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1 Institut National de la Santé et de la Recherche Médicale Unité 356, Institut Fédératif de Recherche 58, Université Pierre et Marie Curie, 75270 Paris Cedex 06; and 2 Hôpital Européen Georges Pompidou, Assistance Publique-Hôpitaux de Paris, 75908 Paris Cedex 15, France
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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In the proximal tubule, the apical Na+/H+ exchanger identified as NHE3 mediates most NaCl and NaHCO3 absorption. The purpose of this study was to analyze the long-term regulation of NHE3 during alkalosis induced by dietary NaHCO3 loading and changes in NaCl intake. Sprague-Dawley rats exposed to a low-NaCl, high-NaCl, or NaHCO3 diet for 6 days were studied. Renal cortical apical membrane vesicles (AMV) were prepared from treated and normal rats. Na+/H+ exchange was assayed as the initial rate of 22Na+ uptake in the presence of an outward H+ gradient. 22Na+ uptake measured in the presence of high-dose 5-(N-ethyl-N-isopropyl) amiloride was not different among models. Changes in NaCl intake did not affect NHE3 activity, whereas NaHCO3 loading inhibited 22Na+ uptake by 30%. AMV NHE3 protein abundance assessed by Western blot analysis was unaffected during changes in NaCl intake. During NaHCO3 loading, NHE3 protein abundance was decreased by 65%. We conclude that proximal NHE3 adapts to chronic metabolic acid-base disorders but not to changes in dietary NaCl intake.
sodium-hydrogen exchanger type 3; epithelial sodium-hydrogen exchanger; proximal tubule; acid-base status; sodium balance; sodium chloride
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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THE PROXIMAL TUBULE
NORMALLY reabsorbs ~80% of the bicarbonate and 60% of the
Na+ filtered at the glomerulus. Most of the transcellular
NaHCO3 reabsorption is mediated by the proximal tubule
apical membrane Na+/H+ exchanger, identified as
NHE3 (see Ref. 2 for a review). Functionally coupled to a
chloride/anion exchange, NHE3 also mediates most of the NaCl
reabsorption (4). This transporter may therefore be
adaptively regulated during both acid-base and Na+ balance
disturbances. In chronic metabolic acidosis, the absorptive capacity of
HCO
Although renal regulation of Na+ excretion, which is central to the control of extracellular volume regulation, occurs chiefly in the distal nephron through effects on specific aldosterone-sensitive Na+ transporters such as the amiloride-sensitive epithelial Na+ channel (EnaC) in the collecting duct and the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal tubule (19), there is abundant evidence that acute extracellular volume expansion (induced by systemic Ringer-NaCl infusion) is associated with a fall in proximal fluid absorption, an index of NaCl proximal reabsorption (see Ref. 23 for a review). In more physiological, chronic changes in Na+ balance secondary to variations in dietary NaCl intake, whether the proximal tubule adapts NaCl absorption and NHE3 activity is not clear.
The purpose of the present study was to examine whether NHE3 transport activity and protein abundance in apical membrane fractions of rat renal cortex were regulated during long-term dietary NaHCO3 loading and changes in NaCl intake. Our data demonstrate that NHE3 in the proximal tubule is appropriately regulated in response to chronic perturbation of acid-base status (i.e, alkalosis inhibits NHE3 protein abundance and activity) but not by chronic changes in NaCl intake, suggesting that this transporter does not directly participate in renal adaptation to chronic changes in NaCl intake.
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EXPERIMENTAL PROCEDURES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Materials.
5-(N-Ethyl-N-isopropyl) amiloride (EIPA)
was purchased from Molecular Probes (Eugene, OR).
4-Isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate
(HOE-642) was kindly provided by Dr. H. J. Lang (Hoechst
Marion Roussel). The rabbit polyclonal antibody to NHE3 was a gift of
Dr. R. J. Alpern (University of Texas Southwestern Medical Center,
Dallas, TX). The rabbit polyclonal antibody to NCC was a gift of Dr. D. Ellison (University of Colorado Health Sciences Center, Denver, CO).
