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cotransporter (NBC-1)
1 Department of Medicine, University of Cincinnati School of Medicine, and 2 Veterans Affairs Medical Center, Cincinnati, Ohio 45267-0585
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The purpose of the present experiments was to examine the effect of
potassium deprivation on the expression of the renal basolateral
Na+-HCO3
cotransporter (NBC-1). Rats were
placed on a K+-free diet for various time intervals and
examined. NBC-1 mRNA levels increased by about threefold in the cortex
(P < 0.04) at 72 h of K+ deprivation and
remained elevated at 21 days. NBC activity increased by ~110% in
proximal tubule suspensions, with the activity increasing from 0.091 in
control to 0.205 pH/min in the K+-deprived group
(P < 0.005). The inner stripe of outer medulla and
cells of medullary thick ascending limb of Henle (mTAL) showed induction of NBC-1 mRNA and activity in K+-deprived rats,
with the activity in mTAL increasing from 0.010 in control to 0.133 pH/min in the K+-deprived group (P < 0.004). K+ deprivation also increased NBC-1 mRNA levels in
the renal papilla (P < 0.02). We conclude that
1) K+ deprivation increases NBC-1 expression and
activity in proximal tubule and 2) K+
deprivation causes induction of NBC-1 expression and activity in mTAL
tubule and inner medulla. We propose that NBC-1 likely mediates
enhanced HCO3 reabsorption in proximal tubule, mTAL,
and inner medullary collecting duct in K+ deprivation and
contributes to the maintenance of metabolic alkalosis in this condition.
acid-base transporters; proximal tubule; medullary thick ascending limb
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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THE MAJORITY
OF THE FILTERED LOAD of HCO3 is reabsorbed in
the kidney proximal tubule (PT) via the luminal
Na+/H+ exchanger NHE3 (reviewed in 44). The
exit of HCO3 across the basolateral membrane of PT is
via the Na+-HCO3 cotransporter (2,
3, 9, 12, 43, 49). A number of acid-base or electrolyte
disorders are associated with decreased or increased
HCO3 reabsorption in various nephron segments.
Alterations in PT acidification in pathological states should
ultimately result from changes in the activity of luminal NHE3 and
basolateral NBC. Although molecular regulation of NHE3 has been
examined and correlated with its functional activity in several
pathophysiological states (30, 41), adaptive molecular
changes in Na+-HCO3 cotransport have been
studied in very few states.
K+ depletion is known to induce and maintain metabolic
alkalosis in rats and humans (16, 23, 26, 28, 36). The
maintenance of metabolic alkalosis in K+ depletion is
predominantly achieved through enhanced
HCO3-absorbing ability of renal tubules. In
K+ deprivation, the luminal Na+/H+
exchanger NHE3 is upregulated (46) and is likely
responsible for enhanced HCO3 reabsorption in the PT
(16, 28, 36). Molecular studies indicated that this
adaptive regulation is likely a posttranscriptional event
(46). In addition to the PT, K+ deprivation is
also associated with enhanced HCO3 reabsorption in
several other nephron segments, including distal convoluted tubules,
outer medullary collecting duct, and inner medullary collecting duct
(33, 34, 48).
A Na+-HCO3 cotransporter (NBC-1) was
recently cloned from human (14), rat (15),
and amphibian kidney (37). A spliced variant of NBC-1 is
expressed in pancreas (14). Cloning experiments have
identified several other NBC isoforms (NBC-2, kNBC-3, and mNBC-3)
(4, 25, 32, 42). NBC-1 is exclusively expressed in the
basolateral domain of PT and is the only NBC isoform that is present in
this nephron segment. (1, 39). The purpose of these
studies was to examine possible functional and molecular adaptive
regulation of the basolateral Na+-HCO3
cotransporter in kidney PT and other nephron segments in K+ deprivation.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Animal Model
Four groups of male Sprague-Dawley rats (125-150g) were used for these experiments. In the first and the second groups, rats were fed either a regular chow (control) or a potassium-deficient (KD) diet (catalog no. 960189; ICN Biochemicals) for up to 21 days. All rats had free access to distilled water. The third and the forth groups were fed control or KD diet for up to 14 days and, in addition, received 0.85% KCl added to their drinking water. Rats were anesthetized by intraperitoneal injection of 50 mg of pentobarbital sodium. Intracardiac blood was obtained for serum K+ and HCO3 concentration ([K+] and
[HCO3], respectively) measurement, and both kidneys
were rapidly removed for RNA isolation or functional studies.
