Renal salt wasting in mice lacking NHE3 Na+/H+ exchanger but not in mice lacking NHE2

Clara Ledoussal1, John N. Lorenz2, Michelle L. Nieman2, Manoocher Soleimani3, Patrick J. Schultheis4, and Gary E. Shull1

Departments of 1 Molecular Genetics, Biochemistry, and Microbiology, 2 Molecular and Cellular Physiology, and 3 Internal Medicine, University of Cincinnati College of Medicine, Cincinnati, Ohio, 45267; and 4 Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky 41099


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To study the role of Na+/H+ exchanger isoform 2 (NHE2) and isoform 3 (NHE3) in sodium-fluid volume homeostasis and renal Na+ conservation, mice with Nhe2 (Nhe2-/-) and/or Nhe3 (Nhe3-/-) null mutations were fed a Na+-restricted diet, and urinary Na+ excretion, blood pressure, systemic acid-base and electrolyte status, and renal function were analyzed. Na+-restricted Nhe2-/- mice, on either a wild-type or Nhe3 heterozygous mutant (Nhe3+/-) background, did not exhibit excess urinary Na+ excretion. After 15 days of Na+ restriction, blood pressure, fractional excretion of Na+, and the glomerular filtration rate (GFR) of Nhe2-/-Nhe3+/- mice were similar to those of Nhe2+/+ and Nhe3+/- mice, and no metabolic disturbances were observed. Nhe3-/- mice maintained on a Na+-restricted diet for 3 days exhibited hyperkalemia, urinary salt wasting, acidosis, sharply reduced blood pressure and GFR, and evidence of hypovolemic shock. These results negate the hypothesis that NHE2 plays an important renal function in sodium-fluid volume homeostasis; however, they demonstrate that NHE3 is critical for systemic electrolyte, acid-base, and fluid volume homeostasis during dietary Na+ restriction and that its absence leads to renal salt wasting.

sodium absorption; sodium/hydrogen exchanger; slc9a2; slc9a3


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

MAINTENANCE OF NA+-fluid volume homeostasis by the kidney requires the tightly regulated activities of a number of Na+ transport proteins. One of the most important, in terms of bulk reabsorption of Na+ with accompanying fluid, is Na+/H+ exchanger isoform 3 (NHE3) (14, 27), which is expressed in brush-border membranes of the proximal tubule and at lower levels in the thick ascending limb (1, 3). By transporting Na+ into the cell in exchange for H+, NHE3 reabsorbs large quantities of both Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> (9, 19, 30). Much less is known about the physiological functions of isoform 2 (NHE2). Its mRNA is expressed at low levels in rat kidney (30) but at high levels in mouse and rabbit kidney (18, 28). NHE2 has been localized to apical membranes of the thick ascending limb and distal convoluted tubule (5, 25), suggesting that it might play a supplementary role in Na+ and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption.

Null mutant homozygous (Nhe3-/-) mice have low blood pressure, high-renin mRNA in kidney, and sharply elevated serum aldosterone levels, consistent with a chronic volume-depleted state (19). Nhe3-/- mice exhibit severe absorptive defects in both the kidney and intestine (9, 19, 29). In situ microperfusion studies of the proximal tubule demonstrated that reabsorption of both fluid, which accompanies Na+, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> was sharply reduced (19, 29). Micropuncture studies showed that fluid reabsorption was reduced in the proximal tubule of both null mutant and heterozygous mutant (Nhe3+/-) mice but that fluid delivery to the distal convoluted tubule was the same as that of wild-type mice (9). In the knockout, this was due largely to a reduction in the glomerular filtration rate (GFR), and in Nhe3+/- mice it was due to increased absorption in the loop segment. Nhe3-/- mice are mildly acidotic, which may be due to both the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption defect in the proximal tubule and the diarrheal state (19, 29). Analysis of isolated perfused tubules demonstrated that increased HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the collecting duct provides partial compensation for the acid-base disorder (13).

The studies discussed above have shown that the loss of NHE3 impairs Na+ handling in the renal proximal tubule and Na+-fluid volume homeostasis. However, at least part of the fluid volume deficit in Nhe3-/- mice is likely to be due to the chronic diarrheal state; the ability of the NHE3-deficient kidney to retain Na+ in vivo has not been rigorously assessed and, therefore, the relative contribution of the renal defect to the Na+-fluid volume deficit is unclear. Also unknown is whether NHE2 plays a significant role in renal Na+ conservation under normal conditions or provides compensation for the loss of NHE3. The phenotype of Nhe2 null mutant (Nhe2-/-) mice has yielded no evidence of a deficit in renal function (18); nevertheless, it is possible that NHE2 plays a supplementary role in Na+ reabsorption. To address these issues, and to further assess any metabolic perturbations caused by the loss of these exchangers, we subjected Nhe2-/- and Nhe3-/- mice to dietary Na+ restriction and analyzed urinary Na+ excretion and renal function.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Production of mutant mice and analysis of genotypes. Homozygous mutant mice with null mutations in the Nhe2 or Nhe3 genes and the corresponding wild-type control mice were obtained by breeding of heterozygous Nhe2 or Nhe3 mutant mice developed in previous studies (18, 19). For the study described in Table 1 and Fig. 2, doubly heterozygous mutant (Nhe2+/-Nhe3+/-) mice were bred to obtain mice that were null mutant or wild-type with respect to the Nhe2 gene but on an Nhe3 heterozygous mutant (Nhe3+/-) background.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Cardiovascular, blood, and renal measurements in Nhe2+/+Nhe3+/- and Nhe2-/-Nhe3+/- mice after dietary Na+ restriction