Rabbit polyclonal antiserum against the Na+-glucose
cotransporter (SGLT1) was purchased from Chemicon (Temecula, CA).
Anti-
-actin mouse monoclonal antibody (clone AC-15) was purchased
from Sigma-Aldrich Fine Chemicals. All other chemicals were of analytic
grade. Stock solutions of EIPA and HOE-642 (100 mM) were prepared in dimethylsulfoxide.
Animal groups. Male Sprague-Dawley rats (IFFA-CREDO, L'Arbresle, France), weighing 250-350 g, were used for this study. For all experiments, a control group and one or two treated groups were handled in parallel over a 6-day period. In the first series of experiments (11 independent experiments), rats drank distilled water with either 0.28 M NaCl (NaCl-loaded group) or NaHCO3 (NaHCO3-loaded group). All of them were fed ad libitum with standard laboratory rat chow (UAR 210, UAR, Villemoisson, France) with a Na+ content of 0.4%. The control rats were fed ad libitum with standard laboratory rat chow and had free access to distilled water. In a second series of experiments (5 independent experiments), rats were fed ad libitum with low (0.009%)-sodium chow (UAR 212 Na, UAR) and had free access to tap water. The control group consisted of rats that were fed ad libitum with standard laboratory rat chow and had free access to distilled water. A third series of experiments (4 independent experiments) was performed to evaluate the effect of changes in NaCl intake on intracellular pH (pHi) sensitivity of brush-border NHE3.
In the first series, 5 of 11 experiments were carried out in rats kept in metabolic cages during the experimental period. All experiments in the second series were carried out in rats kept in metabolic cages. Urine that was spontaneously voided during each 24-h period was collected with light mineral oil in the urine collector to determine daily urinary electrolyte excretion. At the end of the experimental period, blood was collected by aortic exsanguination from animals under anesthesia provided by peritoneal injections of 4.5 and 0.2 g/100 g body wt of ketamine and xylazine, respectively. Kidneys were rapidly removed and immersed in ice-cold Hanks' modified solution containing (in mM) 137 NaCl, 5.4 KCl, 25 NaHCO3, 0.3 Na2HPO4, 0.4 KH2PO4, 0.5 MgCl2, 10 HEPES, 5 glucose, and 1 leucine, as well as 1 mg/ml BSA. Arterial pH, PCO2, and PO2 were measured with a pH/blood-gas analyzer (AVL Compact 1; AVL Instruments Médicaux, Eragny-sur-Oise, France). Serum electrolytes, protein, and creatinine were measured by standard methods with a Beckman CX7 autoanalyzer (Beckman Coulter, Villepintes, France); urine electrolytes were measured with indirect potentiometry for Na+ and K+ (Beckman E2A, Beckman Coulter); and urine creatinine was measured with a Technicon RA1000 autoanalyzer (Bayer Diagnostics, Puteaux, France). Plasma renin activity was measured by radioimmunoassay of angiotensin I generated during incubation of plasma samples in vitro (Cis Bio, Gif-sur-Yvette, France).Renal cortical apical membrane vesicle preparation. In a typical preparation, apical membrane vesicles (AMV) were isolated from whole renal cortex from two rats with the use of Ca2+ aggregation and differential centrifugations, as described previously (7). Protease inhibitors were included in all buffers. Protein contents of cortex homogenate and AMV were determined by the Bradford protein assay. Preparation of AMV was carried out concurrently in matched groups.
Enzymatic characterization of cortical apical membrane
preparations.
Activities of maltase and 
-glutamyltransferase were assayed by
standard methods with commercially available kits from Sigma.
