1Department of Medicine, University of Cincinnati, Cincinnati 45267; and 2Veterans Affairs Medical Center, Cincinnati, Ohio 45220
Submitted 3 March 2003 ; accepted in final form 5 May 2003
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ABSTRACT |
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putative anion transporter 1; chloride/formate exchanger; SLC26A6
Recent molecular studies have identified a large, highly conserved family
of membrane proteins (designated SLC26A), many of which have been shown to
transport anions (7,
13,
15,
16,
23,
32,
37,
40). PAT1 (putative anion
transporter 1; or SLC26A6), which is a member of this family
(23), was recently shown to be
located on the apical membrane of mouse kidney proximal tubule
(20). Expression studies
showed that the mouse ortholog of PAT1 (also called CFEX) mediates
Cl/formate exchange when expressed in oocytes
(20). We have examined the
functional properties of both human and mouse PAT1 (or CFEX). Our expression
studies in oocytes indicate that PAT1 mediates
Cl/OH and
exchange (40). Subsequent
studies have verified the mediation of
exchange by PAT1 (or SLC26A6)
(17,
43). In addition to the mouse
kidney proximal tubule, PAT1 is expressed on the apical membrane of the villi
of mouse duodenum (40) and on
the tubulovesicles of gastric parietal cells
(27).
On the basis of immunolocalization studies indicating the expression of
SLC26A6 (PAT1) on the apical membrane of mouse proximal tubules and functional
studies in in vitro expression systems indicating that SLC26A6 mediates
exchange (17,
40,
43), we hypothesized that
SLC26A6 (PAT1) is an apical
exchanger in the kidney proximal tubule. Accordingly, we performed
immunocytochemical staining and measured apical Cl/base
exchanger activity in rat isolated microperfused proximal tubules in the
presence or absence of bicarbonate. The results demonstrate that rat proximal
tubules express PAT1 along with a robust
exchanger on their apical membrane.
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METHODS |
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Female Sprague-Dawley rats, weighing 100150 g, were used for these studies. Animals were allowed free access to water and food. The use of anesthetics (pentobarbital sodium) and the method of euthanasia (pentobarbital overdose) were according to the institutional guidelines and approved protocols.
RNA Isolation and Northern Hybridization
Total cellular RNA was extracted from whole kidney and cortex, outer
medulla, and inner medulla cortex by the method of Chomczynski and Sacchi
(11), quantitated
spectrophotometrically, and stored at 80°C. Total RNA samples (30
µg/lane) were fractionated on a 1.2% agarose-formaldehyde gel and
transferred to Magna NT nylon membranes (MSI). Membranes were cross-linked by
ultraviolet light and baked for 1 h. Hybridization was performed according to
Church and Gilbert (12). The
cDNA probes (25 ng) were labeled with 32P-labeled deoxynucleotides
by using the Rad-Prime DNA labeling kit (GIBCO-BRL). The membranes were
washed, blotted dry, exposed to PhosphorImager cassette at room temperature
for 2472 h, and read by PhosphorImager (Molecular Dynamics, Sunnyvale,
CA). For PAT1, an 200-bp PCR fragment was amplified from a mouse
expressed sequence tag (EST) (GenBank accession no. AI747461
[GenBank]
) and used as a
probe.
Immunoblotting and Immunocytochemistry of PAT1
Antibodies. Two polyclonal PAT1-specific antibodies were raised. The first has an amino acid sequence MDLRRRDYHMERPLLNQEHL, and the second has the amino acid sequence CRRDYHMERPLLNQE, which is a truncated form of the first one and, in addition, has a cysteine residue incorporated for better purification. The specificity of both antibodies has been verified in our previous studies (27, 40).
Electrophoresis and immunoblotting. Semiquantitative immunoblotting experiments were carried out as previously described (40). Briefly, the solubilized membrane proteins from rat kidney cortex were size-fractionated on polyacrylamide minigels (Novex, San Diego, CA), electrophoretically transferred to nitrocellulose membranes, blocked with 5% milk proteins, and then probed with affinity-purified anti-PAT1 immune serum at an IgG concentration of 0.8 µg/ml. The secondary antibody was donkey anti-rabbit IgG conjugated to horseradish peroxidase (Pierce). The sites of antigen-antibody complexation on the nitrocellulose membranes were visualized using a chemiluminescence method (Super-Signal substrate; Pierce) and captured on light-sensitive imaging film (Kodak).