RNA Isolation and Northern Hybridization
Total cellular RNA was extracted from kidney superficial cortex, inner stripe of outer medulla, inner medulla, and tubular suspensions [PT and medullary thick ascending limb of Henle (mTAL)], by the method of Chomczynski and Sacchi (18), 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) using 10× sodium
chloride-sodium phosphate-EDTA (SSPE) as transfer buffer. Membranes
were cross-linked by ultraviolet light and baked for 1 h.
Hybridization was performed according to Church and Gilbert
(19). Briefly, membranes were preprehybridized for 1 h in 0.1× SSPE-1% SDS solution at 65°C and then prehybridized for
3 h at 65°C with 0.5 M sodium phosphate buffer, pH 7.2, 7% SDS,
1% BSA, 1 mM EDTA, and 100 µg/ml sonicated cDNA. Thereafter, the
membranes were hybridized overnight in the above solution with
32P-labeled DNA probe for NBC-1, NBC-2, or kNBC-3. The cDNA
probes (25 ng) were labeled with 32P deoxynucleotides by
using the Rad-Prime DNA labeling kit (GIBCO-BRL). The membranes were
washed twice in 40 mM sodium phosphate buffer, pH 7.2, 5% SDS, 0.5%
BSA, and 1 mM EDTA for 10 min at 65°C, washed four times in 40 mM
sodium phosphate buffer, pH 7.2, 1% SDS, and 1 mM EDTA for 10 min at
65°C, exposed to PhosphorImager cassette at room temperature for
24-72 h, and read by PhosphorImager (Molecular Dynamics,
Sunnyvale, CA). For NBC-1, the full-length cDNA was used as a specific
probe. For NBC-2, the probe was generated by PCR amplification of an
EST cDNA fragment (GenBank accession no. AA216661) that encodes
nucleotides 8-2808 and is a part of human NBC-2. For kNBC-3, a
700-bp fragment corresponding to nucleotides 900-1600 was used as
a specific probe. The respective NBC-1 and kNBC-3 probes recognize
appropriate-size mRNA on high-stringency human multiple-tissue blots
(7, 14). NBC-2 cDNA probe recognizes an 8.5-kb mRNA in
brain, skeletal muscle, and lymph node tissues on a human
multiple-tissue blot (data not shown). Glyceraldehyde-3-phopshate dehydrogenase (GAPDH) cDNA probe was a generous gift from Drs. P. James
and J. Lingrel at the University of Cincinnati.
Tubular Suspension Preparation
PT and mTAL suspensions were prepared as previously described (5, 7, 24). Briefly, kidneys were removed and bathed in an ice-cold Hanks' solution consisting of (in mM) 115 NaCl, 3 KCl, 1 Na2HPO4, 1 KH2PO4, 1 MgSO4, 1 CaCl2, 25 NaHCO3, and 10 HEPES, which in addition contained 5 mM glucose, 5 mM leucine and 1 mg/ml BSA, and bubbled with 95% O2-5% CO2 at pH 7.40. The superficial cortex or the inner stripe of outer medulla was dissected and cut into small pieces and then subjected to successive 6-min periods of collagenase digestion (0.40g/l) at 37°C. After each period, the supernatant containing tubule fragments was collected after 1 min of gravity sedimentation and stored on ice. The suspension of PT or mTAL tubules was washed twice by centrifugation (1 min, 50 g) and resuspended in an appropriate solution (solution A, Table 1).
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Intracellular pH Measurements
Intracellular pH (pHi) in PT suspension (or mTAL suspensions) was measured with the use of 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) as previously described (5, 7). In brief, aliquots of BCECF-loaded tubules were diluted into glass cuvettes containing 2 ml of the experimental medium, and BCECF fluorescence was monitored with the use of a Delta Scan dual-excitation spectrofluorometer (Photon Technology International, Brunswick, NJ) that was equipped with a water-jacketed, temperature-controlled cuvette holder and a magnetic stirrer. Fluorescence intensity was recorded at an emission wavelength of 525 nm and excitation wavelengths of 500 and 450 nm. The fluorescence ratios (F500/F450) were converted into pHi values with the use of calibration curves that were established daily by Triton X-100 methods, as used before (5, 7).Measurement of NBC Activity
NBC activity in PT (or mTAL) suspensions was determined as the DIDS1-sensitive, Na+ and HCO3-dependent pHi recovery from
intracellular acidosis as previously described (6, 14).
