Rat proximal NHE3 adapts to chronic acid-base disorders but not to chronic changes in dietary NaCl intake

Dominique Eladari1,2, Françoise Leviel1,2, Françoise Pezy1, Michel Paillard1,2, and Régine Chambrey1

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> in the proximal tubule increases, an effect attributed mainly to enhancement of apical Na+/H+ exchange activity associated with an increased NHE3 protein abundance (2). Conversely, in acute metabolic alkalosis induced by systemic NaHCO3 infusion, the proximal absorptive capacity of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> is inhibited independently of the associated extracellular fluid volume expansion, an effect related to inhibition of proton secretion (3). No data are available on proximal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorptive capacity in chronic metabolic alkalosis induced by NaHCO3 administration. In models of chloride-depleted chronic metabolic alkalosis, proximal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption is normal or enhanced (9, 17, 29). However, little information exists regarding the regulation of NHE3 in the proximal tubule during chronic metabolic alkalosis (1).

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.


    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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-beta -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 gamma -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% beta -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-beta -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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration was found and blood pH was higher. A decrease in plasma HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration was also noted in the group fed the low-NaCl diet compared with control rats. In parallel with the increase in plasma HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration, NaHCO3-treated rats exhibited a significant decrease in plasma Cl- 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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Fig. 1.   Daily urinary Na+ excretion in control (), NaCl-loaded (triangle ), NaHCO3-loaded () rats, and rats on a low-NaCl diet (black-triangle) 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.


                              
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Table 1.   Plasma electrolytes, protein, creatinine clearance, and acid-base status in control, NaCl-loaded, NaHCO3-loaded rats, and rats on a low-Na diet after 6 days of treatment



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Fig. 2.   Comparison of plasma renin activity in control, NaCl-loaded, and NaHCO3-loaded rats, and rats on a low-NaCl diet (low Na+). Values were obtained from the plasma collected on day 6 of the treatment. Because natriuresis and plasma renin activity of the 2 control groups were identical, all control values were pooled. Values are means ± SE; n = 21 rats in the control group, 12 in the NaCl-loaded group, 13 in the NaHCO3-loaded group, and 10 in the low-NaCl group. *Statistically significant change vs. control (P < 0.05).

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 gamma -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 gamma -glutamyltransferase. How maltase was affected by alkalosis is not clear. Of note, a similar unexpected effect of acid-base status (acidosis) on brush-border gamma -glutamyl transpeptidase has been reported by Wu et al. (33).

                              
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Table 2.   Effect of NaCl or NaHCO3 loading and low-NaCl diet on enzymatic parameters of membrane vesicle preparations

In the third series of experiments, in which control, NaCl-loaded rats and rats on a low-NaCl diet were compared, AMV preparations had similar specific activities, enrichment factors, and yields for maltase and gamma -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 protein-1 · 9 s-1 (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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Fig. 3.   Effect of NaCl or NaHCO3 loading on Na+/H+ exchanger (NHE)-mediated 22Na+ uptake in apical membrane vesicles (AMV) isolated from rat kidney cortices after 6 days of treatment. Experiments were performed in the absence (open bars) and the presence (filled bars) of 30 µM 4-isopropyl-3-methylsulfonylbenzoyl-guanidine methanesulfonate (HOE-642), a concentration of HOE-642 that has minimal effect on NHE3 and completely inhibits NHE2. Values are means ± SE of 3 determinations in 5 independent sets of membrane preparations. *Statistically significant decrease vs. control (P < 0.05).

Because the inhibition of NHE3 activity could have been the result of the increase in Na+ intake rather than the effect of chronic metabolic alkalosis, we also tested in parallel the effect of chronic NaCl loading. Figure 3 shows that chronic NaCl loading had no effect on NHE3 activity [EIPA-sensitive, H+-stimulated 22Na+ uptake values were 40.4 ± 1.4 (controls) vs. 40.1 ± 2.3 pmol · mg protein-1 · 9 s-1 (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. beta -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%). beta -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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Fig. 4.   NHE3 and sodium-glucose cotransporter (SGLT1) protein abundance in AMV isolated from control, NaCl-loaded, and NaHCO3-loaded rats after 6 days of treatment. A: immunoblots of NHE3 and SGLT1 proteins in representative AMV preparations. B: densitometric analysis of all immunoblots of NHE3 and SGLT1 proteins in AMV from control, NaCl-loaded, and NaHCO3-loaded rats. Values are means ± SE of measurements in 5 independent sets of membrane preparations. *Statistically significant decrease vs. control (P < 0.05).

Taken together, these results indicate that apical Na+/H+ exchange activity in the proximal tubule is inhibited during chronic metabolic alkalosis and that this effect is related to a specific downregulation of NHE3 protein expression.