Immunofluorescence. Animals were killed with an overdose of pentobarbital sodium and perfused through the left ventricle with 200 ml of 0.9% saline followed by 500 ml of cold 4% paraformaldehyde in 0.1 M sodium phosphate buffer (pH 7.4). Kidneys were removed, cut in tissue blocks, and fixed in the same solution overnight at 4°C. For cryosections, tissue blocks were removed from the fixative solution and soaked in 30% sucrose overnight. The tissue was frozen on dry ice, and 5-µm sections were cut on a cryostat and stored at 80°C until used.
For staining, cryosections were washed twice in 0.01 M PBS (pH 7.4) and blocked with 10% goat serum-0.3% Triton X-100-PBS solution for 4560 min. Primary antibodies were diluted 1:40 in 1% BSA-0.3% Triton X-100-PBS solution and applied to sections overnight at room temperature. Sections treated with the primary antibodies were rinsed twice in 0.01 M PBS for 10 min and then incubated with a secondary antibody for 2 h at room temperature. Oregon Green (Molecular Probes, Eugene, OR)-conjugated goat-anti-rabbit IgGs were used as secondary antibodies at 1:150 dilution. Sections were then washed four times, air-dried, and mounted in Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA). Sections were examined and images acquired on the Nikon PCM 2000 laser confocal scanning microscope as 0.5-µm "optical sections" of the stained cell membrane, using a x40 objective. The standard argon laser exciter 488-nm filter and the 515/30-nm emission filter was used for the green emitting dye.
Isolation of Proximal Tubules and In Vitro Microperfusion
Rats were killed by intraperitoneal injection of pentobarbital sodium (100
mg/kg body wt). Kidneys were quickly removed and placed in ice-cold dissection
medium (Table 1, solution
1). Thin coronal slices (1 mm) were cut and transferred to the
dissection chamber. Proximal straight tubules, comprising the ends of the S2
and S3 segments, were isolated from the cortical part of medullary rays and
transferred to the 1-ml temperature-controlled specimen chamber mounted on the
inverted Zeiss Axiovert S-100 microscope (Carl Zeiss, Thornwood, NY).
Experiments were done at 37°C. In vitro microperfusion of the isolated
tubule segments was performed by using concentric glass pipettes according to
the method of Burg (9,
10) as previously described at
5-cm of water pressure (26,
28). Solutions used to perfuse
and bathe the tubules are listed in Table
1. Solutions were delivered to the specimen chamber in
CO2- and O2-impermeable tubing (Cole Palmer, Chicago,
IL) by the peristaltic pump (Peristar; WPI, Sarasota, FL) at a rate of 1
ml/min. The chamber was closed by a lid and constantly superfused with 95%
O2-5% CO2 to keep the pH of the bath fluid constant.
Chamber pH was frequently checked with a Horiba pH meter (model B 213; Horiba,
Kyoto, Japan). Tubules were perfused with 0.15 mg/ml Fast Green dye (Sigma,
St. Louis, MO) at the beginning of each experiment to identify the damaged
cells (normal cells are impermeable, whereas damaged cells take up this dye).
Tubules were carefully inspected and discarded if damaged cells were found in
the tubule wall (28,
39).
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Intracellular pH Measurements
Intracellular pH was measured by using 5 µM 2',7'-bis(3-carboxypropyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCPCF-AM) (22), a close analog of 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF-AM) with improved spectral characteristics, as previously described (26, 28, 39). Briefly, after 1520 min of equilibration in the initial bath and luminal solution, the tubule was perfused with 1 µM BCPCF-AM for 2 min and then 10 min were allowed for dye washout. Fluorescence measurements were done on a Zeiss Axiovert S-100 inverted microscope equipped with an Attofluor RatioVision digital imaging system (Attofluor, Rockville, MD) as previously described (26, 28). An Achroplan x40/0.8 NA water objective with a 3.6-mm working distance was used. Excitation wavelengths were recorded at 488 and 440 nm, and emission was measured at 520 nm. Digitized images were analyzed using the Attograph software. Only one tubule per animal was examined. Intracellular calibration was performed at the end of each experiment by using the high-K+-nigericin method (26, 28, 38, 39).
