From the Department of Medicine, University of
Cincinnati School of Medicine, Cincinnati, Ohio 45267-0585 and the
§ Veterans Affairs Medical Center,
Cincinnati, Ohio 45267
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ABSTRACT |
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Functional properties of a cloned human kidney
Na+:HCO3 cotransporter (NBC-1) were
studied in cultured HEK-293 cells that were transiently transfected
with NBC-1 cDNA. The Na+:HCO3
cotransporter activity was assayed as the Na+ and
HCO3
dependent pHi recovery from
intracellular acidosis with the use of the pH-sensitive dye
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. In acid-loaded cells
and in the presence of amiloride (to block Na+/H+ exchange), switching to a
Na+-containing solution (115 mM) resulted in
rapid pHi recovery only in the presence of
HCO3
. This recovery was completely abolished by 300 µM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid.
Replacing the Na+ with Li+ (115 mM)
caused significant HCO3
-dependent,
DIDS-sensitive pHi recovery from intracellular acidosis, with
Li+ showing lower affinity than Na+. Potassium
(K+) had no affinity for the
Na+:HCO3
cotransporter. The
Na+-dependent HCO3
cotransport was abolished in the presence of 0.2 mM
harmaline. The Na+:HCO3
cotransporter
could also function in Na+:OH
cotransport
mode, although only at high external pH (7.8). Based on functional
similarities with the mammalian kidney experiments, we propose that
NBC-1 is the proximal tubule Na+:HCO3
cotransporter.
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INTRODUCTION |
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The majority of the filtered load of HCO3
is reabsorbed in the kidney proximal tubule via the luminal
Na+/H+ exchanger NHE-3 (1-4). The exit of
HCO3
across the basolateral membrane of proximal
tubule is via the Na+:HCO3
cotransporter (5-8). The
Na+:HCO3
cotransporter
(NBC)1 mediates an
electrogenic process with an stoichiometry of 3 eq of
HCO3
per Na+ (9, 10).
Recent studies have indicated that the actual ionic mechanism involves
the cotransport of Na+,
HCO3
, and
CO32
in a 1:1:1 ratio (11). In
addition to reabsorption of HCO3
in
proximal tubule, NBC also plays an important role in cell pH regulation
in several tissues, including brain, liver, heart, and lung
(12-17).
Functional studies support the presence of more than one NBC isoform as
judged by direction and stoichiometry of the cotransporter. In kidney,
NBC activity leads to cell acidification, whereas in other tissues
(such as liver and heart) its function leads to cell alkalinization
(5-8, 13, 14). Furthermore, NBC has a stoichiometry of 3 eq of
HCO3 per Na+ ion in the
kidney (9, 10) but shows a stoichiometry of 2 eq of
HCO3
per Na+ in other
tissues (12).
We recently cloned and functionally expressed a human kidney
Na+:HCO3 cotransporter
(18). In addition to the kidney, the
Na+:HCO3
cotransporter
(called here NBC-1) is highly expressed in pancreas, with detectable
levels in brain (18). The human NBC-1 (18) shows 80% homology to the
amphibian Na+:HCO3
cotransporter (19). Both the human NBC-1 (18) and the amphibian NBC
(19) mediate Na+-dependent
HCO3
cotransport in a DIDS-sensitive
manner. The purpose of the current study was to examine the functional
properties of the human kidney NBC. Accordingly, cultured HEK-293 cells
were transiently transfected with the NBC-1 cDNA and studied.
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EXPERIMENTAL PROCEDURES |
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Cell Culture Procedures-- HEK-293 cells were cultured in Dulbecco's modified Eagle's medium containing 100 units/ml penicillin-G and supplemented with 10% fetal bovine serum. Cultured cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 in air. The medium was replaced every other day.