PCR genotyping of wild-type and mutant alleles of the Nhe2 and Nhe3 genes was performed in separate reactions using DNA from tail biopsies. Each reaction contained one primer corresponding to sequences from the neomycin resistance gene that would be present only in the mutant allele and two primers from exon sequences flanking the site used for targeted disruption of the gene. For Nhe2, forward (5'-CATCTCTATCACAAGTTGCCCACAATCGTG-3') and reverse (5'-GTGACTGCATCGTTGAGCAGAGACTCG-3') primers corresponding to sequences from near the 5' and 3' ends of exon 2 and a primer from near the 3' end of the neomycin resistance gene (5'-GACAATAGCAGGCATGCTGG-3') were used to amplify 450- and 221-base pair (bp) products from the wild-type and mutant genes, respectively. For the Nhe3 gene, forward (5'-CATCTCTATCACAAGTTGCCCACAATCGTG-3') and reverse (5'-GTGACTGCATCGTTGAGCAGAGACTCG-3') primers corresponding to sequences from near the 5' and 3' ends of exon 6 and a primer from near the 5' end of the neomycin resistance gene (5'-GCATGCTCCAGACTGCCTTG-3') were used to amplify 199- and 113-bp products from the wild-type and mutant genes, respectively. Reactions (40 cycles of amplification) were performed under the following conditions: denaturing at 94°C for 30 s, annealing at 60°C for 30 s, and extension at 72°C for 30 s.

Dietary sodium restriction and analysis of urine and feces. Adult mice were housed in metabolic cages and provided with drinking water and food ad libidum, as described previously (20). The food (Harlan Tecklad, Madison, WI) contained normal (1% NaCl), medium (0.1% NaCl), or low (0.01% NaCl) amounts of Na+. In the studies with Nhe2-/- mice, 1-day urine and fecal samples were collected. In the studies with Nhe2-/-Nhe3+/- mice, urine and fecal samples were each pooled over a 3-day period. In the studies with Nhe3-/- mice, 1-day urine samples were collected; feces were not collected because of diarrhea, and cages were cleaned and urine was collected several times a day to avoid contamination of urine with feces. In each experiment, the volume of urine was measured, urinary Na+ and K+ concentrations were analyzed by flame photometry (Corning model 480), and total excretion of each ion was calculated. Feces were collected and homogenized in 4-10 ml of 0.75 N nitric acid. After overnight incubation, the tubes were centrifuged and the supernatants were analyzed to determine fecal Na+ and K+ excretion.

Analysis of urinary Ca2+ excretion. Urine volumes of adult mice maintained on a diet containing 1% NaCl were measured, Ca2+ concentrations were determined colorimetrically (arsenazo III assay, Sigma), and total urinary Ca2+ excretion was calculated.

Cardiovascular and renal measurements. After either 15 (for Nhe2-/-Nhe3+/- mice) or 3 days (for Nhe3-/- mice) of sodium depletion, the mice were anesthetized with intraperitoneal injections of inactin (100 µg/g body wt) and ketamine (50 µg/g body wt) and surgically instrumented for cardiovascular and renal measurements under baseline conditions and after extracellular volume expansion (ECVE), as described previously (4, 9, 12). Mean arterial blood pressure and heart rate were monitored via a pressure transducer connected to a catheter in the femoral artery, and urine was collected via a catheter in the bladder (9). Urine electrolytes were measured by flame photometry, and plasma electrolytes and acid-base status were analyzed using a pH/blood-gas analyzer (model 348, Chiron Diagnostics; Norwood, MA). Two 30-min baseline clearance measurements were performed in the presence of a maintenance infusion of isotonic saline (0.15 µl/g of body wt/min) and were followed by two additional 30-min clearance periods in which isotonic saline was infused more rapidly (1.0 µl/g of body wt/min) to induce ECVE. GFR under baseline and ECVE conditions was determined using fluorescein isothiocyanate-labeled inulin (1.5 µg/g of body wt/min; 9) and was averaged for each of the two clearance periods. For each mouse, the values for mean arterial pressure and heart rate were an average of the values recorded during the last 2 min of the two collection periods that occurred either before or after ECVE. Blood for analysis of plasma electrolytes and acid-base status was taken at the midpoint of each 30-min collection period; values for individual mice were an average of the two samples taken either before or after ECVE.

Statistics. Data are presented as means ± SE. Student's t-test was used to compare each group of mutant mice to the corresponding control mice.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Loss of NHE2 does not lead to renal salt wasting during dietary Na+ restriction. As an initial test of the hypothesis that NHE2 contributes to renal Na+ conservation, wild-type and Nhe2-/- mice were maintained successively on diets containing high, medium, and low concentrations of sodium (1% NaCl for 6 days, followed by 0.1% NaCl for 5 days, and then 0.01% NaCl for 5 days), and urinary excretion of both Na+ (Fig. 1A) and K+ (Fig. 1B) was analyzed. On each of the three diets, the amount of Na+ and K+ excreted in the urine was similar for Nhe2+/+ and Nhe2-/- mice. Even when fed a 0.01% NaCl diet, Nhe2-/- mice were able to reduce urinary Na+ excretion to very low levels (Nhe2+/+, 7.3 ± 1.6 µmol/day; Nhe2-/-, 6.7 ± 1.7 µmol/day), indicating that the NHE2-deficient kidney is able to retain Na+ as well as that of wild-type mice.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 1.   Urinary excretion of Na+ (A) and K+ (B) by Nhe2+/+ and Nhe2-/- mice during dietary Na+ restriction. Adult Nhe2+/+ (n = 7) and Nhe2-/- (n = 4) mice were fed a 1% NaCl diet for the first 6 days, a 0.1% NaCl diet for the next 5 days, and a 0.01% NaCl diet for the last 5 days. The arrows indicate the points at which the diets were switched. Urine was collected daily, the volume was measured, and Na+ and K+ concentrations were analyzed by flame photometry. Values are means ± SE. There were no significant differences between Nhe2-/- and wild-type mice.