Apical Cl/OH
and
Exchange
Stable basal intracellular pH (pHi) readings were obtained for
at least 5 min before any experimental maneuvers. Tubules were perfused with
either bicarbonate-containing (Table
1, solution 2) or bicarbonate-free solutions
(Table 1, solution 4)
in the presence of 300 µM (0.3 mM) DIDS in the bath and 100 µM EIPA in
the lumen 1015 min before the experiments to inhibit the basolateral
cotransporter and luminal Na+/H+ exchanger,
respectively. Luminal perfusate was switched to a Cl-free
solution (Table 1, solution
3 for bicarbonate-containing experiments and solution 5 for
bicarbonate-free experiments). Apical
exchanger activity was assessed as the rate of pHi acidification
(dpHi/dt) calculated from the slope of the initial pH
change upon switching the luminal perfusate from Cl-free to
Cl-containing solution in either bicarbonate-containing
(Table 1, solution 3
to solution 2) or bicarbonate-free solutions
(Table 1, solution 5
to solution 4). This maneuver causes cell acidification, which is
reversed upon subsequent return to the Cl-free solution. To
examine the effect of DIDS on apical
exchanger, we performed experiments in the absence of luminal EIPA, because
EIPA and DIDS precipitate when introduced in the same solution. To test the
sensitivity of the exchanger to DIDS, we incubated tubules with 0.5 mM DIDS
and 0 mM Cl in the lumen for 1015 min and measured
apical
exchanger activity by switching the perfusate to a
Cl-containing solution.
Intrinsic cell buffer capacity (i) was measured using a
NH4Cl prepulse technique according to established protocols
(21,
29,
35,
36). Measurement of
i was done in Na+-free solutions
(Table 1, solution 6)
with 1 mM DIDS on both the bath and luminal side, to keep Na+- and
Cl-dependent acidification mechanisms inactive. In
NH4Cl-containing solutions, 20 mM NH4Cl replaced an
equimolar concentration of tetramethylammonium (TMA)-Cl. In bicarbonate-free
solutions,
i was 31.8 ± 3.9 mM/pH unit (n =
5). In bicarbonate-containing solutions, the total buffering capacity
(
T) was estimated as the sum of
i and the
bicarbonate buffer capacity
(
).
was
calculated as 2.3 x
(21,
29), where
is the
intracellular bicarbonate concentration. The buffer capacity in the presence
of bicarbonate was 88.9 ± 5 mM (n = 5). Equivalent base flux
(EBF) was calculated as dpHi/dt x
x V
and expressed in picomoles per millimeter per minute
(21), where
is the cell
buffer capacity expressed as
i in the experiments in which
bicarbonate-free solutions were used and as
T in the
experiments in which bicarbonate-containing solutions were used. V designates
the cell volume per tubule length, calculated according to the following
formula V =
[(do)2
(di)2]/4, where do and
di stand for the outer and inner tubule diameter,
respectively. Tubule diameters were measured at x400 magnification using
an eyepiece reticle. Average tubule volume was 8.5 ± 0.16 x
1010 l/mm.
Free oxalate concentration in the perfusate was measured by an oxalate oxidase spectrophotometric enzymatic assay according to the manufacturer's protocol (Sigma).
Cloning and Expression of Mouse PAT1
Full-length mouse PAT1 cDNA was cloned from the duodenum by RT-PCR, according to published reports (20) and as described from our laboratory (40). The following oligonucleotide primers were designed and used for RT-PCR: 5'-CGT CTG CAC TGC TCC CTC CTC CAT TG and 5'-GAG TCC CAG GGC ATC CAT CCA TG (accession no. AY032863 [GenBank] ). These primers encode nucleotides 452498 of the mouse SLC26A6, also known as PAT1 or CFEX (20). Amplification of the mouse PAT1 cDNA by PCR was performed according to the Clontech Advantage 2 PCR kit protocol. The capped PAT1 cRNAs were generated using the mMessage mMACHINE kit (Ambion) according to the manufacturer's instructions.
