Loss of the NHE2 Na+/H+ exchanger has no apparent effect on diarrheal state of NHE3-deficient mice

Clara Ledoussal1, Alison L. Woo1, Marian L. Miller2, and Gary E. Shull1

1 Department of Molecular Genetics, Biochemistry, and Microbiology and 2 Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The expression of NHE2 and NHE3 on intestinal-brush border membranes suggests that both Na+/H+ exchangers serve absorptive functions. Studies with knockout mice showed that the loss of NHE3, but not NHE2, causes diarrhea, demonstrating that NHE3 is the major absorptive exchanger and indicating that any remaining absorptive capacity contributed by NHE2 is not sufficient to compensate fully for the loss of NHE3. To test the hypothesis that NHE2 provides partial compensation for the diarrheal state of NHE3-deficient mice, we crossed doubly heterozygous mice carrying null mutations in the Nhe2 and Nhe3 genes and analyzed the phenotypes of their offspring. The additional loss of NHE2 in NHE3-deficient mice caused no apparent reduction in viability, no further impairment of systemic acid-base status or increase in aldosterone levels, and no apparent worsening of the diarrheal state. These in vivo phenotypic correlates of the absorptive defect suggest that the NaCl, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and fluid absorption that is dependent on apical Na+/H+ exchange is due overwhelmingly to the activity of NHE3, with little contribution from NHE2.

sodium absorption; diarrhea; Slc9a2; Slc9a3


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

ABSORPTION of NaCl, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and fluid in the intestinal tract is dependent, in part, on apical Na+/H+ exchange (17). Among the five plasma membrane Na+/H+ exchangers, three isoforms (NHE1, NHE2, and NHE3, which are products of the Slc9a1, Slc9a2, and Slc9a3 genes, respectively) are known to be expressed in the intestine (11, 26, 29, 33, 34, 37). Although low levels of NHE4 mRNA have been reported in rat intestine (29), other studies (3) suggested that detection of this isoform could have been due to cross-hybridization with NHE2. On the basis of Northern analyses (G. Shull, unpublished observations), we agree with this interpretation. NHE2 and NHE3 are present in apical membranes of the intestinal epithelium (1, 14), whereas NHE1 is restricted to basolateral membranes (1). The apical location of NHE2 and NHE3 and the results of studies with brush-border membrane vesicles (8, 9, 40) are consistent with the possibility that both of these isoforms might contribute to the absorptive capacity of the intestine.

There is compelling evidence (6, 8, 24, 25, 34) that NHE3 serves an important absorptive function in the mammalian intestine. Na+/H+ exchange activity and NHE3 mRNA are induced in rabbit ileum in response to glucocorticoids (41), and aldosterone has been shown to induce Na+/H+ exchange and NHE3 mRNA and protein in colon (6). NHE3 has a substantial reserve capacity, as a considerable fraction of the protein is present in endosomal vesicles and can be recruited rapidly to the plasma membrane (7, 10, 15) if additional Na+/H+ exchange activity is needed. Absorption studies (24, 25) performed in vivo in dogs suggested that NHE3 plays a major role in basal and meal-stimulated ileal Na+ and water absorption. The critical absorptive function of NHE3 in the mammalian intestine in vivo was conclusively demonstrated by the occurrence of diarrhea in gene-targeted mice lacking the exchanger (34). In contrast, mice lacking NHE2 did not have diarrhea (33) and displayed no obvious intestinal phenotype. Similarly, the studies (24, 25) that revealed a major absorptive function for NHE3 in the dog intestine in vivo failed to reveal any absorptive activity that could be attributed to NHE2. Also, several recent studies (19, 23, 30) indicate that in other epithelial tissues, NHE2 does not contribute to Na+ absorption.