It was shown previously that fluid reabsorption is reduced in the proximal tubule of mice carrying a null mutation in one copy of the Nhe3 gene (10). Thus, to allow a more rigorous assessment of a potential role for NHE2 in renal Na+ reabsorption, we mated mice carrying null mutations in the Nhe2 and Nhe3 genes to obtain Nhe2+/+ and Nhe2-/- mice on an Nhe3 heterozygous mutant (Nhe3+/-) background and then subjected them to dietary Na+ restriction. We reasoned that under Na+-restricted conditions a reduction in NHE3-mediated Na+ reabsorption in the proximal tubule might stress the more distal Na+ reabsorption mechanisms and reveal a contribution by NHE2. In addition to urinary Na+ and K+ excretion, fecal excretion of these ions was also analyzed to determine whether the loss of two copies of the Nhe2 gene in an Nhe3+/- mouse had an affect on the absorption of these ions in the intestinal tract. Compared with control mice (Nhe2+/+Nhe3+/-), Nhe2-/-Nhe3+/- mice did not exhibit significant differences in fecal or urinary excretion of Na+ or K+ (Fig. 2). During the last 6 days of dietary Na+ restriction, urinary excretion of Na+ was 3.3 ± 0.7 µmol/day for Nhe2-/-Nhe3+/- mice and 2.3 ± 0.3 µmol/day for Nhe2++Nhe3+/- mice, indicating that the loss of NHE2 does not lead to significant urinary salt wasting.


View larger version (34K):
[in this window]
[in a new window]
 
Fig. 2.   Urinary and fecal excretion of Na+ and K+ by Nhe2+/+ Nhe3+/- and Nhe2-/-Nhe3+/- mice during dietary Na+ restriction. The mice had 1 wild-type and 1 mutant allele of Nhe3 and were either wild-type or null mutant with respect to Nhe2. Nhe2+/+ Nhe3+/- and Nhe2-/-Nhe3+/- mice (n = 8 for each genotype) were fed a 1% NaCl diet for 6 days and then switched (arrow) to a 0.01% NaCl diet for 15 days. Urine and feces were collected over 3-day intervals, and the Na+ and K+ content of each sample was determined. Values for urinary (A) and fecal (C) excretion of Na+ and for urinary (B) and fecal (D) excretion of K+ are means ± SE. There were no significant differences between the 2 groups of mice.

Acid-base and electrolyte status, mean arterial pressure, and renal function in Na+-restricted NHE2-deficient mice. After the 15-day period of dietary Na+ restriction, the Nhe2+/+Nhe3+/- and Nhe2-/-Nhe3+/- mice were anesthetized and their blood pressure, acid-base and electrolyte status, and renal function were assessed under baseline conditions and after ECVE. As shown in Table 1, baseline values for mean arterial blood pressure, heart rate, plasma Na+, plasma K+, and blood pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations were similar in Nhe2+/+Nhe3+/- and Nhe2-/-Nhe3+/- mice. The GFR was similar in both groups under baseline conditions and did not change significantly after volume expansion (Table 1). Renal K+ handling was similar in the two groups under baseline conditions, and K+ excretion increased in a similar fashion in both groups after volume expansion. There was no major difference in renal Na+ handling between the two groups under either experimental condition (Table 1). Under baseline conditions, the mean value for fractional Na+ excretion was slightly higher in Nhe2-/-Nhe3+/- mice than in Nhe2+/+Nhe3+/- mice; however, the difference was not statistically significant, and both values were very low, with greater than 99.9% of the filtered Na+ being reabsorbed. After ECVE, fractional Na+ excretion increased sharply to similar values in both groups of mice. These data provide no evidence of an important role for NHE2 in either renal Na+ reabsorption or the response to ECVE.

Nhe3-/- mice exhibit mild renal salt wasting and do not tolerate a Na+-restricted diet. Micropuncture (10) and in situ microperfusion (30) studies of Nhe3-/- mice have shown that the loss of NHE3 causes a severe impairment of Na+ reabsorption in the proximal tubule; however, it is unclear whether this leads to significant renal salt wasting. To examine this issue, we maintained Nhe3+/+ and Nhe3-/- mice on a Na+-replete diet for 4 days and then switched them to a Na+-restricted (0.01% NaCl) diet for 3 days. Their body weights and urinary Na+ and K+ excretion were measured daily. Fecal excretion of Na+ and K+ were not determined because the diarrheal state of Nhe3-/- mice makes it difficult to obtain accurate measurements, and these data were not necessary for assessment of renal salt wasting.

Immediately after the switch to a low Na+ diet on day 4, Nhe3-/- mice began losing weight (Fig. 3A), and some of them died during the study. Among the 10 Nhe3-/- mice subjected to dietary Na+ restriction, 1 was severely dehydrated after 1 day of Na+ restriction and was euthanized and another died after 2 days of Na+ restriction. By day 7 (third day of Na+ deprivation), the average body weight of Nhe3+/+ mice was 102.0 ± 3.0% of the body weight on day 4, whereas the body weight of surviving Nhe3-/- mice had dropped to 82.5 ± 4.5% of that on day 4, (P < 0.01). The poor viability and loss of body weight indicates that dietary Na+ restriction leads to severe hypovolemia in Nhe3-/- mice. As shown in Fig. 3B, when the mice were fed a Na+-replete diet, urinary Na+ excretion was lower in Nhe3-/- mice than in the wild-type controls (223 ± 48 vs. 649 ± 73 µmol/day; P < 0.0001), apparently a reflection of the impaired absorption of Na+ by the Nhe3-/- intestine. By days 6 and 7 (second and third days of Na+ depletion), urinary Na+ excretion was three- to fourfold higher in Nhe3-/- mice than in Nhe3+/+ mice. The rate of decrease in urinary Na+ excretion was substantially greater in Nhe3+/+ mice than in Nhe3-/- mice. For example, between the first and second days of Na+ depletion, urinary Na+ excretion dropped approximately sevenfold in Nhe3+/+ mice (145 ± 23 to 20 ± 4 µmol/day) and only approximately twofold in Nhe3-/- mice (123 ± 26 to 68 ± 15 µmol/day). Urinary K+ excretion was significantly lower in the knockout under both Na+-replete and Na+-depleted conditions. This is probably due, at least in part, to a reduction in net intestinal K+ recovery as a result of the diarrheal state.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   Body weight (A) and urinary excretion of Na+ (B) and K+ (C) for Nhe3+/+ and Nhe3-/- mice during dietary Na+ restriction. The mice were fed a 1% NaCl diet on days 1-4 (D1-D4) and a 0.01% NaCl diet on days 5-7 (D5-D7). Urine was collected daily, and Na+ and K+ content was analyzed by flame photometry. A: body weights of Nhe3+/+ and Nhe3-/- mice (n = 5 for each genotype; a subset of the mice used in B and C) were determined each day and expressed as a percentage of body weight on day 4. Nhe3-/- mice lost about 20% of their body weight after 3 days of Na+ restriction. Urinary Na+ (B) and K+ (C) excretion (in mmol/day) was determined for Nhe3+/+ (open bars; n = 12) and Nhe3-/- (filled bars; n = 10) mice. Values are means ± SE. *P < 0.05 when compared with Nhe3+/+ mice.