Transient Transfection-- Cultured HEK-293 cells were plated on coverslips and transfected at 60% of confluence with 8 µg of the full-length human NBC-1 cDNA construct (in the cloning/expression vector pCMV.SPORT1) by calcium phosphate-DNA coprecipitation (20). Cells were assayed 44-52 h after transfection.
Intracellular pH Measurement--
Changes in intracellular pH
(pHi) were monitored using the acetoxymethyl ester of the
pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF-AM) as described (21-23). HEK-293 cells were grown to confluence on
coverslips and incubated in the presence of 5 µM BCECF in
a Na+-free solution consisting of 115 mM
tetramethylammonium-Cl and 25 mM KHCO3, pH 7.4 (solution A, Table I). pHi was measured in a thermostatically
controlled holding chamber (37 °C) in a Delta Scan dual excitation
spectrofluorometer (Photon Technology International, South Brunswick,
NJ). The monolayer was then perfused with the appropriate solutions
(Table I). The fluorescence ratio at excitation wavelengths of 500 and
450 nm was utilized to determine intracellular pH values in the
experimental groups by comparison with the calibration curve that was
generated by KCl/nigericin technique. The fluorescence emission was
recorded at 525 nm. The Na+:HCO3 cotransporter
activity was determined as the initial rate of the DIDS-sensitive,
Na+-dependent pHi recovery
(dpHi/dt, pH/min) in a
HCO3
-containing solution following an
acid load induced by
NH3/NH4+ withdrawal. The
experiments were performed in the presence of 1 mM
amiloride to block the Na+/H+ exchanger
activity. The experiments were repeated in the absence of
HCO3
and CO2
and bubbled with O2 to determine whether NBC can
mediate Na+:OH
(hydroxyl) cotransport. The
dpHi/dt was calculated by fitting to a linear
equation the first 2 min of the time course of intracellular pH
recovery. Correlation coefficients for these linear fits averaged
0.984 ± 0.004
Materials-- Dulbecco's modified Eagle's medium was purchased from Life Technologies, Inc. BCECF-AM was from Molecular Probes Inc. Nigericin, DIDS, amiloride, and other chemicals were purchased from Sigma.
Statistics-- Results are expressed as means ± S.E. Statistical significance between experimental groups was determined by Student's t test or by one-way analysis of variance.
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RESULTS |
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Na+ and HCO3
Dependence of the Cloned Human Kidney NBC (Influx Mode)--
In the
first series of experiments, cells were incubated and loaded with BCECF
in Na+-free solution (solution A, Table
I), exposed to
NH4+ for 10 min (solution B, Table I),
and acid-loaded by switching to an
NH4+-free solution (solution A, Table
I). The base-line pHi in sodium-free solution was 7.11 ± 0.02. In the presence of 1 mM amiloride (to block
Na+/H+ exchange), switching to a
Na+ (115 mM) and
HCO3
-containing solution (solution C,
Table I) resulted in rapid pHi recovery from acidosis in
transfected cells (Fig. 1A),
which was completely inhibited by 300 µM DIDS. The rate
of pHi recovery was 0.190 ± 0.013 pH/min in transfected cells (n = 5). This recovery was
HCO3
-dependent, as shown
by lack of significant pHi recovery in the absence of
HCO3
(solution D, Table I) (Fig.
1B). In HCO3
-free solution,
pHi recovery was 0.015 ± 0.003 pH/min in transfected
cells (p < 0.001 versus
HCO3
-containing solution,
n = 5). Nontransfected cells showed little Na+-dependent
HCO3
cotransport (Fig. 1C)
with pHi recovery of 0.017 ± 0.002 pH/min
(p < 0.001 versus transfected
cells, n = 5).
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Na+ and
HCO3 Dependence of
NBC (Efflux Mode)--
NBC activity in transfected cells was also
measured in the efflux mode. As shown in Fig.