Briefly, PT suspensions were acidified by the NH4+
pulse technique in a Na+-free solution (solution
A, Table 1). Acidification of mTAL tubular suspensions was
achieved by subjecting the cells to two cycles of wash and gentle
centrifugation in a Na+-free solution (solution
B, Table 2). Cells were then allowed to recover from acidosis in the presence of Na+ and
HCO3 ± DIDS. In both PT and mTAL suspensions,
experiments were performed in the presence of 2 mM amiloride (to block
the luminal and basolateral Na+/H+ exchangers)
and 50 nM bafilomycin (to inhibit H+-ATPase; see
RESULTS for details). The initial rate of DIDS-sensitive, Na+-dependent, HCO3-mediated
pHi recovery from acidosis (dpHi/dt)
was measured as an index of NBC activity. This method has previously
been used to determine the activity of NBC in HEK 293 cells transiently transfected with NBC-1 cDNA (6, 14). The initial rates of pHi recovery (dpHi/dt) from acidosis
were estimated from the slopes of pHi vs. time
(t) and were expressed as pH units per minutes as previously
described (4).
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Buffering Power
The intrinsic buffering power (mMH+/pH unit) was measured in PT and mTAL tubules by using the NH4+ pulse method according to the formula
i=
[NH4+]i/pHi, and as
described (38), where i is buffering power. In both PT and mTAL tubules, 50 nM bafilomycin (to inhibit
H+-ATPase) and 500 µM ouabain (to block
NH4+ entry into the cells via the
Na+-K+-ATPase pump) (29) were
present. In mTAL, 10 mM barium was added to the solution (to block
K+/NH4+ antiport) (5, 8).
BCECF-loaded cells (proximal convoluted tubule or mTAL) were initially
incubated in Na+- and HCO3-free solution
(solution A, Table 1) and monitored for pHi. At steady-state pHi, addition of 12 mM NH4SCN
caused a rapid initial increase in cell pH due to the influx of
NH3 and subsequent generation of NH4+. This
alkalinization was followed by a plateau (no acidification was observed
in both proximal convoluted tubule and mTAL tubules). Buffering power
was determined in both control and KD rats according to the formula.
Materials
32P-dCTP was purchased from New England Nuclear (Boston, MA). Nitrocellulose filters and other chemicals were purchased from Sigma (St. Louis, MO). RadPrime DNA labeling kit was purchased from GIBCO-BRL. BCECF was from Molecular Probes (Eugene, OR).Statistics
Results are expressed as means ± SE. Statistical significance between experimental groups was determined by paired or unpaired Student's t-test. P < 0.05 was considered significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Serum [K+] and [HCO3] in
K+ Deprivation
] in rats on a
K+-free diet for 2 wk was not significantly different from
control (HCO3 was 27 ± 1.9 meq/l in KD vs.
25 ± 1.7 meq/l in C, n = 4, P > 0.05).
Effect of K+ Deprivation on NBC-1 mRNA Expression in Superficial Cortex
The expression of NBC-1 mRNA is predominantly restricted to the PT under normal conditions (1, 39). In the first series of experiments, the effect of K+ deprivation on NBC-1 mRNA levels in the superficial cortex of KD animals was studied. As shown in Fig. 1A (top), NBC-1 mRNA levels increased as early as 72 h (by ~3-fold, P < 0.04, n = 3, Fig. 1B) and remained elevated at 3 wk of K+ deprivation. Neither NBC-2 nor NBC-3 mRNA was detected in the cortex or outer medulla of rats on a normal or K-free diet for 14 days.
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Measurement of NBC Activity in PT Cells
Role of Na+/H+ exchangers and
H+-ATPase in pHi recovery from acid load in PT
cells.