As shown in Fig. 4, water intake and urinary output were significantly greater in Nhe3-/- mice than in wild-type mice when maintained on either the Na+-replete or Na+-restricted diets (water intake, Na+-replete diet: 6.9 ± 0.3 vs. 2.8 ± 0.2 ml/day, P < 0.00001; Na+-restricted diet: 4.1 ± 0.2 vs. 2.4 ± 0.2 ml/day, P < 0.00003; urinary output, Na+-replete diet: 1.64 ± 0.09 vs. 1.36 ± 0.12 ml/day, P < 0.05; Na+-restricted diet: 1.94 ± 0.18 vs. 0.98 ± 0.16 ml/day, P < 0.0003).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 4.   Water intake (A) and urinary output (B) of Nhe3+/+ and Nhe3-/- mice. The mice (n = 5 of each genotype; same animals as in Fig. 3A) were fed a 1% NaCl diet on days 1-4 (D1-D4) and a 0.01% NaCl diet on days 5-7 (D5-D7). dagger P < 0.00001, *P < 0.0001 when compared with values for wild-type mice.

Acid-base and electrolyte status, mean arterial pressure, and renal function in Na+-restricted NHE3-deficient mice. The sharp drop in body weight by day 3 of dietary Na+ restriction indicated that Na+-restricted Nhe3-/- mice were unable to maintain Na+-fluid volume homeostasis. The mice were anesthetized and surgically prepared for analysis of mean arterial pressure, systemic acid-base and electrolyte status, and renal function (Figs. 5-7 and Table 2). Three of the eight Nhe3-/- mice that survived during the 3-day period of dietary Na+ restriction died shortly after being anesthetized for surgery, presumably as a result of hypovolemic shock.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 5.   Mean arterial blood pressure and heart rate of Nhe3+/+ and Nhe3-/- mice after dietary Na+ restriction. Mice were maintained on a 0.01% NaCl diet for 3 days, anesthetized, and then surgically instrumented for analysis of blood pressure and renal function. Mean arterial pressure (A) and heart rate (B) are expressed as means ± SE for Nhe3+/+ (n = 4) and Nhe3-/- (n = 5) mice before (baseline; open bars) and after extracellular fluid volume expansion (ECVE; filled bars). *P < 0.05 compared with the corresponding Nhe3+/+ mice.



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 6.   Acid-base status of Nhe3+/+ and Nhe3-/- mice after dietary Na+ restriction. The mice used in this experiment are the same as those in Figs. 5 and 7. Arterial blood was collected from anesthetized Nhe3+/+ (n = 4) and Nhe3-/- (n = 5) mice, and the pH (A) and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations (B) were determined. Values are means ± SE before (baseline; open bars) and after ECVE (filled bars). *P < 0.05 compared with Nhe3+/+ mice.



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 7.   Plasma Na+ (A) and K+ (B) concentrations in Nhe3+/+ and Nhe3-/- mice after dietary Na+ restriction. The mice are the same as those in Figs. 5 and 6. Plasma Na+ and K+ concentrations are means ± SE from anesthetized Nhe3+/+ (n = 4) and Nhe3-/- (n = 5) mice before (baseline; open bars) and after ECVE (filled bars). *P < 0.05 compared with the Nhe3+/+ mice.


                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Renal measurements in Nhe3+/+ and Nhe3-/- mice following dietary Na+-restriction

Under both baseline conditions and after ECVE, the mean arterial pressure of Na+-restricted Nhe3-/- mice was about 25 mmHg lower than that of Nhe3+/+ mice (Fig. 5). Blood pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> levels were sharply reduced in the Nhe3-/- mice (pH: 7.17 ± 0.05 vs. 7.35 ± 0.01 in wild-type, P < 0.03; HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>: 14.3 ± 2.5 vs. 24.9 ± 0.5 mM in wild-type; P < 0.01) under baseline conditions and dropped slightly after ECVE (Fig. 6). Before ECVE, the plasma Na+ concentration of Na+-restricted Nhe3-/- mice was reduced to 140.9 ± 3.6 mM, compared with 152.9 ± 3.4 mM in wild-type mice (Fig. 7A). Plasma K+ concentrations were elevated in the Na+-restricted knockout (Nhe3-/-, 6.4 ± 0.8 mM; Nhe3+/+, 4.4 ± 0.2 mM; P < 0.01) (Fig. 7B), as observed previously in Na+-replete Nhe3-/- mice (19).

The average GFR of anesthetized Na+-restricted Nhe3-/- mice, under both baseline conditions and after ECVE, was reduced to ~30-40% of that observed in wild-type controls (Table 2). Under baseline conditions, the mean value for fractional excretion of Na+ was approximately fourfold higher in Nhe3-/- mice than in wild-type mice (Table 2). Although the differences were not statistically significant, this was apparently due to the high variability in the small group of knockouts that survived during the anesthesia and surgery (see DISCUSSION). For several of the knockouts, fractional excretion of Na+ exceeded 1-2% under baseline conditions, whereas values in this range were not observed in the controls or in any of the Na+-restricted mice analyzed in Table 1. ECVE led to an increase in fractional excretion of Na+ in both groups, although the fold-increase was much less in the knockout. Despite the increase in fractional excretion of Na+ in anesthetized Na+-restricted Nhe3-/- mice, the mean value for Na+ excretion under baseline conditions in these mice was lower than in wild-type mice (Table 2). This result is opposite than that obtained in awake mice, in which a higher level of Na+ excretion was observed in the knockout (Fig. 3B). The discrepancy was due, at least in part, to the very low GFR observed in some of the anesthetized knockouts, which appeared to be due to hypovolemic renal failure (see below). In response to ECVE, urinary K+ excretion increased in Nhe3-/- mice but not in the wild-type.