2, switching the cells from a
Na+ and HCO3
-containing
medium (solution C, Table I) to a Na+-free solution
(solution A, Table I) resulted in rapid cell
acidification,2 with
pH of
0.27 pH unit (n = 3). This cell acidification was reversible, with pHi returning to base line upon switching back
to the Na+-containing solution (solution A, Table I) (Fig.
2A), with
pH of 0.31 pH unit (n = 3). The
pHi recovery back to base line was completely inhibited in the
presence of 300 µM DIDS (Fig. 2A)
(n = 3). Neither cell acidification nor pHi
recovery were observed in the absence of
HCO3
(Fig. 2B), indicating
the dependence of the transporter on Na+ and
HCO3
. Taken together with Fig. 1,
these studies indicate that the cloned cDNA encodes a
Na+:HCO3
cotransporter.
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Interaction of Lithium (Li+) with the Cloned
NBC--
The ability of NBC to mediate
Li+:HCO3 cotransport was
next tested. As shown in Fig.
3A, exposing the cells to a
Li+-containing solution (solution E, Table I) caused
significant recovery from intracellular acidosis. Switching from the
Li+-containing solution to the Na+-containing
solution (solution A, Table I) further increased the rate of
pHi recovery (Fig. 3A). At comparable acidic pHi (nadir pH of 6.282 ± 0.039 and 6.32 ± 0.027 for
Li+ and Na+ experiments, respectively,
p > 0.05, n = 4) the rate of
pHi recovery caused by Na+ was 4-fold higher then
Li+ (Fig. 3, A and B) (pHi
recovery was 0.185 ± 0.003 pH/min in the presence of
Na+ and 0.045 ± 0.003 in the presence of
Li+, p < 0.001, n = 4 for
each group). The Li+-dependent
HCO3
cotransport was completely
inhibited in the presence of 300 µM DIDS (Fig.
3C) and was not detected in nontransfected cells (Fig. 3D). These results indicate that Li+ can
substitute for Na+ on NBC, with Li+ showing
lower rates in mediating HCO3
transport. This is in contrast to the proximal tubule luminal NHE,
where Li+ has much stronger affinity than Na+
(1).
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Inhibition of the Cloned NBC by Harmaline--
The purpose of the
next series of experiments was to examine the interaction of harmaline
with NBC. As indicated in Fig. 4, the
presence of 0.2 mM harmaline completely blocked the
Na+-dependent pHi recovery in
HCO3-containing media (the experiments
were performed in the presence of 1 mM amiloride to block
Na+/H+ exchange). The inhibition by harmaline
was reversible as shown by recovery from cell acidosis upon switching
to a harmaline-free solution (Fig. 4). pHi recovery from
acidosis was 0.184 ± 0.005 pH/min in the absence of harmaline and
0.007 ± 0.002 in the presence of harmaline (p < 0.001, n = 4 for each group). pHi recovery upon
removal of harmaline was 0.178 ± 0.006 pH/min (n = 4).
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Effect of External pH on the Cloned NBC--
NBC in rabbit kidney
cortex is absolutely dependent on HCO3
(24) and does not demonstrate any affinity for OH
whereas
in colon it can also accept OH
(25). To examine the
interaction of human kidney NBC with OH
, cells
were transfected and assayed for pHi recovery from an acid load
in the presence of varying pHo and the absence of
HCO3
. As shown in Fig.
5A, in the absence of
HCO3
and at pHo 7.4, transfected cells showed little recovery from an acid load, indicating
that at normal pH, NBC-1 has very low affinity for OH
(pHi recovery from acidosis was 0.020 ± 0.004 pH/min in
the absence of HCO3
(p < 0.001 versus
HCO3
-containing solution,
n = 5 for each group). Increasing the inward OH
gradient by increasing the external pH to 7.8 caused
significant Na+-dependent pHi recovery
from an acid load (0.085 ± 0.005, p < 0.001 versus pHo 7.4, n = 5) that was
abolished in the presence of DIDS (Fig. 5B).