To assay NBC activity in PT cells, Na+-dependent and
-independent H+ transporters (NHEs and
H+-ATPase, respectively) were verified and
inhibited in the presence of specific inhibitors. BCECF-loaded PT cells
were acidified in a HCO3-free solution by
NH4+ pulse (EXPERIMENTAL PROCEDURES) and
then exposed to a Na+-containing solution (solution
B, Table 1). As shown in Fig. 2A, exposure of PT cells to
Na+ resulted in a rapid pHi recovery from
acidosis, with an initial rate of 0.519 ± 0.041 pH/min (Fig.
2B, n = 6). This recovery was reduced to
0.046 ± 0.014 pH/min in the presence of 2 mM amiloride (P < 0.002 vs. no amiloride, n = 4)
and to 0.017 ± 0.006 pH/min in the presence of both 2 mM
amiloride (an NHE inhibitor) and 50 nM bafilomycin A1
(H+-ATPase inhibitor) (P < 0.05 vs.
amiloride alone, n = 4, Fig. 2B). Nadir
pHi was not different between the three groups (Fig. 2A). In the presence of 2 mM amiloride and 50 nM
bafilomycin, the pHi recovery from acidosis was comparable
in the presence (solution B, Table 1) or the absence of
Na+ (solution A, Table 1, n = 4, P > 0.05, data not shown). These results indicate that
in the absence of CO2/HCO3 in the media,
the pHi recovery from an acid load was completely abolished
by 2 mM amiloride and 50 nM bafilomycin.
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Role of NBC in pHi recovery from acid load in PT cells.
To determine NBC activity in PT cells the experiments were repeated in
the presence of CO2/HCO3 in the media. In
the presence of 2 mM amiloride and 50 nM bafilomycin A1, the switch to
a Na+-containing solution (solution D, Table 1)
resulted in an initial pHi recovery of 0.157 ± 0.013 pH/min (Fig. 3, n = 6),
which was significantly higher than in the absence of bicarbonate
(P < 0.04 vs. Fig. 2). The presence of DIDS inhibited
the Na+- and HCO3-dependent
pHi recovery in a dose-dependent manner (Fig.
3B). The pHi recovery decreased to 0.022 ± 0.008 pH/min in the presence of 300 µM DIDS (P < 0.001 vs. vehicle, n = 3, Fig. 3B). The
DIDS-insensitive pHi recovery from acidosis in the presence
of Na+ (solution D, Table 1, Fig. 3B)
is comparable to the pHi recovery in the absence of
Na+ and the presence of 2 mM amiloride and 50 nM
bafilomycin [solution C, Table 1,
dpHi/dt = 0.022 ± 0.008 pH/min in the
presence of Na+ (Fig. 3B) vs. 0.017 ± 0.009 in the absence of Na+, P > 0.05, n = 5]. These results indicate that the presence of Na+ is required for the DIDS-sensitive
HCO3-dependent pHi recovery in PT cells.
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Effect of K+ Deprivation on NBC Activity in PT Cells
To examine the effect of K+ deprivation on NBC activity, PT suspensions were harvested from rats on a K+-free diet for 6 days, loaded with BCECF, and assayed for Na+- and HCO3-dependent pHi
recovery from intracellular acidosis (see EXPERIMENTAL PROCEDURES). The pHi recovery was significantly
increased in KD rats, with dpHi/dt increasing
from 0.107 ± 0.017 in control to 0.226 ± 0.021 pH/min in KD
rats (P < 0.02, n = 6). The
NBC-mediated pHi recovery was blocked by 300 µM DIDS in
control and KD rats. These results are summarized in Fig.
4A and indicate that the DIDS-sensitive NBC activity is significantly increased in PT cells of
KD rats (Fig. 4A). Taken together, these results demonstrate that enhanced NBC-1 mRNA expression correlates with increased NBC
activity in K+ deprivation.
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Cl Independence of
Na+-HCO3
Cotransporter in PT Cells
/HCO3 exchanger has been reported in
the PT cells (3). To determine whether a possible
upregulation of this transporter was contributing to enhanced NBC
activity in K+ deprivation, the experiments described above
(Fig. 4A) were repeated in the absence of Cl.