Transport measurements. Na+/H+ exchange activity was assayed by measurement of 22Na+ uptake at 20-25°C by the rapid filtration technique. AMV were preincubated for 2 h at room temperature with a pH 6.0 medium consisting of (in mM) 100 mannitol, 60 N-methyl-D-glucamine (NMG)-nitrate, 40 NMG-gluconate, 3 EGTA, and 100 Mes/Tris. A 10-µl aliquot of AMV (20-100 µg protein) was added to 200 µl of a pH 8.0 medium containing 1.5 µCi/ml of 22Na+ and (in mM) 100 mannitol, 60 NMG-nitrate, 40 NMG-gluconate, 3 EGTA, and 100 Tris-HEPES, as well as the indicated concentrations of inhibitors. Incubation periods of 9 s were used to estimate initial rates. Uptakes were terminated by adding 1.4 ml of an ice-cold stop solution containing 20 mM Tris-HEPES, pH 7.4, and the desired potassium-D-gluconate concentration to maintain isosmolarity (equilibration medium, incubation medium, and stopping medium were always kept isosmotic). This suspension was rapidly filtered on the center of a 0.45-µm prewetted Millipore cellulose filter (HAWP) and washed with an additional 18 ml of the same ice-cold stop solution. For all experiments, nonspecific isotopic binding to the filter was measured with appropriate blanks and the results were subtracted from values of the incubated samples. The filters were dissolved in 3 ml of scintillant (Filter-count, Packard), and radioactivity was determined using a beta scintillation counter.
In studies examining NHE3 activity as a function of intravesicular pH (pHi), AMV were preincubated for 2 h at room temperature with a medium consisting of (in mM) 100 mannitol, 60 NMG-nitrate, 40 NMG-gluconate, and 3 EGTA with either 100 Mes/Tris at pH 5.5, 5.8, 6.1, and 6.5 or 100 Tris-HEPES at pH 7.0 and 7.5. A 10-µl aliqot of AMV (20 µg protein) was added to 200 µl of a pH 7.5 buffer containing 1.5 µCi/ml of 22Na+ and (in mM) 100 mannitol, 60 NMG-nitrate, 40 NMG-gluconate, 3 EGTA, and 100 Tris-HEPES, as well as 30 µM HOE-642 or 500 µM EIPA. The methods of 22Na+ uptake measurements were identical to those described above. Measurements of 22Na+ influx specific to the Na+/H+ exchanger were determined as the difference between the initial rates of H+-activated 22Na+ influx in the absence and presence of 500 µM EIPA and expressed as the Na+/H+ exchanger-mediated 22Na+ uptake. The background levels of 22Na+ influx that were not attributable to the Na+/H+ exchanger were <10%. To specifically study NHE3 activity, putative NHE2 activity was inhibited by 30 µM HOE-642, a compound that inhibits NHE2 with a much greater affinity than NHE3 [inhibition constant (Ki) values of 3 µM and 1 mM, respectively] (24).SDS-PAGE and immunoblotting.
Cortical apical membrane proteins were diluted 3:5 in 2.5× SDS loading
buffer (10 mM Tris · HCl, pH 6.8, 1% SDS, 2%
-mercaptoethanol, 13% glycerol and bromophenol blue), heated at
95°C for 10 min, and then frozen at 
20°C until use. Proteins were
separated by SDS-PAGE using a 7.5% polyacrylamide gel, transferred
electrophoretically for 1 h at 4°C from the gel to
nitrocellulose membranes (Amersham, Arlington Heights, IL), and then
stained with 0.5% Ponceau S in acetic acid to confirm equality of
loading in all lanes. Immunoblotting was performed as follows. Strips
of nitrocellulose were incubated first in 10% nonfat dry milk in PBS,
pH 7.4, for 1 h at room temperature to block nonspecific binding
of the antibody, followed by an overnight incubation at 4°C with
either rabbit polyclonal anti-NHE3 antiserum, rabbit polyclonal
antiserum against SGLT1, rabbit polyclonal antiserum against NCC, or a
mouse anti--actin antibody diluted 1:10,000, 1:30,000, 1:10,000 and
1:300,000, respectively, in PBS containing 1% nonfat dry milk.
Membranes were then washed five times with PBS containing 0.1% Tween
20 for 5 min each before incubation with a 1:3,000 dilution of goat
anti-rabbit IgG conjugated to horseradish peroxidase (Bio-Rad
Laboratories, Hercules, CA). Blots were washed as above, and
luminol-based enhanced chemiluminescence (Amersham) was used to
visualize bound antibodies on Polaroid film. Photographs of immunoblots
were digitized by using a laser scanner (ScanJet IIcx;
Hewlett-Packard), and quantification of each band was performed by
using the National Institutes of Health's Image software
(http://rsb.info.nih.gov/nih-image). Band densities were
normalized as a percentage of control values.