The presence of diarrhea in mice lacking NHE3 and the absence of diarrhea in mice lacking NHE2 shows that NHE3 is the major absorptive Na+/H+ exchanger in the intestine but does not rule out a similar, albeit less prominent, absorptive role for NHE2. If NHE2 does play an important supplementary role in the absorption of NaCl, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and fluid in the intestinal tract, then it should provide some degree of compensation for the congenital diarrhea resulting from the loss of NHE3. In such a role, NHE2 would contribute to the maintenance of acid-base balance, Na+-fluid volume homeostasis, and viability of the NHE3 knockout. To test this hypothesis, we developed mice carrying various combinations of wild-type and mutant Nhe2 and Nhe3 genes and analyzed their viability, acid-base status, serum aldosterone levels, and intestinal contents. Analysis of these in vivo correlates of an absorptive defect provides no support for the hypothesis that NHE2 serves as an important compensatory mechanism in the NHE3-deficient intestine.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Mice. The use of animals in these experiments was approved by the University of Cincinnati Animal Care and Use Committee. The development of mice carrying mutations in the Nhe2, Nhe3, and the gastric H+-K+-ATPase alpha -subunit genes was performed as described previously (33-35). To produce mice with mutations in both the Nhe2 and Nhe3 genes, a female Nhe2 homozygous mutant (Nhe2-/-) mouse was bred repeatedly with a male Nhe3 heterozygous (Nhe3+/-) mouse to generate offspring that were heterozygous at both loci. The Nhe3+/- mouse used for the initial matings was of a mixed background consisting of 129/SvJ and Black Swiss strains, and the Nhe2-/- mouse was on a non-Swiss albino background. Seven pairs of doubly heterozygous (Nhe2+/-Nhe3+/-) mice were then mated to generate offspring that included all nine genotypes expected for the genetic transmission of two independent genes. Before the study was completed, four additional pairs of doubly heterozygous mice, derived from the first set of breeding pairs, were mated to generate the mice needed to complete the experiments.

To produce mice with mutations in both the Nhe3 gene and the gastric H+-K+-ATPase alpha -subunit gene (needed as a control for the achlorhydria that also occurs in NHE2-deficient mice), a female Nhe3+/- mouse was crossed with a gastric H+-K+-ATPase heterozygous male (gHKA+/-) mouse. Both heterozygous mice were on a mixed background consisting of 129/SvJ and Black Swiss strains. Doubly heterozygous (Nhe3+/-gHKA+/-) offspring from the initial cross were then mated to obtain all nine possible genotypes.

Analysis of genotypes. PCR genotyping 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. Genotyping of Nhe2 and Nhe3 alleles was performed as described previously (19). For gHKA, forward (5'-GCCTGTCACTGACAGCAAAGAGG-3') and reverse (5'-GGTCTTCTGTGGTGTCCGCC-3') primers corresponding to sequences from near the 5' and 3' ends of exon 8 and a primer from near the 3' end of the neomycin-resistance gene (5'-CTGACTAGGGGAGGAGTAGAAGG-3') were used to amplify 117- and 299-bp products from the wild-type and mutant genes, respectively. For all PCR 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.

Analysis of blood pH, gases, and electrolytes. Mice were placed on a 37°C warming pad to stimulate peripheral blood circulation. Blood (50 µl) was collected from the tail vein in a heparinized capillary tube (Ciba-Corning, Medfield, MA) and immediately analyzed using a pH and blood gas analyzer (model 348, Chiron Diagnostics, Oberlin, OH).

Analysis of size and contents of intestinal segments. Mice were anesthetized with an intraperitoneal injection of 2.5% Avertin (0.02 ml/g body wt) and euthanized. The lengths of the small intestine and colon and the surface area of the cecum were measured, and the weights of each segment and its contents were determined. The contents of each segment were mixed with saline to a total volume of 1 ml and centrifuged, and the pH of the supernatant was determined. These measurements were carried out on all nine genotypes for the Nhe2/Nhe3 mice (n = 5 for each genotype). For the gHKA/Nhe3 mice, only four experimental groups were analyzed. The genotypes of these groups were 1) gHKA+/+ and Nhe3+/+, 2) gHKA-/- and Nhe3+/+ or Nhe3+/-, 3) gHKA+/+ or gHKA+/- and Nhe3-/-, and 4) gHKA-/- and Nhe3-/- (n = 5 for each group).

Serum aldosterone levels. Mice were anesthetized with an intraperitoneal injection of 2.5% Avertin (0.02 ml/g body wt), and blood was drawn by cardiac puncture. Serum was separated from blood cells and frozen at -70°C until analysis was performed. Serum aldosterone concentrations were determined using an RIA kit according to the manufacturer's suggested protocol (Diagnostics Products, Los Angeles, CA).

Northern blots. Total RNA was isolated from mouse intestinal segments using the Tri-Reagent kit (Molecular Research Center, Cincinnati, OH) according to the manufacturer's suggested protocol. Northern blots were prepared, hybridized, and washed as described previously (33). Total RNA (10 µg) was denatured with glyoxal and dimethyl sulfoxide, fractionated in 1% agarose, and transferred to a nylon membrane. Hybridization was carried out using 32P-labeled cDNA probes for rat NHE2 (a fragment spanning nt 449 to 3561; Ref. 39), rat NHE3 (a fragment spanning nt -44 to 4228; Ref. 29), and the mouse L32 ribosomal subunit as a loading control. Signal intensities were quantitated by PhosphorImager analysis.