There was evidence that the short period of Na+ restriction was leading to renal failure in Nhe3-/- mice, most likely in response to hypovolemia and reduced blood pressure, and exacerbated by the anesthesia. For one of the five mice that survived during the procedure, urine flow ceased after ECVE, indicating that GFR was severely reduced (baseline mean arterial pressure for this mouse was only 54 mmHg and decreased further after ECVE); for two other mice, GFR was very low (~50 µl/min) and fractional excretion of Na+ was quite high (>1%) under baseline conditions, and the kidneys were very pale (almost white) at the end of the experiment. For the other two mice that survived during the procedure, GFR was ~300 µl/min under baseline conditions, and both blood pressure and fractional excretion of Na+ were within the range observed in wild-type mice after 3 days of Na+ restriction; after ECVE, however, GFR in one of these mice unexpectedly dropped to ~25 µl/min.

Urinary calcium excretion is similar in Nhe2-/-, Nhe3-/-, and wild-type mice. Loss of the apical Na+-K+-2Cl- cotransporter of the thick ascending limb or the thiazide-sensitive NaCl cotransporter of the distal convoluted tubule causes hypercalciuria or hypocalciuria, respectively (20-22, 26). To explore the possibility that null mutations in NHE2 or NHE3 might affect renal calcium handling, we maintained null mutants and wild-type controls on a 1% NaCl diet and analyzed urinary calcium excretion. As shown in Fig. 8, there were no significant differences between the knockouts and their wild-type controls.


View larger version (19K):
[in this window]
[in a new window]
 
Fig. 8.   Urinary calcium excretion in Nhe3-/- and Nhe2-/- mice. Urinary Ca2+ excretion was determined for Nhe3+/+ (n = 11), Nhe3-/- (n = 9), Nhe2+/+ (n = 6), and Nhe2-/- (n = 8) mice. Values are means ± SE for 24-h urine samples. Urinary Ca2+ excretion in Nhe3-/- and Nhe2-/- mice was not significantly different from that of their wild-type controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Previous studies of Nhe2-/- and Nhe3-/- mice showed that NHE3 is the major absorptive Na+/H+ exchanger in the kidney (9, 19, 29) but did not reveal a renal function for NHE2 (18). It was unclear, however, whether the loss of NHE3 caused significant renal salt wasting and whether NHE2 might play a supplementary role in Na+ reabsorption. To gain a better understanding of the relative roles of NHE2 and NHE3 in renal Na+ conservation, we used the NHE2 and NHE3 knockouts to determine whether the loss of either exchanger would result in urinary salt wasting or metabolic disturbances when the mice were fed a Na+-restricted diet.

NHE2-deficient mice exhibit a stomach phenotype characterized by achlorhydria and the loss of gastric parietal and chief cells (18). Although NHE2 mRNA is expressed in mouse kidney at levels comparable to those observed in stomach and is expressed at high levels in the small intestine and colon, renal and intestinal phenotypes were not observed in Nhe2-/- mice. Because NHE2 has been localized to apical membranes of the thick ascending limb and distal convoluted tubule (5, 25), a renal absorptive function seemed likely. NHE2 has also been identified in basolateral membranes of the inner medullary collecting duct cell line (IMCD3) (23), consistent with functions such as secretion or cell volume and pH regulation. Nhe2-/- mice maintained on a low-Na+ diet were able to retain Na+ as well as wild-type mice; however, similar studies of mice lacking the thiazide-sensitive Na+-Cl- cotransporter (NCC) also failed to reveal a salt-wasting phenotype (20), despite the known function of NCC in NaCl absorption in the distal convoluted tubule. Therefore, these results did not exclude the possibility that the loss of NHE2 causes a mild impairment of Na+ reabsorption but that other Na+ transporters provide compensation for the defect.

It was shown previously that Na+ reabsorption is reduced in the proximal tubule of Nhe3+/- mice and that compensation occurs via increased absorption in the loop segment rather than a decrease in GFR, as in Nhe3-/- mice (9). This suggested that under Na+-depleted conditions a reduction in NHE3-mediated Na+ reabsorption in the proximal tubule might stress the more distal Na+ reabsorption mechanisms and reveal a contribution by NHE2. However, Nhe2-/- Nhe3+/- mice subjected to dietary Na+ restriction retained Na+ as well as Nhe2+/+Nhe3+/- mice and exhibited none of the metabolic defects observed in Nhe3-/- mice. Measurements of renal function revealed no alterations in GFR, fractional excretion of Na+, or the natriuretic response to ECVE. Also, in contrast to mice lacking NCC (20), when fed a Na+-restricted diet they did not exhibit a decrease in blood pressure indicative of a defect in Na+-fluid volume homeostasis. On the basis of these results, which are in sharp contrast to those obtained with the NHE3 knockout, we conclude that NHE2 plays little if any role in Na+-fluid volume homeostasis.

Similarly, there was no indication of a role for NHE2 in renal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption. Systemic acid-base homeostasis, as judged by blood pH and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations, was normal in both Nhe2-/- and Nhe2-/- Nhe3+/- mice. Previous studies of Nhe3-/- mice, in which inhibitors of Na+/H+ exchange were utilized, also yielded no evidence of a role for NHE2 in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption (7, 29), although those experiments focused on the proximal tubule. In a separate study (Ledoussal C and Shull G, unpublished observations) designed to assess the relative absorptive functions of NHE2 and NHE3 in the intestine, we found that the loss of both copies of the Nhe2 gene caused no further impairment of acid-base balance in Nhe3-/- mice, which normally exhibit a mild acidosis (19, 29). If NHE2 played a major role in renal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption, then Nhe2-/-Nhe3-/- mice would have been expected to have a more severe acidosis. Although additional studies will be needed to assess the possible function of NHE2 in renal HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> handling, the available data suggest that it does not play a significant role in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption.