Statistical methods. All data are represented as means ± SE. Comparisons among groups were assessed by ANOVA or an unpaired Student's t-test. Na+/H+ exchanger-mediated 22Na+ uptake values determined at pHi 5.5-7.5 were fit to a four-parameter logistic sigmoid equation using Prism 3.0 (GraphPad Software). Comparisons of curves were assessed by an F-test. Statistical significance was established as P < 0.05.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Whole-animal data during treatments. Rats from the three groups tolerated all treatments well. At the end of the experiments, body weights of rats in the treated groups were similar but slightly lower than in control rats.
The time course of changes in daily urinary Na+ excretion during the experimental period is depicted in Fig. 1. As expected, urinary Na+ excretion was markedly decreased in rats on a low-NaCl diet, whereas rats exposed to high-Na+ intake (NaCl or NaHCO3 loading) exhibited markedly elevated natriuresis. Importantly, at the end of the experiment there was no difference between Na+ excretion in NaCl-loaded rats compared with NaHCO3-loaded rats. Natriuresis was identical in control groups from the two series of experiments. Plasma electrolytes, protein, and acid-base status of the different groups are summarized in Table 1. As expected, in rats subjected to dietary NaHCO3 loading, a significant increase in plasma HCO
concentration. Serum K+
concentration was normal in all groups and ranged from 4.1 to 4.7 mM.
There was no difference between serum K+ concentration in
NaHCO3-loaded and control rats, but significant differences
in serum K+ concentration were noted in the low- and
high-NaCl-diet groups compared with control rats. Plasma
Na+ concentrations were normal and identical in all groups.
There was no significant difference in creatinine clearance in any
experimental group, suggesting that the adaptation to the experimental
maneuver did not involve changes in glomerular filtration rate but
rather tubular processes. Plasma protein concentrations were not
altered in all treated groups compared with the control group. However, lower plasma protein concentration was found in NaCl-loaded rats than
in rats on a low-NaCl diet, probably reflecting a slight increase or
decrease in extracellular volume, respectively. As shown in Fig.
2, plasma renin activity consistently
increased in rats on a low-NaCl diet and decreased with NaCl loading.
NaHCO3 loading had no effect on plasma renin activity,
indicating no change in extracellular volume during this challenge.
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Enzymatic characterization of cortical apical membrane
preparations.
The quality of the AMV preparations isolated from the renal cortex of
control, NaCl-loaded, NaHCO3-loaded animals and animals on
a low-NaCl diet is summarized in Table 2.
Relative to their respective homogenates, membranes obtained from
control, NaCl-loaded, NaHCO3-loaded animals and animals on
a low-NaCl diet showed an identical degree of enrichment in luminal
enzyme markers maltase or -glutamyltransferase. A significantly
lower maltase-specific activity was noted in the AMV isolated from
NaHCO3-loaded rats compared with control rats. For the
following reasons, this decrease in maltase activity was likely
attributable to a specific inhibitory effect of metabolic alkalosis on
maltase rather than to a difference in membrane fractionation procedure
induced by the chronic NaHCO3 loading. First, numerically
lower maltase activity was also noted in homogenates of the same group.
Second, no difference in specific activities or enrichment factor was
noted for -glutamyltransferase. How maltase was affected by
alkalosis is not clear. Of note, a similar unexpected effect of
acid-base status (acidosis) on brush-border -glutamyl transpeptidase
has been reported by Wu et al. (33).
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-glutamyltransferase (data not shown).
Effect of chronic NaCl or NaHCO3 loading on NHE3 activity and protein abundance in cortical AMV. To determine whether chronic metabolic alkalosis would regulate NHE3, we first tested the effect of chronic NaHCO3 loading on NHE3 activity and protein abundance assayed in cortical AMV compared with control.