Statistics. A chi 2-square test was performed to determine whether the observed distribution of nine genotypes among mice derived by mating doubly heterozygous mice with null mutations in the Nhe2 and Nhe3 genes (Nhe2/Nhe3 colony) or the gHKA and Nhe3 genes (gHKA/Nhe3 colony) deviated significantly from the expected distribution. In other experiments, a nonpaired Student's t-test was used to analyze the data.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Our working hypothesis was that NHE2 provides a supplementary absorptive function that blunts the severity of the diarrheal state in NHE3-deficient mice. Because there is a significant incidence of morbidity and death among Nhe3-/- mice (see DISCUSSION), which have a severe impairment of Na+-fluid volume homeostasis and a mild perturbation of acid-base balance (22, 34, 38), we reasoned that the additional loss of a major compensatory mechanism that contributes to the recovery of NaCl, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and accompanying fluid from the intestinal lumen would cause a further increase in morbidity and death. To begin testing of this hypothesis in vivo, a colony was established by breeding mice that were heterozygous for both mutant genes (Nhe2+/-Nhe3+/-).

Genotype ratios, body weight, and survival of offspring from matings of Nhe2/Nhe3 doubly heterozygous mice. The genotypes of 444 offspring were determined by PCR analysis, and all nine expected genotypes were obtained. The observed and expected numbers of doubly homozygous mutant (Nhe2-/-Nhe3-/-) mice were very similar (Table 1), indicating that the absence of both isoforms simultaneously does not cause major perturbations of embryonic or fetal development. Although several genotypes seemed to be underrepresented, notably the Nhe2 and Nhe3 null mutants that were wild type for the other isoform, the distribution of genotypes did not differ significantly from the normal Mendelian ratio for the transmission of two independent genes. When transmission of each gene was analyzed separately, they were found to be transmitted at ratios close to the normal 1:2:1 Mendelian ratio (Nhe2: 19.1% -/-, 56.3% +/-, and 24.6% +/+; Nhe3: 20.7% -/-, 53.8% +/-, and 25.5% +/+), as observed in previous studies (33, 34).

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Production of Nhe2/Nhe3 mutant mice

At 7-8 wk of age, the mean body weight of Nhe2-/- Nhe3-/- mice (23.8 ± 1 g, n = 7) was slightly less than that of Nhe3-/- mice carrying one or two functional copies of the Nhe2 gene (26.3 ± 1.4 g, n = 17) and was also slightly less than that of mice with at least one copy of both genes (26 ± 0.7 g, n = 27). The differences, however, were not significant.

Most of the mice that lacked both NHE2 and NHE3 grew to adulthood, and on the basis of their outward appearance and behavior, the double-null mutants could not be distinguished from mice that lacked only NHE3. Some of the Nhe3-/- mice, regardless of the Nhe2 allelic dosage, died at ages ranging from 3 to 24 wk. Deaths were most common during the week following weaning, but others occurred in adult animals, which generally presented with bloating of the abdomen. Full lethality curves extending over many months and including all of the Nhe3 null mice could not be constructed, because some of the mice were used for experiments beginning at 7 wk of age and because many of the mice that were not needed for experiments were euthanized before weaning. However, among the 75 Nhe3-/- mice (carrying 0, 1, or 2 copies of the wild-type Nhe2 gene) not euthanized before 7 wk of age, there were 10 deaths. All of these occurred at 3-4 wk of age and included 5 of 24 Nhe2-/-Nhe3-/- mice, 3 of 34 Nhe2+/-Nhe3-/- mice, and 2 of 17 Nhe2+/+ Nhe3-/- mice. Contrary to our hypothesis, the death of Nhe3 null mutants was not strongly associated with the loss of the Nhe2 gene (see DISCUSSION).