Accordingly, PT cells harvested from control or KD rats were incubated
in a Cl-free solution for 30 min (Cl
replaced by gluconate in solution C, Table 1) and assayed for NBC
activity as described above. The results depicted in Fig. 4B
show that the Na+-and HCO3-dependent
pHi recovery from acidosis was increased in PT cells of KD
rats (dpHi/dt was 0.079 ± 0.015 pH/min in
control, n = 5, and 0.195 ± 0.028 pH/min in KD
rats, n = 6, P < 0.04). In the presence of 300 µM DIDS, dpHi/dt in PT cells
of KD rats was reduced to 0.016 ± 0.009 pH/min (n = 4). The DIDS-sensitive dpHi/dt (NBC activity)
was increased from 0.079 ± 0.015 (n = 5) in
control to 0.195 ± 0.028 (n = 6) in KD rats
(P < 0.04, Fig. 4C). Taken together, the
results depicted in Fig. 4, A and C, indicate
that enhanced NBC activity (expressed as DIDS-sensitive
dpHi/dt) in KD is mediated via a
Cl-independent DIDS-sensitive process.
Effect of K+ Deprivation on Buffering Power in PT Cells
The intracellular buffering power was measured according to EXPERIMENTAL PROCEDURES in PT cells of rats on normal or KD diet for 6 days. PT cells were incubated in HCO3/CO2 and Na+-free media, loaded with BCECF, and monitored for pHi changes. The buffering power in PT cells was 31 ± 1.27 mM H+/pH unit (at pHi =7.13 ± 0.016) in control and 33 ± 1.89 mM H+/pH unit (at pHi = 7.09 ± 0.029) in KD rats (P > 0.05, n = 6 for each group). These results indicate that K+ deprivation did not alter the intracellular buffering power in PT cells.Effect of K+ Supplement on Cortical NBC mRNA Expression
To determine whether the KD-mediated NBC mRNA upregulation is specific to the lack of K+ in the diet, rats were placed on control or K+-free diet and supplemented with 0.85% of KCl in their drinking water.1 Animals were killed after 6 days and examined for the expression of NBC. As shown in Fig. 5, the upregulation of NBC-1 mRNA levels in the KD group was blocked in the presence of KCl in the drinking water (Fig, 5, A and B, P > 0.05, n = 4 rats in each group).
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Effect of K+ Deprivation on NBC-1 mRNA Expression in Outer Medulla and mTAL
NBC-1 mRNA levels are undetectable in the rat medulla under normal conditions (14). We tested the possibility that K+ deprivation may induce the expression of NBC-1 mRNA in various medullary segments of rat kidney. The inner stripe of outer medulla was dissected from the kidneys of control and KD animals and utilized for Northern hybridization. Figure 6 demonstrates that NBC-1 mRNA was heavily induced at 2 days of K+ deprivation (P < 0.05, Fig. 6B, n = 3), further increased at 3 days, reached a maximal plateau at 6 days (P < 0.04 compared with 3 days, n = 3 for each, Fig. 6B), and remained elevated at 21 days of K+ deprivation. To determine the nephron segment that could be the source of enhanced NBC-1 mRNA in the inner strip of outer medulla, mTAL suspensions were harvested from the inner stripe of outer medulla of both control and KD rats and utilized for Northern hybridization. As shown in Fig. 7, NBC-1 mRNA expression was sharply induced in mTAL cells of rats on K+-free diet for 2 wk. It is worth mentioning that the induction of NBC-1 mRNA in the outer medulla of KD rats was prevented when the KD animals were supplemented with 0.85% KCl in their drinking water (data not shown).
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Effect of K+ Deprivation on NBC Activity in mTAL Cells
To assay NBC activity in mTAL cells Na-dependent and -independent H+ transporters were first determined and inhibited in the presence of specific inhibitors.Inhibition of H+-dependent transporters in mTAL cells.
mTAL cells express both H+-ATPase and NHE3 on their apical
membrane (13, 22) and NHE1 on their basolateral membrane
(47). In addition, a K+/ H+
antiport (4, 8), a K+-HCO3
cotransport (10, 31), and a
Cl/HCO3 exchanger (20) are
also expressed in mTAL cells. The experiments were therefore performed
in mTAL tubules depleted of Cl and K+ by
prolonged incubation (25 min at least) in Cl- and
K+-free solution (solution A, Table 2) to keep
the Cl/HCO3 exchanger and
K+/H+ antiporter inactive. In the presence of
CO2/ HCO3, but in the absence of amiloride and
bafilomycin A1, exposure of the acid-loaded mTAL cells to the
Na+-containing solution (solution C, Table 1)
resulted in a rapid pHi recovery from intracellular
acidosis with an initial rate of 1.198 ± 0.070 pH/min (Fig.