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 protein-1 · 9 s-1 (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). beta -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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Fig. 5.   Effect of low-Na diet on NHE-mediated 22Na+ uptake in AMV isolated from rat kidney cortices after 6 days of treatment. Experiments were performed in the absence (open bar) and the presence (filled bar) of 30 µM HOE-642. Values represent means ± SE of 3 determinations in 4 independent pairs of membrane preparations.



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Fig. 6.   NHE3 and SGLT1 protein abundance in AMV isolated from control rats and rats on a low-NaCl diet after 6 days of treatment. A: immunoblots of NHE3 and SGLT1 proteins in representative AMV preparations. B: densitometric analysis of all immunoblots of NHE3 and SGLT1 proteins in AMV from control rats and rats on a low-NaCl diet. Values are means ± SE of measurements in 4 independent pairs of membrane preparations.

However, as depicted in Fig. 7, we were able to detect on the same cortical AMV preparations changes in NCC abundance, a protein recently described to be markedly upregulated during Na+ restriction (14). Statistical analysis of relative densities revealed that a low-NaCl diet resulted, as expected, in a marked increase in NCC protein abundance in cortical AMV compared with control conditions (normalized band density: 166.3 ± 69%). Interestingly, a significant decrease in NCC protein expression was also found during high-NaCl intake (normalized band density: 31.5 ± 11%).


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Fig. 7.   Thiazide-sensitive Na-Cl cotransporter (NCC) protein abundance in AMV isolated from control, NaCl-loaded rats, and rats on a low-NaCl diet after 6 days treatment. A: immunoblots of NCC protein in representative AMV preparations. B: densitometric analysis of all immunoblots of NCC protein in AMV from control, NaCl-loaded rats, and rats on a low-NaCl diet. Values are means ± SE of measurements in 4 independent sets of membrane preparations.

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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Fig. 8.   Transport activity of NHE3 as a function of intravesicular pH (pHi). NHE3-mediated 22Na+ uptake in AMV isolated from control (open circle ), NaCl-loaded (black-triangle) rats, and rats on a low-NaCl diet () after 6 days of treatment was measured as a function of intravesicular H+ concentration of pHi in the 5.5-7.5 range. Values represent the average of 3 determinations in 4 independent sets of membrane preparations.

These results, taken together, indicate that NHE3 activity and protein abundance are not regulated by chronic changes in dietary NaCl intake.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Most of the reabsorption of filtered NaCl and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> occurs in the proximal tubule. The main apical Na+- and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>-absorptive pathway is related to the Na+/H+ exchanger NHE3. It has been suggested that adaptation of Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the proximal tubule could participate in the overall regulation of Na+ and acid-base balance by the kidney. The results of the present study provide evidences that NHE3 in brush-border membranes is appropriately regulated in response to chronic NaHCO3 loading-induced metabolic alkalosis but was not altered during chronic high- or low-NaCl intake. The responses of proximal tubule NHE3 expression and transport activity to chronic variations in NaCl and NaHCO3 intake extend previous data obtained in medullary thick ascending limb with similar models. It thus appears that acid-base status is a critical determinant of NHE3 expression and activity in the kidney.

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<UP><SUB>3</SUB><SUP>−</SUP></UP> concentration of NaHCO3-loaded rats was only 3 mM higher than that of control rats. Despite chronic NaCl loading, there was no indication of extracellular fluid volume expansion in rats with chronic NaHCO3 loading as plasma renin activity remained normal. In addition, because we observed no significant difference in creatinine clearance between NaHCO3-loaded rats and control rats, it is likely that no change in glomerular filtration rate occurred after the 6-day administration of NaHCO3. It is thus expected that decreased NHE3 transport activity, observed in AMV from rats with chronic NaHCO3 loading, occurs with a similar change in proximal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption. A decrease in proximal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption was previously observed in alkalosis induced by acute systemic NaHCO3 infusion independently of associated extracellular volume expansion using in vivo microperfusion (3). This proximal response is adapted to prevent the occurrence of severe metabolic alkalosis. Similar changes in NHE3 were observed in medullary thick ascending limbs in response to chronic NaHCO3 loading (15). These results may explain why metabolic alkalosis remains mild to moderate during chronic NaHCO3 loading.

However, NHE3 may not simply respond to plasma HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>. Indeed, in contrast to chronic dietary NaHCO3 loading, in rats with Cl--depletion alkalosis, proximal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption was normal to enhanced, depending on the filtered load of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (9, 17, 18, 29). Severe volume depletion (12) and K+ depletion (25), which accompany Cl--depletion alkalosis, have been implicated as factors stimulating proximal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption and/or Na+/H+ exchange activity. Therefore, the effect of alkalemia per se to decrease proximal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption could be counterbalanced by these stimulatory effects on NHE3. Consistently, Na+/H+ exchange activity measured in rabbit AMV was unchanged in this model of alkalosis (1). Thus the normal-to-increased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in the proximal tubule, together with the enhanced net HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in distal tubule and cortical collecting duct secondary to both K+ and Cl- 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.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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