Expression of Mouse PAT1 in Xenopus Oocytes
Xenopus oocytes were injected with mouse PAT1 cRNA, as described previously (40). pHi in oocytes was measured with the pH-sensitive fluorescent probe BCECF-AM (Molecular Probes, Eugene, OR) as described previously (37, 40). Oocytes were loaded with 10 µM BCECF-AM, and cell pHi was monitored by ratiometric fluorescence using the Attofluor digital imaging system (40). Data analyses were performed using the Attograph and Attoview software packages provided with the imaging system. The ratios were obtained from the submembrane region of the oocytes that were visualized with a x40 water objective. Measured excitation ratios were converted to pHi by using a calibration curve that was constructed with the high-K+/nigericin method at the end of each experiment.
Materials
32P-dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma. The RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCPCF-AM was from Molecular Probes. All the other chemicals, including the oxalate oxidase enzymatic assay, were from Sigma. Nigericin was dissolved in ethanol and diluted 1:1,000 for the final concentration of 10 µM. EIPA was dissolved in methanol and diluted 3:1,000 for the final concentration of 100 µM. DIDS was dissolved directly in the solutions used for perfusion.
Statistics
Results are expressed as means ± SE. Statistical significance between experimental groups was determined by Student's t-test, as required. Significance was asserted if P < 0.05.
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RESULTS |
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Northern hybridization. In the first series of experiments, the mRNA expression of PAT1 was examined in various rat kidney zones by Northern hybridization. As shown in Fig. 1A, PAT1 mRNA is predominantly expressed in the kidney cortex. There were very faint bands in the outer and inner medulla.
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Immunoblot analysis of PAT1 in rat kidney. In the next series of
experiments, Western blot analysis of PAT1 on microsomal membranes isolated
from rat kidney cortex was performed. As shown in
Fig. 1B,
left, PAT1 immune serum detects an 90-kDa band in rat kidney
cortex. Preadsorption of the immune serum with the synthetic peptide prevented
the labeling (Fig. 1B,
right), indicating the specificity of the immune serum.
Immunofluorescence labeling of PAT1. To examine the cell distribution and subcellular localization of PAT1 in rat kidney, we performed immunocytochemical staining. As shown in Fig. 1C, PAT1 immune serum labeled the apical membrane of rat proximal tubules (top). The labeling was specific, because no staining was detected with the preadsorbed immune serum (Fig. 1C, bottom). Occasionally, faint labelings on basolateral membranes were detected, but these were not consistent. No labeling was detected in the medulla (not shown). The localization of PAT1 on the apical membrane of rat kidney proximal tubule is in agreement with recent results in mouse kidney (20).
and Cl/OH Exchange
in Rat Straight Proximal Tubules
PAT1 expression in oocytes causes mediation of
exchange, as examined using the pH-sensitive dye BCPCF-AM
(28). Because
immunofluorescence studies localized PAT1 to the apical membrane of rat
proximal tubule (Fig. 1), we
next tested whether an apical
exchanger is present in the proximal tubule. Tubules were first bathed and
perfused with solutions containing 25 mM
at pH 7.4. DIDS and EIPA were
introduced to the bath and perfusate to inhibit basolateral
cotransport and luminal Na+/H+ exchange, respectively,
and Cl was removed from the lumen. Representative
pHi tracings are shown in Fig.
2 demonstrating the induction of cell acidification in response to
luminal Cl addition in the presence (A) or absence
of
(B). In the presence of
,
addition of Cl in the lumen decreased the pHi
from 7.46 ± 0.01 to 7.08 ± 0.02 at a rate of 0.39 ± 0.01
pH unit/min. In the absence of
,
addition of Cl resulted in pHi reduction from
7.39 ± 0.03 to 7.16 ± 0.05 at the rate of 0.2 ± 0.05 pH
unit/min. When expressed in EBF, the rate of Cl/base
exchange was significantly higher in the presence of
(Fig. 3), with the equivalent
base transport of 30.9 ± 6.3 pmol ·
mm1 · min1
(n = 5) in the presence and 5.7 ± 1.1 pmol ·
mm1 · min1
(n = 5) in the absence of
(P < 0.05). These results demonstrate that rats express an apical
exchanger in the proximal tubule, with
Cl/OH exchange being a minor portion of
the observed Cl/base exchange.