8A). This recovery was
decreased to 0.106 ± 0.007 pH/min in the presence of 2 mM
amiloride and 50 nM bafilomycin A1 (P < 0.001, Fig. 8,
A and B). These results demonstrate the viability
of mTAL cells in Na+-, K+-, and
Cl-free medium. To examine whether the residual recovery
from acidosis was Na+ dependent, mTAL tubules were acid
loaded and exposed to a Na+-free but
HCO3-containing media (solution B, Table
2) that in addition contained 2 mM amiloride and 50 nM bafilomycin A1.
As shown in Fig. 7 (A and B), the pHi
recovery from acidosis was negligible in the absence of
Na+, indicating that the residual pHi recovery
from acidosis in the presence of 2 mM amiloride and 50 nM bafilomycin
(Fig. 8, A and B) was Na+ dependent.
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Lack of NBC activity in mTAL cells under normal conditions.
To determine whether the amiloride-resistant pHi recovery
from acidosis (Fig. 8) was HCO3 dependent, mTAL
tubules were acid loaded in a solution that was buffered with HEPES and
was free of Na+, K+, Cl, and
HCO3 (solution D, Table 2). In the absence
of HCO3, the switch to a Na+-containing
solution (solution E, Table 2) that contained 2 mM amiloride
and 50 nM bafilomycin A1 resulted in a significant recovery from
acidosis, with the pHi recovery of 0.099 ± 0.009 pH/min (Fig. 9A). This
HCO3-independent pHi recovery was
inhibited in the absence of Na+ in the media
(solution D, Table 2), with recovery rates of 0.003 ± 0.002 pH/min (Fig. 9, A and B). The
Na+-dependent pHi recovery from acidosis was
comparable in the presence or absence of HCO3 in the
solution (P > 0.05, Fig. 9C). Taken
together, these results indicate that under normal conditions, mTAL
cells do not exhibit any Na+ dependent
HCO3-cotransport activity. This result is consistent
with published reports (31). They further correlate with
Northern hybridization experiments indicating the absence of NBC-1 mRNA
expression in mTAL cells under normal conditions (Fig. 7).
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Induction of NBC activity in mTAL cells of KD rats.
To determine whether induction of NBC-1 mRNA expression correlates with
NBC activity, mTAL tubule suspensions were harvested from animals on
the control or K+-free diet for 14 days. mTAL cells were
incubated and acid loaded in a HCO3-containing,
Na+-, K+-, and Cl-free solution
(solution B, Table 2) and assayed for DIDS-sensitive, Na+- and HCO3-dependent pHi
recovery. Amiloride at 2 mM and bafilomycin at 50 nM were present. The
results are summarized in Fig. 10 and
indicate that in the presence of HCO3,
Na+-dependent pHi recovery was significantly
higher in mTAL cells of KD rats (Fig. 10A), with the
recovery rate of 0.227 ± 0.007, n = 5, vs.
0.110 ± 0.009 pH/min in control rats, n = 4 (P < 0.003). The increased pHi recovery in
KD animals was completely blocked by 300 µM DIDS (the recovery rate
was 0.101 ± 0.005 pH/min, n = 4, P < 0.001, vs. no DIDS). Figure 10B depicts
the NBC activity as DIDS-sensitive Na+-dependent,
HCO3-mediated pHi recovery and
demonstrates a significant induction of NBC activity in mTAL tubules of
KD rats (Fig. 10B).
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i) of mTAL cells was measured in control and KD rats
for 14 days. The experiments were performed in Na+-,
K+-, Cl-, and
HCO3/CO2-free solution (solution D,
Table 2). The i was comparable in both groups (49 ± 2.15 mM H+/pH unit at pHi = 9.84 ± 0.021 in control and 45 ± 1.58 mM H+/pH unit at
pHi = 9.79 ± 0.032 in KD animals,
P > 0.05). Figure 10C indicates that in the
absence of Na+ (solution B, Table 2), no
pHi recovery was observed in acidified mTAL cells harvested
from KD rats, confirming the results in Fig. 10A that the
pHi recovery in KD animals is Na+ dependent.