NHE3 Na+/H+ exchange was defined as the initial rate of 22Na+ uptake stimulated by an outward H+ gradient [pHi = 6.0; extravesicular pH (pHo) = 8.0] and sensitive to high-dose EIPA (500 µM). Because another isoform (i.e., NHE2) could have contributed to the Na+/H+ exchange activity measured in cortical AMV, we also tested the effect of 30 µM HOE-642, a compound that should have specifically inhibited NHE2 without inhibiting NHE3 (24). Figure 3 shows that chronic NaHCO3 loading resulted in a marked decrease (~30%) in EIPA-sensitive, H+-stimulated 22Na+ uptake [40.4 ± 1.4 (controls) vs. 28.6 ± 1.3 pmol · mg protein1 · 9 s1
(NaHCO3-loaded group)]. This was attributable to a
specific inhibition of NHE3 activity as the HOE compound had no effect
on 22Na+ uptake, demonstrating that no
significant NHE2 activity was present in cortical apical membranes in
basal and NaHCO3-loaded conditions.
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1 · 9 s1 (NaCl-loaded
group)], demonstrating that the inhibition of NHE3 activity observed
under NaHCO3-loading conditions was specific to alkalemia.
Of note, still no NHE2 activity could be detected after NaCl loading.
We next evaluated the effects of these treatments on NHE3 protein
abundance. Immunoblot analysis was performed on membrane protein
samples kept apart from membrane vesicles used in
22Na+ influx assays. To ascertain the
specificity of the effects on NHE3 protein abundance observed during
treatments, we tested whether SGLT1, another apical membrane protein in
the proximal tubule, was affected. -Actin protein abundance was also
determined in these membrane preparations as a loading control. Figure
4 shows immunoblots obtained of
representative AMV preparations and the mean results for the whole
series. Statistical analysis of densitometric data revealed that
NaHCO3 loading led to a marked decrease in NHE3 protein
relative abundance (~65%) in cortical apical membrane vesicles
compared with controls (normalized band density of 34.8 ± 6.9%),
whereas no significant difference was seen in the expression of NHE3
protein after NaCl loading (normalized band density of 87.6 ± 1.4%). This inhibitory effect of metabolic alkalosis on NHE3 protein
expression could not be attributable to a generalized effect of
alkalemia on apical membrane proteins because SGLT1 protein abundance
was unaffected by the different treatments (normalized band densities:
NaHCO3-loaded, 102.5 ± 6.4%; NaCl-loaded, 100.0 ± 3.8%). -Actin protein abundance in membranes from treated groups was not different from that of control (normalized band densities: NaHCO3-loaded, 108.7 ± 11.2%; NaCl-loaded, 86.4 ± 4.4%). It should be noted that percentages of inhibition of NHE3
activity and NHE3 protein abundance by chronic NaHCO3
loading were different (30 vs. 65% inhibition for activity and protein
abundance, respectively). This difference could be explained by the
existence of two forms of NHE3 in brush-border membranes
(6), active and inactive, that are both measured by
immunoblotting.
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Effect of chronic Na+ restriction on
NHE3 activity and protein abundance in cortical AMV.
Dietary restriction of NaCl intake is normally associated with a
decrease in natriuresis, and Moe et al. (22) have
demonstrated that apical Na+/H+ exchange
activity is slightly stimulated in cortical AMV isolated from rats
after chronic Na+ restriction compared with high-NaCl
intake. As high-NaCl intake did not lead to a decrease in apical
Na+/H+ exchange in our studies, we wanted to
test the effect of chronic low-NaCl intake. We therefore compared rats
fed chow with low-NaCl content with control rats fed with standard
laboratory rat chow. Figure 5 shows that
there was no effect of chronic Na+ restriction on
EIPA-sensitive, H+-stimulated 22Na+
uptake [28.0 ± 2.9 (controls) vs. 25.3 ± 2.6 pmol · mg protein1 · 9 s1
(Na+-restricted group)]. The HOE compound had no effect on
22Na+ uptake, demonstrating that no significant
NHE2 activity was present in cortical apical membranes in basal and
Na+-restricted conditions. Figure
6 shows that a low-NaCl diet did not
change the level of expression of either NHE3 or SGLT1 protein, as
determined by semiquantitative immunoblotting (normalized band densities: 97.7 ± 11.4% for NHE3 and 93.6 ± 9.6% for
SGLT1). -Actin protein abundance in membranes from rats fed a
low-NaCl diet was not different from that in the control group
(normalized band densities: 101.5 ± 13.3%).