The previous demonstration of an absorptive defect in Nhe3-/- proximal tubules (19, 29) suggested that at least part of the fluid volume deficit was due to renal salt wasting, with an additional component resulting from the intestinal defect. However, because a defect in bulk reabsorption of Na+ in the proximal tubule can be sensed by the macula densa, it remained possible that tubuloglomerular feedback (TGF) might bring about a sufficient reduction in GFR to enable the Na+-transport mechanisms of the more distal segments of the nephron to reduce urinary Na+ losses to the low levels observed in wild-type mice. Previous studies showed that TGF was intact in Nhe3-/- mice, that GFR was reduced to about 65-70% of normal, and that fluid delivery to the distal tubule was the same as in wild-type mice (4, 9). Surprisingly, absorption in the loop segment and apical Na+-K+-2Cl- cotransporter (NKCC2) protein levels was reduced in the knockout (4, 9), rather than upregulated as would be expected if NKCC2 provided partial compensation. Thus it was unclear whether the loss of NHE3 caused severe renal salt wasting, as observed in null mutants for NKCC2 (21, 26) and the epithelial Na+ channel (ENaC) (2, 6, 8, 11, 15, 25).

To examine this issue, we subjected NHE3-deficient mice to dietary Na+ restriction. During the control period, water intake was higher in Nhe3-/- mice than in wild-type mice and, although it decreased during Na+ restriction, it remained higher than wild-type levels. Part of the elevation in water intake was undoubtedly secondary to the diarrheal state, which was noticeably diminished during Na+ restriction. Mean urinary volume was significantly higher in the knockout during Na+ restriction, and the greatest increase was observed in Nhe3-/- mice that exhibited relatively strong Na+ retention. Na+ restriction could not be extended beyond 3 days because of severe weight loss, indicative of volume depletion, and morbidity (2/10 mice died) that occurred in the NHE3 knockout. After the switch to the Na+-restricted diet, the rate of reduction in urinary Na+ excretion was much less in the knockout than in wild-type mice. This may be a reflection of the fact that the more distal Na+-conserving mechanisms were already upregulated in the knockout mice when the switch in diet was made, whereas wild-type mice have a substantial reserve capacity that can be activated during the days after the switch. By the third day of Na+ restriction, urinary losses of Na+ were four to five times greater in the knockout mice, demonstrating that the loss of NHE3 causes urinary salt wasting.

When we analyzed renal function after 3 days of Na+ restriction, three of the eight surviving knockout mice died during anesthesia and surgical instrumentation. Given the magnitude of the weight loss and reduced blood pressure, the observed deaths were probably due to hypovolemic shock. Among the remaining mice, the response was variable. Some of the knockouts had very low GFRs and were in hypovolemic renal failure; however, under baseline conditions two of them had GFRs within the range observed for knockouts on a normal- Na+ diet (4), but with blood pressure and fractional Na+ excretion within the range observed for wild-type mice after 3 days of Na+ restriction. It should be noted that 3 days of Na+ restriction is a quite brief period and not long enough for Na+-conserving mechanisms to be fully activated in wild-type control mice (for example, see Table 1). Thus the degree of renal Na+ conservation in the knockout, in which Na+-conserving mechanisms are sharply elevated even when maintained on a normal diet (19), falls far short of that of wild-type mice. NHE2-deficient or wild-type mice exhibit no ill effects when maintained for several weeks on the low-Na+ diet used in this study, whereas Nhe3-/- mice have little tolerance for even a brief period of Na+ restriction. These observations demonstrate that NHE3, in sharp contrast to NHE2, is critically important for Na+-fluid volume homeostasis during salt deprivation.

Defective Na+-fluid volume homeostasis resulting from genetic defects in renal Na+ transporters leads to a number of metabolic disorders. NCC and NKCC2 mutations are associated with hypokalemic alkalosis (21, 22), and ENaC mutations are associated with hyperkalemic acidosis (6). Previous studies showed that Na+-replete NHE3-deficient mice are mildly acidotic and hyperkalemic (19, 29), and the present study shows that dietary Na+ restriction leads to reduced plasma Na+ and a worsening of both the metabolic acidosis and hyperkalemia. In contrast, no metabolic disturbances were observed in NHE2-deficient mice under either Na+-replete or Na+-restricted conditions, consistent with the lack of an effect on Na+-fluid volume homeostasis.

Hyperkalemia in ENaC-deficient mice has been attributed to reduced K+ secretion via apical channels in the collecting duct, which are normally coupled with Na+ reabsorption via ENaC (11). Similarly, hypokalemia in humans with NKCC2 or NCC mutations has been proposed to be due to a compensatory increase in ENaC activity, with a coupled increase in K+ secretion (21, 22). However, NHE3-deficient mice would be expected to have increased ENaC activity, as indicated by increased aldosterone levels (19) and increased abundance of the 70-kDa form of the gamma -subunit (4). Because this would be expected to lead to increased K+ secretion and subsequent hypokalemia, the basis for the hyperkalemia in Nhe3-/- mice is unclear.

Loss of the apical Na+-K+-2Cl- cotransporter of the thick ascending limb or the thiazide-sensitive NaCl cotransporter of the distal convoluted tubule causes hypercalciuria or hypocalciuria, respectively (20-22, 26). It has been suggested that the loss of Na+ uptake activity in the distal convoluted tubule as a result of NCC null mutations increases the driving force for Ca2+ absorption, thereby leading to reduced urinary Ca2+ excretion (21). Because NHE2, like NCC, has been reported to be expressed in apical membranes of distal convoluted tubule cells (5), it seemed possible that hypocalciuria would be observed, but this was not the case. The observation that NHE2-deficient mice have normal urinary Ca2+ excretion provides suggestive evidence that NHE2 is not involved in Na+ reabsorption in the distal convoluted tubule. With regard to NHE3, we anticipated that its absence might lead to hypercalciuria as a result of compensatory upregulation of Na+ reabsorption via NCC in the distal convoluted tubule or reduced paracellular transport of Ca2+ in the proximal tubule due to the observed reduction in fluid transport (29); however, urinary Ca2+ excretion in Nhe3-/- mice was the same as that of wild-type mice.