Taken together, these results indicate that the increase in
dpHi/dt in mTAL of KD rats is independent of changes in
i, and is mediated via a DIDS-sensitive,
Na+-dependent HCO3 cotransporter.
Effect of K+ Deprivation on NBC-1 mRNA Expression in the Papilla of Rat Kidney
In the last series of experiments, NBC-1 mRNA expression was examined in the inner medulla of rats that were fed control or K+-free diet for 2 wk. The results of these experiments depicted in Fig. 11 demonstrate that NBC-1 mRNA abundance was increased by ~3-fold in K+ deprivation (P < 0.02, n = 3, Fig. 11B).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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The above-mentioned studies demonstrate that K+
deprivation increases the mRNA expression and activity of the
basolateral Na+-HCO3 cotransporter
(NBC-1) in PT (Figs. 1 and 4). The time course studies reveal that the
expression level of NBC-1 mRNA in the superficial cortex is upregulated
at 72 h of K+ deprivation (Fig. 1) and precedes the
onset of hypokalemia. K+ deprivation induces the mRNA
expression and activity of NBC-1 in mTAL (Figs. 7 and 10) and enhances
the expression of NBC-1 in the inner medulla (Fig. 11).
K+ depletion enhances HCO3 reabsorption
in the PT (16, 17, 23, 26, 28, 36). Functional studies
have demonstrated that the activity of the cortical luminal
Na+ /H+ exchanger (NHE3) and the basolateral
Na+-HCO3 cotransporter is increased in
K+-depleted animals (41, 46). NHE3 mRNA
expression remained unchanged (46), indicating that
enhanced NHE3 activity in K+ deprivation is likely a
posttranslational event (46). Enhanced expression of NBC-1
mRNA (Fig. 1) in the PT, however, indicates that increased NBC activity
in K+ deprivation (Fig. 4 and Ref. 41) is transcriptionally
regulated. The rats were not alkalotic despite significant increase in
NBC activity. It has been shown that the generation of metabolic
alkalosisin rat is time dependent, with animals demonstrating elevated
serum bicarbonate at 4 wk of K+ deprivation
(41). It is likely that the involvement of other bicarbonate-absorbing transporters (such as apical
Na+/H+ exchanger) is necessary for enhanced
vectorial transport of bicarbonate.
K+ deprivation is associated with a sharp induction of
NBC-1 in the inner stripe of outer medulla of rat kidney (Fig. 6). This is predominantly due to the induction of NBC-1 in mTAL cells (Figs. 7
and 10). The signal responsible for the induction of NBC-1 in mTAL
cells remains unknown. Whether NBC induction indicates enhanced HCO3 reabsorption or reflects a compensatory adaptive
regulation remains unknown. HCO3 exit across the
basolateral membrane of mTAL cells under normal conditions is
predominantly mediated via the K+-HCO3
cotransporter and Cl/HCO3 exchanger
(10, 20, 31). The K+-HCO3
cotransporter has a Michaelis-Menten coeffecient of 70 mM for the
intracellular K+, and its efflux mode is dependent on a
high intracellular [K+] (10). It is possible
that the reduction of intracellular [K+] (if it exceeds
the magnitude of extracellular [K+] reduction) decreases
the outward favorable driving force for the
K+-HCO3 cotransporter in K+
deprivation. Whether NBC-1 induction in K+ deprivation is
in response to possible downregulation of
K+-HCO3 cotransport or reflects enhanced
HCO3 reabsorption in mTAL cells remains unknown.
Additional experiments are needed to evaluate the membrane localization
of NBC in mTAL and its role in transepithelial HCO3
reabsorption in K+ deprivation.