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Metabolic production of CO2 can result in the generation of
cytosolic levels that are probably
greater than those of hydroxyl (OH). As such, it is
plausible that even in the absence of exogenous
, the
intracellular base species that is transported in exchange for luminal
Cl (Fig.
2B) is actually
and not OH. To
determine whether OH is indeed the base species transported
in Fig. 2B, we assayed
the apical Cl/base exchanger in isolated microperfused
kidney proximal tubules in the reverse mode and in the absence of
. This
means that the cell pH was assayed in response to the removal (rather than the
addition) of luminal Cl. This maneuver results in the
movement of luminal (extracellular) OH into the cell via
exchange and, as a result, bypasses the contribution of the endogenous
CO2 production to the anion exchanger activity. As shown in
Fig. 3B, removal of
luminal Cl resulted in intracellular alkalinization, which
returned to baseline upon switching back to the
Cl-containing perfusate. The results of multiple experiments
demonstrated that removal of luminal Cl caused significant
intracellular alkalinization in isolated microperfused proximal tubule cells
(with the rate of 0.095 ± 0.03, P < 0.05 vs. baseline
pHi; n = 5). Taken together, the results of experiments in
Figs. 2 and
3 demonstrate the presence of
Cl/OH exchange and
exchange in the apical membrane of kidney proximal tubule.
In the next series of experiments, we tested the effect of DIDS on the
apical
/OH
exchanger. In the experiments shown in Figs.
2 and
3, DIDS was present at 0.3 mM
in the bath to inhibit the basolateral
cotransporter. To examine the effect of luminal DIDS on the apical
Cl/base exchanger, we added 0.5 mM DIDS to the lumen. No
EIPA was added to the lumen in these experiments. The reason for removing EIPA
from the lumen in these series of experiments is that DIDS and EIPA
coprecipitate when added to the same solution. Under these circumstances, the
activity of the luminal
exchanger measured upon addition of luminal Cl decreased
from 0.23 ± 0.03 pH unit/min in the absence of luminal DIDS to 0.09
± 0.02 pH unit/min in the presence of 0.5 mM of DIDS in the lumen
(P < 0.01; n = 3).
Figure 4A summarizes
the results of these experiments and demonstrates that DIDS inhibited the
apical Cl/
exchange
by 61% (P < 0.05).
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Next, we examined the effect of DIDS on the activity of the apical Cl/OH exchange in a manner similar to the above experiments. The activity of the luminal Cl/OH exchanger measured upon addition of luminal Cl decreased from 0.16 ± 0.03 pH unit/min in the absence of luminal DIDS to 0.08 pH ± 0.01 pH unit/min in the presence of 0.5 mM DIDS in the lumen (P < 0.05; n = 3). Figure 4B summarizes the results of these experiments and demonstrates that DIDS inhibited the apical Cl/OH exchange by 50% (P < 0.05).
A recent study indicated that the presence of physiological concentrations
of oxalate inhibits the
exchange mode of PAT1 in oocytes injected with PAT1 cRNA
(17). To examine whether
oxalate can inhibit the apical
and
Cl/OH exchangers in rat kidney proximal
tubule, we determined the rate of cell acidification in response to luminal
Cl addition in isolated microperfused proximal tubule in the
presence of 200 µM of oxalate added to the lumen. Experiments were
performed in the presence or absence of bicarbonate at pH 7.4, with DIDS in
the bath and EIPA in the lumen. Representative pHi tracings for
bicarbonate-containing solutions are shown in
Fig. 5A and
demonstrate comparable cell acidification in response to luminal
Cl addition in the presence or absence of oxalate in the
lumen. In the presence of bicarbonate in the bath and lumen and oxalate in the
lumen (Fig. 5Aa),
addition of Cl to the lumen decreased the pHi
from 7.41 ± 0.01 to 6.88 ± 0.02 at a rate of 0.37 ± 0.03
pH unit/min (n = 3). When expressed in EBF, the results translated
into a rate of 29.8 ± 3.9
pmol·mm1·min1.