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pHi dependence of NHE3 transport activity in cortical
AMV during chronic Na+ restriction and NaCl loading.
The number of transporters is not the only determinant of the maximal
rate of Na+/H+ exchange. Aronson et al.
(5) have described that internal protons could exert a
stimulatory effect in addition to their effect as a substrate, an
apparent allosteric effect in which protons were proposed to act at an
internal modifier site. It has also been described that
Na+/H+ activity in opossum kidney cells can be
modulated through changes in pHi sensitivity
(21). We thus tested whether regulation of NHE3 in
response to chronic changes in NaCl intake could involve a shift in
half-maximal intracellular H+ activation value. We
therefore determined pHi dependence of NHE3-mediated 22Na+ influx in cortical AMV isolated from
NaCl-loaded, low-NaCl-intake, and control rats.
22Na+ influx was measured as a function of
pHi 5.5-7.5 by equilibrating membrane vesicles in
media with the appropriate pH. As expected, 22Na+ uptake exhibited a sigmoidal pattern of
inhibition when pHi was raised, consistent with the
presence of an internal H+ modifier site (Fig.
8). The curves of pHi
dependence of NHE3 activity were not different among the three groups.
The half-maximal intracellular H+ activation values were
6.11 ± 0.44, 6.31 ± 0.05, and 6.25 ± 0.09 in
low-NaCl-intake, NaCl-loaded, and control rats, respectively (not
significant). Note that in agreement with the results shown in Figs. 3
and 5, initial rates of 22Na+ uptake specific
to NHE3 measured in the presence of a transmembrane proton gradient
(pHi = 5.5 ; pHo = 7.5) were not
significantly different among the groups.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Most of the reabsorption of filtered NaCl and
HCO
In situations of chronic NaHCO3 loading, metabolic
alkalosis remains mild to moderate and glomerular filtration rate
usually remains normal (27, 30). In the present
study, plasma HCO
However, NHE3 may not simply respond to plasma HCO-depletion alkalosis, proximal
HCO-depletion alkalosis, have been implicated as factors
stimulating proximal HCO
depletion (31), may explain the severity of metabolic
alkalosis in these models.
Chronic modifications of NaCl intake were accompanied by mild
modifications of extracellular fluid volume. Plasma protein concentration did not significantly change in these situations compared
with controls, but the values were significantly lower in rats with
high-NaCl intake compared with rats with low-NaCl intake (52 ± 0.7 vs. 56 ± 0.8 g/l). Plasma renin activity was increased in
rats with low-NaCl intake and decreased in rats with high-NaCl intake
compared with controls. In situations of chronic variations in NaCl in
the diet, glomerular filtration rates usually remain normal (22,
26, 32). In our study, it is likely that no change in glomerular
filtration rate occurred after 6 days of treatment because no
significant difference in creatinine clearance among NaCl-loaded,
NaCl-restricted, and control rats was observed. In the present study,
AMV NHE3 protein abundance and transport activity were the same in rats
with normal, high-, and low-NaCl intake. Our results are consistent
with two previous studies, one by Holtbäck et al.
(13), which indicated that high-NaCl intake did not
influence Na+/H+ exchange activity in AMV, and
the other by Masilamani et al. (19), who found no change
in NHE3 protein abundance in crude membrane fractions from rat kidney
cortex after chronic NaCl restriction. Interestingly, the absence of
regulation of NHE3 after chronic increase in NaCl intake was also
observed in rat medullary thick ascending limbs (15).