Despite the fact that NHE3 is a major bulk transporter of Na+ in the kidney, the salt-wasting defect seems mild when compared with that of NKCC2 and ENaC null mutant mice (2, 8, 11, 15, 26). Although ENaC is responsible for only a small fraction of total Na+ reabsorption, its activity in the collecting duct is critical because there are no downstream transporters that can compensate for its absence and it is located beyond the macula densa, where TGF can adjust GFR (16). The severe phenotype of the NKCC2 knockout is due to the loss of NaCl reabsorption in the thick ascending limb and the urinary concentrating defect, and it is conceivable that an impairment of the TGF mechanism (10, 16, 17) also contributes to the defect. The mild perturbation of Na+-fluid volume homeostasis in NCC mutants (20) is presumably because this transporter does not reabsorb large quantities of Na+ and because absorption via ENaC can compensate for the defect. The results of the present and previous studies of Nhe3-/- mice (9, 19, 29) suggest that the loss of NHE3, which normally mediates bulk reabsorbtion of Na+, is tolerated when mice are maintained on a normal diet because reduced blood pressure and TGF limits the amount of Na+ that is filtered and distal mechanisms of Na+ reabsorption are intact. However, when they are fed a Na+-restricted diet, the reduction in GFR and induction of Na+-conserving mechanisms in more distal segments of the nephron are not sufficient to fully compensate for the defect in bulk Na+ reabsorption in the proximal tubule.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-50594, DK-57552, and DK-54430.


    FOOTNOTES

Address for reprint requests and other correspondence: G. E. Shull, Dept. of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, 231 Albert Sabin Way, ML 524, Cincinnati, OH 45267-0524 (E-mail: shullge{at}ucmail.uc.edu).

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.

Received 20 March 2001; accepted in final form 23 March 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Amemiya, M, Loffing J, Lotscher M, Kaissling B, Alpern RJ, and Moe OW. Expression of NHE-3 in the apical membrane of rat renal proximal tubule and thick ascending limb. Kidney Int 48: 1206-1215, 1995[ISI][Medline].

2.   Barker, PM, Nguyen MS, Gatzy JT, Grubb B, Norman H, Hummler E, Rossier B, Boucher RC, and Koller B. Role of gamma ENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseuhypoaldosteronism. J Clin Invest 102: 1634-1640, 1998[Abstract/Free Full Text].

3.   Biemesderfer, D, Pizzonia J, Abu-Alfa A, Exner M, Reilly R, Igarashi P, and Aronson PS. NHE3: a Na+/H+ exchanger isoform of renal brush border. Am J Physiol Renal Fluid Electrolyte Physiol 265: F736-F742, 1993[Abstract/Free Full Text].

4.   Brooks, HL, Sorensen AM, Terris J, Schultheis PJ, Lorenz JN, Shull GE, and Knepper MA. Profiling of renal tubule Na+ transporter abundances in NHE3 and NCC null mice using targeted proteomics. J Physiol (Lond) 530: 359-366, 2001[Abstract/Free Full Text].

5.   Chambrey, R, Warnock DG, Podevin RA, Bruneval P, Mandet C, Belair MF, Bariety J, and Paillard M. Immunolocalization of the Na+/H+ exchanger isoform NHE2 in rat kidney. Am J Physiol Renal Physiol 275: F379-F386, 1998[Abstract/Free Full Text].

6.   Chang, SS, Grunder S, Hanukoglu A, Rosler A, Mathew PM, Hanukoglu I, Schild L, Lu Y, Shimkets RA, Nelson-Williams C, Rossier BC, and Lifton RP. Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1. Nat Genet 12: 248-253, 1996[ISI][Medline].

7.   Choi, JY, Shah M, Lee MG, Schultheis PJ, Shull GE, Muallem S, and Baum M. Novel amiloride-sensitive sodium-dependent proton secretion in the mouse proximal convoluted tubule. J Clin Invest 105: 1141-1146, 2000[Abstract/Free Full Text].

8.   Hummler, E, Barker P, Talbot C, Wang Q, Verdumo C, Grubb B, Gatzy J, Burnier M, Horisberger JD, Beermann F, Boucher R, and Rossier BC. A mouse model for the renal salt-wasting syndrome pseuhypoaldosteronism. Proc Natl Acad Sci USA 94: 11710-11715, 1997[Abstract/Free Full Text].

9.   Lorenz, JN, Schultheis PJ, Traynor T, Shull GE, and Schnermann J. Micropuncture analysis of single-nephron function in NHE3-deficient mice. Am J Physiol Renal Physiol 277: F447-F453, 1999[Abstract/Free Full Text].

10.   Lorenz, JN, Weihprecht H, Schnermann J, Skott O, and Briggs JP. Renin release from isolated juxtaglomerular apparatus depends on macula densa chloride transport. Am J Physiol Renal Fluid Electrolyte Physiol 260: F486-F493, 1991[Abstract/Free Full Text].

11.   McDonald, FJ, Yang B, Hrstka RF, Drummond HA, Tarr DE, McCray PB, Stokes JB, Welsh MJ, and Williamson RA. Disruption of the beta  subunit of the epithelial Na+ channel in mice: hyperkalemia and neonatal death associated with pseudohypoaldosteronism phenotype. Proc Natl Acad Sci USA 96: 1727-1731, 1999[Abstract/Free Full Text].

12.   Meneton, P, Schultheis PJ, Greeb J, Nieman ML, Liu LH, Clarke LL, Duffy JJ, Doetschman T, Lorenz JN, and Shull GE. Increased sensitivity to K+ deprivation in colonic H,K-ATPase-deficient mice. J Clin Invest 101: 536-542, 1998[Abstract/Free Full Text].