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DISCUSSION |
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The results of current experiments indicate that the cloned human
kidney Na+:HCO3
cotransporter (NBC-1) accepts Na+ and
HCO3
and is inhibited by DIDS (Figs. 1
and 2). The results further indicate that Li+ can
substitute for Na+ on NBC-1, with Li+ showing
decreased capacity to mediate
HCO3
-dependent pHi
recovery compared with Na+ (Fig. 3). NBC-1 is inhibited by
DIDS and harmaline (Fig. 4) and can accept OH
in place of
HCO3
, with
HCO3
showing much higher affinity than
OH
(Fig. 5). NBC does not function in
K+:HCO3
cotransport mode
(Fig. 6).
We recently cloned the human kidney NBC-1 (18). Based on the deduced
amino acid sequence, the cDNA encodes a protein with a molecular
mass of ~116 kDa. Northern blot analysis reveals that NBC-1 encodes a
7.6-kilobase mRNA that is highly expressed in kidney and pancreas,
with lower levels of expression in the brain (18). NBC-1 mRNA was
not detected in the liver, lung, and heart (18) despite functional
studies indicating the presence of
Na+:HCO3 cotransport in
these tissues. These results strongly suggest that the
Na+:HCO3
cotransport in
these latter tissues might be another isoform from this family. The
human kidney NBC cDNA is highly homologous with the amphibian
kidney NBC and shares striking similarities at both structural and
functional levels with that cotransporter (19).
We have cloned rat NBC-1 and examined its mRNA distribution in different tissues as well as various nephron segments (28). Rat NBC-1 is highly expressed in kidney and brain but shows low levels of expression in stomach and colon (28). Nephron segment distribution studies revealed that NBC-1 is predominantly expressed in proximal tubules (28).
The results in Fig. 1 demonstrate that the
Na+:HCO3 cotransport does
not mediate complete pHi recovery from acidosis to baseline
values in the presence of amiloride. Whereas in the absence of
amiloride, intracellular pHi returns to base-line values (data
not shown), further studies are needed to determine whether NHE
activity is indeed essential for complete pHi recovery from
acidosis.
The results of Fig. 3 demonstrate the interaction of Li+
with NBC and indicate that the human kidney NBC can accept
Li+ in place of Na+, with
Li+:HCO3 cotransport
demonstrating lower transport rate than
Na+:HCO3
cotransport (Fig.
3). This is very similar to the NBC in basolateral membranes of rabbit
kidney proximal tubule (11, 29) and is opposite to the luminal NHE,
which has much higher affinity for Li+ than Na+
(1).
The human kidney NBC was completely inhibited in the presence of
harmaline (Fig. 4) and DIDS (Fig. 1), indicating that it has an
inhibitory profile similar to the mammalian NBC in basolateral membranes of rat or rabbit kidney proximal tubule (11, 30). Harmaline
is known to inhibit the rabbit kidney proximal tubule NBC by binding to
the Na+ site (11), whereas DIDS binds to the
HCO3 site (5-8). This latter
conclusion is based on the fact that DIDS inhibits both
Na+-dependent as well as
Na+-independent HCO3
transporters.
The anion exchanger AE1 (band-3) in red blood cells can mediate the
transport of Na+ and HCO3
via ion pair (Na+:CO32
)
(31-33). According to this mechanism, the positive charge of the
Na+ ion is being shielded by a
CO32
(31), allowing the cotransport to
be mediated as an anion via AE1. In support of the ion pair functioning
as an anion, we find that cation inhibitors do not inhibit the
transport of Na+ via band-3 (31). NBC was cloned based on
homology to anion exchanger isoforms (18), raising the possibility that
the Na+:CO32
ion pair may
be the actual substrate for transport on this protein. The results of
the experiments in Figs. 3 and 4, however, argue against
Na+:CO32
ion pair as the
substrate. The first argument against ion pair formation concerns the
relative affinities of Na+ and Li+. The
association constant of Li+ for ion pair formation with
CO32
is 4-fold greater than of
Na+ (32), consistent with Li+ having higher
affinity than Na+ for transport via red cell band-3 (33).
