Specific primers (see Table
1) and RT-PCR were used to amplify cDNAs corresponding to
OAT1-like, OAT3-like, MRP2 and MRP4 transporters from chick PTC mRNA
(Fig. 5). In each case, a
single cDNA product of the appropriate size (OAT1=483 bp; OAT3=344 bp;
MRP2=586 bp; MRP4=565 bp) was identified on 2% agarose gels. No product was
detected when the template mRNA was omitted.
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Discussion |
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Lithium inhibits Na+/dicarboxylate transport
(Kekuda et al., 1999) and, in
the present study, reduced net urate secretion by about one-half, through
inhibition of the unidirectional secretory flux although the reabsorptive flux
was unaffected. These data, in conjunction with the aforementioned PAH and
probenecid sensitivity, are consistent with the presence of
Na+/dicarboxylate cotransport coupled with urate/
-KG
exchange in the BLM as was demonstrated in mammals
(Pritchard, 1990
;
Shimada et al., 1987
) and
confirmed in turkey BLMV (Grassl,
2002b
). The physiological saline solution used for the present PTC
transport studies was not supplemented with
-KG, so the lithium
inhibition of urate secretion further supports urate exchange for
intracellularly produced
-KG and recycling of the
-KG from the
interstitial bath to the cell by Na+/dicarboxylate cotransport. In
the absence of extracellular
-KG, rabbit proximal tubule S2 segment
uptake of OA is inhibited 25% by lithium, and the overall contribution of
metabolically produced
-KG to OA secretion in rabbits is about 40%
(Dantzler, 2002
). In the chick
PTCs, no stimulation of urate secretion by glutarate was observed, and it is
possible that endogenous
-KG accumulates to a saturating concentration,
precluding an effect of additional accumulation of glutarate. Lithium,
however, would lead to depletion of intracellular
-KG and slowing of
urate uptake. The inhibitory effect of high levels of interstitial glutarate
(1 mmol l1, see Table
3) is consistent with glutarate/urate interaction.
Asymmetrical addition of 10 mmol l1 PAH, 2.5 mmol
l1 ES, or 0.5 mmol l1 MTX to the
interstitial side of PTCs not only inhibited unidirectional secretory flux, it
increased unidirectional reabsorptive flux with no nonspecific effects. This
behavior (i) obviously cannot be due to increased non-mediated leak flux; (ii)
is consistent with urate efflux, i.e. cell to interstitium, driven by reversal
of an anion exchange mechanism and (iii) indicates that these substrates and
urate interact with the same exchanger(s). These findings are consistent with
the facts that PAH is accumulated intracellularly to twice the concentration
of urate in isolated chicken proximal tubule fragments
(Brokl et al., 1994) and
PAH/urate exchange exists in turkey BLMV as demonstrated by Grassl
(Grassl, 2002b
). The mechanism
by which lumen-to-cell flux was altered in this circumstance is not clear,
however, urate must enter from the lumen against an electrochemical gradient,
a phenomenon observed for PAH, but not urate, in perfused chicken proximal
tubule (Brokl et al., 1994
).
This is apparently a very small component and perhaps not like the
reabsorption process in mammals, in which luminal Cl- exchanges for
intracellular urate on URAT1 (Enomoto et
al., 2002
; Kahn and Aronson,
1983
), because the present study revealed no effect of
Cl- removal from the luminal side on urate transport (see
Table 4); and as already noted,
urate/anion exchange could not be demonstrated in a recent study of avian BBMV
(Grassl, 2002a
).
A possible cell-to-lumen exit step in urate secretion has been
characterized in turkey kidney (Grassl,
2002a). In isolated BBMV from these Galliforms a conductive
uniporter facilitates diffusion of urate down its electrochemical gradient and
is trans-stimulated and cis-inhibited by PAH. Likewise, PAH
transport is driven by the electrical gradient and blocked by urate.
Cl- and pH gradients have no effect on BBMV uptake except
secondarily through membrane diffusion potentials
(Grassl, 2002a
). There is no
consensus as to the transporter(s) responsible for this step. In
urate-secreting mammals the most likely transporters were recently reviewed
(Hediger et al., 2005
;
Wright and Dantzler, 2004
) and
include MRP4 and Oatv1 (Jutabha
et al., 2003
). The latter is 65% identical to human NPT1.
Oatv1 and NPT1 are located in proximal tubule BBM of pig and human,
respectively, and can mediate electrogenic transport of PAH and urate
(Busch et al., 1996
;
Uchino et al., 2000
). However,
no chicken orthologs of these two transporters have yet been identified; they
are Cl- sensitive and they do not have the BBMV urate transport
properties, i.e. they are not electrogenic antiporters
(Roch-Ramel et al., 1994
).
MRP4 is a primary active export pump localized to the proximal tubule BBM
and it mediates transport of MTX and urate
(Van Aubel et al., 2005). In
PTCs urate transport was inhibited by MTX from either the interstitial side or
the luminal side. Luminal application of MK-571, a leukotriene C4
receptor antagonist, dramatically reduced both urate secretory and
reabsorptive flux. This compound is a known substrate for MRP2, MRP4 and the
organic anion transporter oatp1 (Sundkvist
et al., 2000
), also known to be present in mammalian BBM; however,
urate is not transported by MRP2 (Van
Aubel et al., 2005
). Interestingly, amino acid alignment of the
chicken MRP4-like sequence against human MRP4 indicates 86% sequence
similarity. However, the chicken MRP4-like sequence is 303 amino acids longer
than human MRP4, containing additional amino acids at the extreme N terminus
and from amino acid 1068 to amino acid 1148. If MRP4 is involved in the urate
efflux step in the chicken proximal tubule it may be beneficial to investigate
the structurefunction relationship of MRP4 in this uricotelic species.
The sensitivity of other possible urate transport pathways to MK-571 is not
known. MK571 had no effect on urate transport from the interstitial side of
the PTCs, providing, for the first time, a means for discrimination of BLM and
BBM urate transport processes in intact epithelium.
In conclusion, the chick PTC system served to characterize urate transport in a predominantly secretory system amenable to the examination of the transepithelial aspect of urate transport. The chicken PTC culture system maintained excellent urate secretory capacity and should prove a useful tool for further study of this process, unclouded by the coexistence of a highly expressed mediated reabsorptive transport process. The pharmacological data and RT-PCR demonstration of expression in the PTCs of genes associated with OA and urate transport, presented here, should aid the ultimate identification of the participants in active tubular urate secretion. The data support the conclusion that avian renal urate secretion is mediated by an OAT-like transporter in the BLM and provide a means of distinguishing the apical and basolateral membrane steps in proximal tubule epithelium.
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Acknowledgments |
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References |
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