13.   Nakamura, S, Amlal H, Schultheis PJ, Galla JH, Shull GE, and Soleimani M. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in renal collecting duct of NHE3-deficient mouse: a compensatory response. Am J Physiol Renal Physiol 276: F914-F921, 1999[Abstract/Free Full Text].

14.   Orlowski, J, Kandasamy RA, and Shull GE. Molecular cloning of putative members of the Na/H exchanger gene family. J Biol Chem 267: 9331-9339, 1992[Abstract/Free Full Text].

15.   Pradervand, S, Barker PM, Wang Q, Ernst SA, Beermann F, Grubb BR, Burnier M, Schmidt A, Bindels RJM, Gatzy JT, Rossier BC, and Hummler E. Salt restriction induces pseudohypoaldosteronism type 1 in mice expressing low levels of the beta -subunit of the amiloride-sensitive epithelial sodium channel. Proc Natl Acad Sci USA 96: 1732-1737, 1999[Abstract/Free Full Text].

16.   Schnermann, J, and Briggs JP. Function of the juxtaglomerular apparatus. Control of glomerular hemodynamics and renin secretion. In: The Kidney: Physiology and Pathophysiology, edited by Seldin DW, and Giebisch G.. New York: Raven, 1991, p. 1249-1289.

17.   Schnermann, J, Ploth DW, and Hermle M. Activation of tubuloglomerular feedback by chloride transport. Pflügers Arch 362: 229-240, 1976[ISI][Medline].

18.   Schultheis, PJ, Clarke LL, Meneton P, Harline M, Boivin GP, Stemmermann G, Duffy JJ, Doetschman T, Miller ML, and Shull GE. Targeted disruption of the murine Na+/H+ exchanger isoform 2 gene causes reduced viability of gastric parietal cells and loss of net acid secretion. J Clin Invest 101: 1243-1253, 1998[Abstract/Free Full Text].

19.   Schultheis, PJ, Clarke LL, Meneton P, Miller ML, Soleimani M, Gawenis LR, Riddle TM, Duffy JJ, Doetschman T, Wang T, Giebisch G, Aronson PS, Lorenz JN, and Shull GE. Renal and intestinal absorptive defects in mice lacking the NHE3 Na+/H+ exchanger. Nat Genet 19: 282-285, 1998[ISI][Medline].

20.   Schultheis, PJ, Lorenz JN, Meneton P, Nieman ML, Riddle TM, Flagella M, Duffy JJ, Doetschman T, Miller ML, and Shull GE. Phenotype resembling Gitelman's syndrome in mice lacking the apical Na+ Cl- cotransporter of the distal convoluted tubule. J Biol Chem 273: 29150-29155, 1998[Abstract/Free Full Text].

21.   Simon, DB, Karet FE, Hamdan JM, DiPietro A, Sanjad SA, and Lifton RP. Bartter's syndrome, hypokalaemic alkalosis with hypercalciuria, is caused by mutations in the Na-K-2Cl cotransporter NKCC2. Nat Genet 13: 183-188, 1996[ISI][Medline].

22.   Simon, DB, Nelson-Williams C, Bia MJ, Ellison D, Karet FE, Molina AM, Vaara I, Iwata F, Cushner HM, Koolen M, Gainza FJ, Gitleman HJ, and Lifton RP. Gitelman's variant of Bartter's syndrome, inherited hypokalaemic alkalosis, is caused by mutations in the thiazide-sensitive Na-Cl cotransporter. Nat Genet 12: 24-30, 1996[ISI][Medline].

23.   Soleimani, M, Singh G, Bizal GL, Gullans SR, and McAteer JA. Na+/H+ exchanger isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells. Expression, functional localization, and differential regulation. J Biol Chem 269: 27973-27978, 1994[Abstract/Free Full Text].

24.   Strautnieks, SS, Thompson RJ, Gardiner RM, and Chung E. A novel splice-site mutation in the gamma  subunit of the epithelial sodium channel gene in three pseudohypoaldosteronism type 1 families. Nat Genet 13: 248-250, 1996[ISI][Medline].

25.   Sun, AM, Liu Y, Dworkin LD, Tse CM, Donowitz M, and Yip KP. Na+/H+ exchanger isoform 2 (NHE2) is expressed in the apical membrane of the medullary thick ascending limb. J Membr Biol 160: 85-90, 1997[ISI][Medline].

26.   Takahashi, N, Chernavvsky DR, Gomez RA, Igarashi P, Gitelman HJ, and Smithies O. Uncompensated polyuria in a mouse model of Bartter's syndrome. Proc Natl Acad Sci USA 97: 5434-5439, 2000[Abstract/Free Full Text].

27.   Tse, CM, Brant SR, Walker MS, Pouyssegur J, and Donowitz M. Cloning and sequencing of a rabbit cDNA encoding an intestinal and kidney-specific Na+/H+ exchanger isoform (NHE-3). J Biol Chem 267: 9340-9346, 1992[Abstract/Free Full Text].

28.   Tse, CM, Levine SA, Yun CHC, Montrose MH, Little PJ, Pouyssegur J, and Donowitz M. Cloning and expression of a rabbit cDNA encoding a serum-activated ethylisopropylamiloride-resistant epithelial Na+/H+ exchanger isoform (NHE-2). J Biol Chem 268: 11917-11924, 1993[Abstract/Free Full Text].

29.   Wang, T, Yang CL, Abbiati T, Schultheis PJ, Shull GE, Giebisch G, and Aronson PS. Mechanism of proximal tubule bicarbonate absorption in NHE3 null mice. Am J Physiol Renal Physiol 277: F298-F302, 1999[Abstract/Free Full Text].

30.   Wang, Z, Orlowski J, and Shull GE. Primary structure and functional expression of a novel gastrointestinal isoform of the rat Na/H exchanger. J Biol Chem 268: 11925-11928, 1993[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 281(4):F718-F727
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society