The results of Fig. 3 indicate that the ability of Li+ to
mediate HCO3
transport via NBC is
significantly lower than Na+. This difference is opposite
to that for ion pair formation with CO32
. The other argument against ion
pair formation is inhibition of NBC by alkaloid harmaline (Fig. 4).
Harmaline is an organic cation known to compete at the Na+
site of several Na+-coupled transport systems in kidney
proximal tubules such as the Na+/H+ exchanger
and the Na+/glucose cotransporter. Although we have not
studied the kinetics of NBC inhibition by harmaline, we suggest that
based on similarities with the renal basolateral
Na+:HCO3
cotransporter in
rabbit and rat kidney (11, 30), harmaline inhibits the cloned human NBC
by interaction with the Na+ site. These results, in
conjunction with the results of Li+ studies, strongly argue
that Na+ directly interacts at a distinct site on the NBC
rather than via ion pair formation with
CO32
.
The human kidney NBC accepts OH in place of
HCO3
, with OH
demonstrating much lower
Na+-dependent transport rates than
HCO3
(Fig. 5). These results are in
agreement with functional studies in rabbit colon, indicating lower
affinity of NBC for OH
(25). The rabbit kidney NBC on the
other hand shows absolute dependence on
HCO3
and does not demonstrate any
affinity for OH
(24). It is worth mentioning that
functional studies in rabbit kidney cortex were performed at
physiologic pH (pHo 7.4) (24). As such, the affinity of rabbit
kidney cotransporter for OH
at external pH values similar
to current experiments (i.e. pHo 7.8) remains
unknown.
Kidney NBC functions in an outwardly directed mode under physiologic
conditions (5-8), resulting in trans-vectorial transport of
HCO3 from lumen to blood. As such, the
physiologic role of the Na+:OH
cotransport
mode remains speculative, as carbonic anhydrase activity in the kidney
proximal tubule cells couples the CO2 with OH
to generate HCO3
, thereby diminishing
the concentration of intracellular OH
. We propose that
the physiologic significance of Na+:OH
cotransport mode may be in the colon, where secretion of acid into the
lumen via NHE-3 may not lead to the reabsorption of
HCO3
, as the luminal
[HCO3
] is negligible (34), but
rather results in generation of intracellular OH
. The
Na+:OH
cotransporter will then exit the cell
across the basolateral membrane, thereby regulating cell pH.
In conclusion, based on functional properties (Figs. 1-6) and nephron
segment distribution studies (28), we propose that NBC-1 is the kidney
proximal tubule Na+:HCO3
cotransporter.
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ACKNOWLEDGEMENT |
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The technical assistance of Holli Shumaker is greatly appreciated.
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FOOTNOTES |
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* This work was supported by the National Institutes of Health Grants DK 46789 and DK 52821 and by funds from Dialysis Clinic Inc. (to M. S.).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.
¶ To whom correspondence should be addressed: University of Cincinnati Medical Center, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585. Tel.: 513-558-5462; Fax: 513-558-4309; E-mail: manoocher.soleimani{at}uc.edu.
1
The abbreviations used are: NBC,
Na+:HCO3 cotransporter;
DIDS, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid; BCECF,
2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein.
2
For the
Na+:HCO3 cotransporter to
work in the efflux mode, the cells were loaded with a high
sodium-containing solution (140 mM) for 30 min as compared
with regular solution (Table I, solution A), which has 115 mM sodium. Although the difference between the sodium
concentrations in the two solutions is only 25 mM, we
observe that NBC can function in the efflux mode only at higher
Na+ concentration, indicating that incubation with high
Na+ solution increases intracellular Na+ and
therefore allows for the
Na+:HCO3
cotransporter to
function in the efflux mode.
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REFERENCES |
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