Systemic acid-base and electrolyte status. Because diarrhea often impairs acid-base and electrolyte homeostasis, we evaluated blood pH, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, gases, and electrolytes from awake animals of the nine different genotypes and performed multiple pairwise comparisons. As shown in Table 2 (see the first 3 rows), blood pH and plasma HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> concentrations in mice lacking both NHE2 and NHE3 did not differ significantly from those of Nhe3-/- mice with one or two functional copies of the Nhe2 gene (Nhe2+/- or Nhe2+/+). When the values for Nhe2-/- mice with one or two functional copies of the Nhe3 gene were compared with those of Nhe3+/- or Nhe3+/+ mice that were wild type with respect to Nhe2 (Table 2, row 4 vs. 6 and row 7 vs. 9), no significant differences were observed, consistent with previous experiments (19, 33) showing that the loss of NHE2 alone does not lead to acid-base or electrolyte abnormalities. Surprisingly, there were also no significant differences in acid-base values between any of the Nhe3-/- groups and the Nhe3+/- or wild-type groups, although there was a suggestive trend. However, as noted in DISCUSSION, when the number of mice being compared was increased by combining the Nhe3 knockout groups and ignoring the Nhe2 genotype, a small reduction in blood pH was apparent in Nhe3-/- mice, consistent with the mild acidosis reported previously (34, 38).

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Blood pH and gases and plasma electrolytes in Nhe2/Nhe3 mice

Loss of NHE2 does not lead to further increase in serum aldosterone in mice lacking one or two copies of Nhe3 gene. Serum aldosterone levels are known to increase in response to NaCl wasting or volume depletion, thereby stimulating more efficient Na+ and fluid absorption in the kidney and colon. Thus it was of interest to determine whether the loss of NHE2 in Nhe3+/- or Nhe3-/- mice would lead to an increase in serum aldosterone levels beyond that resulting from the loss of NHE3. As shown in Fig. 1, the loss of one Nhe3 allele on an Nhe2 wild-type background (Nhe3+/- Nhe2+/+ mice) led to a modest but significant increase in serum aldosterone relative to that of wild-type mice, but the additional loss of two Nhe2 alleles (Nhe2-/- Nhe3+/- mice) caused no further increase. Similarly, a large increase in serum aldosterone was observed in mice lacking both functional copies of the Nhe3 gene, but the additional loss of one or both Nhe2 alleles had no further effect.


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 1.   Serum aldosterone levels in adult Nhe2/Nhe3 mice. Genotypes with respect to the Nhe2 (2+/+, 2+/-, and 2-/- for Nhe2+/+, Nhe2+/-, and Nhe2-/-, respectively) and Nhe3 genes (3+/+, 3+/-, and 3-/- for Nhe3+/+, Nhe3+/-, and Nhe3-/-, respectively) are indicated below the corresponding values for serum aldosterone. The mice were 7- to 12-wk old, and the no. of mice for each genotype is shown in parentheses. Relative to wild-type mice, serum aldosterone increased in both Nhe3+/- (* P < 0.05) and Nhe3-/- mice (** P < 0.01). Loss of the wild-type Nhe2 gene caused no further increase in aldosterone levels in either Nhe3+/- or Nhe3-/- mice. Values are means ± SE.

Absence of NHE2 in NHE3-deficient mice causes no increase in quantity or pH of intestinal contents. In a previous study, we (34) showed that the quantity and pH of the contents of the small intestine, cecum, and colon of Nhe3-/- mice were increased relative to those of wild-type mice due to the accumulation of alkaline fluid and that the size of each intestinal segment was increased. Also, we have observed that morbidity and death of Nhe3-/- mice is preceded by bloating of the abdomen due to an increase in the contents of the intestine. This suggests that the intestinal absorptive defect is a major factor in the observed morbidity and that the quantity and pH of the luminal contents might provide a rough indication of the severity of the absorptive defect in mice with various combinations of mutant and wild-type Nhe2 and Nhe3 alleles.

Relative to that of wild-type mice, the weights of the intestinal segments were not significantly changed in Nhe2-/- mice but were significantly increased in both Nhe2+/+Nhe3-/- and Nhe2-/-Nhe3-/- mice (Table 3). The mean length of the small intestine and colon and the surface area of the cecum were also increased in Nhe2+/+Nhe3-/- and Nhe2-/-Nhe3-/- mice. However, there were no significant differences between Nhe3-/- and double knockout mice (Table 3).

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Size of intestinal segments in Nhe2/Nhe3 mice

In general, the quantity of the contents of each intestinal segment was similar for Nhe3+/+ or Nhe3+/- mice (Fig. 2) regardless of the Nhe2 gene dosage, whereas the loss of both copies of the Nhe3 gene caused fluid accumulation in all three segments. The additional loss of the Nhe2 gene caused no further increase in the quantity of the contents of the cecum or colon. The mean weight of the small intestinal contents of Nhe2-/-Nhe3-/- mice (0.48 ± 0.09 g) was lower than that of Nhe2+/+Nhe3-/- mice (0.73 ± 0.11 g, P = 0.06). This trend is the opposite of what would be expected if the loss of NHE2 enhanced the diarrheal state of Nhe3-/- mice.