Recent microperfusion studies from our laboratory demonstrated that
K+ deprivation enhances net HCO3
reabsorption in outer and inner medullary collecting duct of rat kidney
(33, 34). Molecular and functional studies indicated that
enhanced HCO3 reabsorption in these nephron segments
was due to the induction of colonic
H+-K+-ATPase at the luminal membrane (33,
34). Enhanced expression of the basolateral NBC-1 in inner
medullary collecting duct cells (Fig. 11) indicates that this
transporter works in concert with the luminal colonic
H+-K+-ATPase to increase the transepithelial
reabsorption of HCO3 in this nephron segment in
K+ deprivation. Whether K+ deprivation also
enhances the expression of NBC-1 in outer medullary collecting duct
cells remains speculative.
The rat mTAL absorbs up to 15% of the filtered load of
HCO3 (21). This is predominantly
accomplished via the luminal Na+/H+ exchanger
NHE-3 (21, 22) and H+-ATPase (13)
and the basolateral K+-HCO3 cotansporter
and Cl/HCO3 exchanger (10, 20,
31). Our results demonstrate the presence of an
amiloride-resistant, Na+-dependent pHi recovery
(presumably an NHE) in mTAL cells of rat kidney (Figs. 8 and 9). The
identity of this transporter and its membrane localization remain
speculative. One plausible candidate is NHE4, which is resistant to
amiloride and is likely expressed in the basolateral membrane of kidney
tubules (11, 35). This transporter does not show adaptive
regulation in K+ deprivation (see RESULTS).
The upregulation of Na+-HCO3
cotransport in the PT in K+ deprivation is of physiological
importance in that it may contribute to acid-base homeostasis by the
kidney. In addition to its role in vectorial transport of
HCO3 in kidney PT, NBC likely plays an important role
in transporting the intracellular HCO3 that is
generated in response to enhanced ammoniagenesis in K+
depletion (45). In this regard, increased NBC activity in
K+ deprivation will promote NH4+ secretion
and Na+ retention via the apical
Na+/H+ exchanger (27) and maintain
cell pH in a physiological range by extruding the
HCO3 load that is originated from both tubular fluid
and ammoniagenesis.
The mechanism(s) responsible for the upregulation or induction of NBC in various nephron segments in K+ deprivation remains unclear. The time course of the effect of K+ deprivation demonstrates that enhanced expression of NBC-1 (Figs. 1 and 5) is an early event and precedes the onset of hypokalemia. These results strongly suggest that the signal is likely activated in response to intracellular K+ depletion that occurs early in the course of K+ deprivation. The identity of this signal(s) remains speculative. K+ deprivation is associated with kidney hypertrophy, indicating activation of one or more growth factors (40). It is possible that the growth factor(s) that is activated in K+ deprivation is responsible for the enhancement of NBC-1. There is also evidence in support of a role for the pituitary gland in the adaptive regulation of ion transporters in the kidney in K+ deprivation. In rats subjected to hypophysectomy, there was blunting of kidney enlargement and colonic H+-K+-ATPase induction in K+ deprivation (46), indicating a possible role for certain pituitary hormones mediating the adaptive changes in this condition. Whether these pituitary factors are also involved in the regulation of NBC by K+ deprivation remains to be examined. It is plausible that other hormones (such as ANG II, insulin, adrenal steroids, or catecholamins) could play a role in mediating the effect of K+ deprivation on NBC mRNA expression and activity.
In conclusion, K+ deprivation enhances or induces the
expression of the basolateral Na+-HCO3
cotransporter (NBC-1) in PT, mTAL, and inner medullary collecting duct.
We propose that NBC-1 likely mediates enhanced HCO3
reabsorption in PT, mTAL, and inner medullary collecting duct and
contributes to the maintenance of metabolic alkalosis in K+ deprivation.
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ACKNOWLEDGEMENTS |
|---|
These studies were supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-46789, DK-52281, and DK-54430 and funds from Dialysis Clinic Incorporated (M. Soleimani).
| |
FOOTNOTES |
|---|
Address for reprint requests and other correspondence: M. Soleimani, Univ. of Cincinnati Medical Center, 231 Bethesda Ave., MSB 5502, Cincinnati, OH 45267-0585 (E-mail: Manoocher.Soleimani{at}uc.edu).
1 The concentration of KCl in the drinking water (0.85%) was calculated on the basis of dietary K+ intake and the volume of drinking water in control rats.
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. §1734 solely to indicate this fact.
Received 15 February 2000; accepted in final form 9 May 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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, no inhibitors,
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indicated n runs.