This was not different from the rate of cell acidification in the absence of
oxalate, which was 0.39 ± 0.09 pH unit/min (n = 5)
(Fig. 5Ab). When
expressed in EBF, the results translated into a rate of 30.9 ± 6.3 pmol
· mm1 ·
min1. When compared, the results indicate that
the apical
exchanger is not inhibited by oxalate (Fig.
5B) (P > 0.05).
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In the absence of bicarbonate but presence of oxalate in the lumen,
addition of Cl to the lumen decreased the pHi
from 7.43 ± 0.02 to 7.04 ± 0.04 at a rate of 0.31 ± 0.03
pH unit/min (n = 4). Representative pHi tracings are shown
in Fig. 6Aa. When
expressed in EBF, the results translated into a rate of 8.9 ± 0.9 pmol
· mm1 ·
min1. Compared with the rate of cell
acidification in the absence of oxalate (0.2 ± 0.05 pH unit/min and an
EBF of 5.7 ± 1.1 pmol · mm1
· min1)
(Fig. 6Ab), the
results indicate the presence of a minor, albeit nonsignificant, stimulatory
effect of oxalate on the apical Cl/OH
exchanger (Fig. 6B).
Taken together, the results of the above experiments indicate that the
presence of physiological concentrations of luminal oxalate does not inhibit
the apical
and
Cl/OH exchangers in rat kidney proximal
tubule (and indeed it may have a minor stimulatory effect on
Cl/OH exchanger).
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Measurement of Free Oxalate in Oxalate-Containing Experiments
To determine the concentration of free oxalate in the lumen under experimental conditions, we measured free oxalate in the perfusate by spectrophotometric enzymatic assay (see METHODS). The results indicated that there was no precipitation or chelation of oxalate by other ions (i.e., Ca2+ or Mg2+) in perfusate solutions at the physiological concentration of oxalate (200 µM) used in our experiments.
The above experiments demonstrate the localization of SLC26A6 as well as
Cl/OH and
exchange on the apical membranes of rat kidney proximal tubule. In the last
series of these experiments, we tested whether SLC26A6 (PAT1) mediates both
Cl/OH and
exchange in oocytes. Accordingly, oocytes were injected with PAT1 cRNA, and
their pHi was monitored in response to sequential removal and addition of
Cl according to published methods from our laboratory
(40).
The representative pHi tracings in
Fig. 7A demonstrate
that in the absence of
in the
perfusate, switching to the Cl-free solution resulted in
intracellular alkalinization in PAT1-expressing oocytes, which returned to
baseline upon switching back to the Cl-containing solution.
In the presence of
,
switching to the Cl-free solution resulted in an
intracellular alkalinization that was faster than in the absence of
in
PAT1-expressing oocytes (Fig.
7B). Switching back to the
Cl-containing solution caused the pHi to return
to normal. Control (water injected) oocytes did not demonstrate any
pHi alteration in response to exposure to the
Cl-free medium in the absence of
(Fig. 7C). The rate of
Cl/base exchanger activity was 0.045 ± 0.001 pH
unit/min (n = 3) in the absence of
(mediated via Cl/OH exchange) and 0.128
± 0.002 pH unit/min (n = 4) in the presence of
(mediated via
exchange) in oocytes injected with mouse PAT1 cRNA (P < 0.02 for
absence vs. presence of bicarbonate). Taken together, these results indicate
that PAT1 mediates both Cl/OH and
exchange.