However, these data are at variance with those from a previous study by
Moe et al. (22) showing a mild increase in AMV
Na+/H+ exchange activity after chronic low-NaCl
intake when compared with chronic high-NaCl intake. Another study
showed a downregulation of AMV Na+/H+ exchange
activity with a high-NaCl diet in salt-resistant Rapp/Dahl rats,
which was not seen in salt-sensitive Dahl/Rapp rats (16). The reasons for these discrepancies are unknown. One explanation could
be a difference between the methods employed to monitor the initial
rate of Na+/H+ exchange. In the present study
and that of Holtbäck et al. (13), Na+/H+ exchange activity was directly assayed
by measurement of 22Na+ uptake, whereas, in the
study by Moe et al. (22), changes in acridine orange
fluorescence were used as an estimate of Na+/H+
exchange activity. As NHE3 is responsible for most of the
Na+ reabsorption in the proximal tubule, it is predicted to
exert major effects on proximal fluid reabsorption. The absence of
changes in brush-border NHE3 protein abundance and transport activity observed in the present study after chronic variations in NaCl intake
suggests that proximal Na+ and fluid absorption should
remain unchanged unless regulation of other transporters involved in
proximal Na+ reabsorption, such as the apical
Cl/base exchanger or basolateral
Na+/K+-ATPase, occurs. Evidence that chronic
changes in Na+ balance do not affect Na+
reabsorption in the proximal tubule was first provided by Willis and
co-workers (32), who demonstrated, using micropuncture, that Na+ reabsorption in normal Na+ balance was
not different from Na+ reabsorption in dogs on a high- or
low-NaCl diet. Similarly, proximal tubule fluid reabsorption was
identical in normal rats and animals with a low-NaCl diet that had
moderate extracellular volume depletion (26). In contrast,
proximal reabsorption was affected in situations with profound
modifications of extracellular fluid volume. In extracellular fluid
volume expansion obtained by acute and massive systemic infusion of
isotonic Ringer or NaCl solutions, with attendant obvious decreases in
hematocrit and plasma protein concentration, NaCl absorption was
reduced in the proximal tubule (8, 10, 20), an effect
attributable to a reduction of transcellular, rather than a passive
component of, NaCl reabsorption (8). Conversely,
severe chronic extracellular fluid volume contraction induced by
furosemide administration and a low-NaCl diet, with the attendant
increase in hematocrit and decrease in glomerular filtration rate, was
associated with an increased fluid absorption capacity in the proximal
tubule (26, 28). The latter effect is consistent with
upregulation of brush-border NHE3 protein, recently shown in a similar
model of chronic volume contraction developed in rabbits by using a furosemide/low-NaCl diet (12). It thus appears that the
response of the proximal tubule after changes in Na+
balance may depend on the degree of extracellular volume depletion or expansion.
The important role of the superficial distal tubule in the regulation of urinary NaCl excretion during chronic variations in NaCl intake has been previously shown (11, 26). Indeed, the ability of the distal tubule to absorb NaCl in in vivo microperfusion studies is appropriately adapted to changes in dietary NaCl (i.e., increased in rats receiving a low-NaCl diet and decreased in rats on a high-NaCl diet) (11). The variations in extracellular fluid volume observed in the present study are presumed to modify aldosterone secretion and thus to alter aldosterone-sensitive tubular transporters in the distal nephron. Recent studies have shown that TSC/NCC and ENaC protein expression is increased in rats with low-NaCl diet (14, 19). In the present study, we confirmed the increased TSC/NCC protein expression in rats on a low-NaCl diet and, conversely, we showed decreased TSC/NCC protein expression in rats on a high-NaCl diet.
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FOOTNOTES |
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Address for reprint requests and other correspondence: R. Chambrey, INSERM Unité 356, Institut de Recherche des Cordeliers, 15 rue de l'Ecole de Médecine, 75270 Paris Cedex 06, France (E-mail: chambrey{at}ccr.jussieu.fr).
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.
First published November 20, 2001;10.1152/ajprenal.00188.2001
Received 20 June 2001; accepted in final form 16 November 2001.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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), NaCl-loaded (
),
NaHCO3-loaded (
) rats, and rats on a
low-NaCl diet (
) as a function of duration of
treatment. Values were obtained from 24-h urine collections. Because
natriuresis of the 2 control groups was identical, all control values
were pooled. Values are means ± SE; n = 20 rats
in the control group, 10 in the NaCl-loaded group, 10 in the
NaHCO3-loaded group, and 10 in the low-NaCl-diet group.
), NaCl-loaded (