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DISCUSSION |
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A new family of anion exchangers has been identified that is referred to as
SLC26A. Several members of this family function as an apical
exchanger in distinct epithelial tissues. One member of this family, SLC26A3,
or DRA (downregulated in adenoma), is an apical
exchanger in the colon and pancreatic ducts
(16,
24). Another is SLC26A4, or
pendrin, which is an apical
exchanger (36,
37) in the kidney cortical
collecting duct (30,
36). Whereas the mRNA
expression of pendrin is detected in both proximal tubule and cortical
collecting duct (37),
immunocytochemical and immunohistochemical staining localizes this exchanger
only to cortical collecting ducts
(28,
36,
41).
SLC26A6, or PAT1 (or CFEX), is an apical
exchanger in the duodenum
(40). The duodenum and
proximal tubule share remarkable similarity with each other with respect to
their role in fluid and electrolyte absorption. Both are responsible for the
bulk of fluid and electrolyte absorption in their respective organs, which is
accomplished via identical apical membrane protein repertoire. These include
Na+/H+ exchanger (NHE3) and Na+-glucose
cotransporter (SGLT) to absorb Na+ and apical
exchanger (PAT1) to absorb Cl
(8,
14,
25,
33,
40). The secretion of
bicarbonate in exchange for Cl (via
exchange) does not conflict with the role of the duodenum and kidney proximal
tubule in
absorption via the
apical Na+/H+ exchanger NHE3. The role of this latter
exchanger in
absorption in both
tissues has been well documented in NHE3 null mice, which develop an alkaline
luminal fluid in their duodena and show reduced
absorption in the kidney proximal
tubule (31). Taken together,
the results of the above studies demonstrate the coordinated regulation of
Na+ and
absorption via
apical Na+/H+ exchanger as well as Cl
absorption and
secretion via the
apical
exchanger in a model epithelial cell. For the apical NHE3 and PAT1 (SLC26A6)
to mediate net
and
Cl absorption in the duodenum, the stoichiometry of NHE3 and
PAT1 may not be one to one. In other words, whereas the activation of the
apical
exchanger results in the absorption of Cl and secretion of
, the small intestine may
demonstrate a net absorption of
secondary to the activation of the apical Na+/H+
exchanger. This has become very clear in NHE3 null mice
(31). Similar to the duodenum,
the parallel arrangement of apical
exchanger and Na+/H+ exchanger should accomplish the
reabsorption of Cl and
via PAT1 and NHE3, respectively,
in the kidney proximal tubule.
Two recent studies confirmed that PAT1 indeed mediates
exchange (17,
43). Both studies, in
addition, indicated that PAT1 can also mediate Cl/oxalate
exchange in an oocyte expression system
(17,
43). One of these studies
(17) found that the presence
of oxalate can inhibit the mediation of
exchange by PAT1. In our studies, physiological concentrations of oxalate in
the lumen did not inhibit the apical
exchange in isolated microperfused proximal tubule, strongly suggesting that
exchange is a major mechanism for the reabsorption of Cl
under physiological conditions. Our functional studies demonstrate that PAT1
mediates Cl/OH and
exchange in an in vitro expression system
(Fig. 7). Our microperfusion
studies demonstrate the presence of both
Cl/OH and
exchangers on the apical membranes of rat kidney proximal tubule (Figs.
2 and
3). On the basis of
immunocytochemical studies demonstrating the expression of PAT1 on the apical
membranes of rat kidney proximal tubule, we suggest that PAT1 is mediating the
apical Cl/OH and
exchanges in rat kidney proximal tubule.
The electrogenicity of SLC26A6 (20, 43) does not conflict with the historical observations indicating that reabsorption of NaCl in the proximal tubule was electroneutral and did not alter the transepithelial membrane potential. The presence of apical K+ channels in kidney proximal tubule likely offsets any alterations in membrane potential otherwise resulting from the operation of electrogenic apical Cl/base exchanger.
In conclusion, SLC26A6 (also referred to as PAT1 or CFEX) is located on the
apical membrane of rat kidney proximal tubule. Functional studies in isolated
microperfused rat proximal tubule demonstrated the presence of a robust apical
exchange that is not inhibited by oxalate. We propose that SLC26A6 (PAT1) is
likely responsible for the apical
(and
Cl/OH) exchanger in kidney proximal
tubule.
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DISCLOSURES |
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FOOTNOTES |
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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.
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REFERENCES |
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