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 2.   The weight and pH of the contents of the small intestine, cecum, and colon of 7- to 12-wk-old Nhe2/Nhe3 mice of all 9 genotypes. The Nhe3 genotypes are indicated by histograms (wild type, 3+/+; heterozygous, 3+/-; homozygous mutant, 3-/-). Nhe2 genotypes (wild type, 2+/+; heterozygous, 2+/-; homozygous mutant, 2-/-) are indicated below columns. Values are means ± SE; n = 5 for each genotype. * P <=  0.05, dagger  P < 0.01, significantly different from wild type.

As shown in Fig. 2, the mean pH of the intestinal contents of Nhe2-/- mice with at least one functional Nhe3 gene (n = 10; small intestine, pH 6.6 ± 0.1; cecum, pH 6.6 ± 0.2; colon, pH 6.8 ± 0.1) was more acidic than in the corresponding Nhe2+/+ mice (n = 10; small intestine, pH 7.2 ± 0.1, P < 0.0001; cecum, pH 7.0 ± 0.1, P < 0.05; colon, pH 7.3 ± 0.1, P < 0.003). The loss of NHE3, regardless of Nhe2 gene dosage, led to alkalinization of the intestinal contents. Minor differences between Nhe2+/+Nhe3-/- and Nhe2-/- Nhe3-/- mice occurred in the small intestine, where the pH of the contents was slightly lower in the double knockout mice (P < 0.05), and in the cecum, where the pH was slightly higher in the double knockout mice (P < 0.02).

It seemed possible that the acidity of the intestinal contents of Nhe2-/- mice and the lack of any apparent worsening of the diarrheal phenotype might be secondary to their stomach phenotype. The absence of NHE2 impairs the viability of the gastric parietal cell, leading to necrotic cell death and a progressive loss of acid secretion as the animals age; adult mice are achlorhydric, with stomach contents ranging in pH from 7.0 to 8.9 (33). Because achlorhydria is known to lead to a sharp reduction in pancreatic HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and fluid secretion into the duodenum (20), the achlorhydria resulting from the loss of NHE2 could indirectly reduce the need for absorption via apical Na+/H+ exchange in the intestine. Also, a reduction in overall fluid secretion in the stomach resulting from achlorhydria would be expected to further reduce fluid and electrolyte delivery to the small intestine. As a control for the confounding effects of achlorhydria, mice with mutations in both the Nhe3 and gastric H+-K+-ATPase alpha -subunit (gHKA) genes were prepared and analyzed. We reasoned that these mice should exhibit the same reductions in fluid and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> delivery to the small intestine as might occur in the Nhe2-/- mice. If NHE2 provided a supplementary absorptive capacity, then the diarrheal state of achlorhydric gHKA-/-Nhe3-/- mice, with intact NHE2 function in the intestine, should be less severe than that of achlorhydric Nhe2-/-Nhe3-/- mice. Contrary to our hypothesis, the phenotype of the gHKA-/- Nhe3-/- mutants was similar to that of the Nhe2/Nhe3 double knockouts.

Analysis of intestinal contents of mice with mutations in Nhe3 and gHKA genes. When mice that were doubly heterozygous for null mutations in the Nhe3 and gHKA genes were bred, all nine genotypic categories were observed (Table 4), and both mutant alleles were transmitted with a normal 1:2:1 Mendelian ratio. The viability of the double knockout mice was impaired, with 6 of 11 mice dying before 7 wk of age. The body weights of surviving double-knockout mice were not significantly reduced when compared with wild-type controls. The weight and pH of the intestinal contents of the five surviving gHKA-/-Nhe3-/- mutants and other genotypic categories for the gHKA/Nhe3 line were determined and compared with those of the Nhe2/Nhe3 mice (Figs. 3 and 4). Overall, the patterns observed for intestinal content weight (Fig. 3) and pH (Fig. 4) for gHKA/Nhe3 mice were similar to those of the corresponding Nhe2/Nhe3 mice.

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   Production of gHKA/Nhe3 mutant mice



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 3.   Quantity of the intestinal contents of Nhe2/Nhe3 and gHKA/Nhe3 mice. The weight of the contents of the small intestine (Sm. Int.), cecum, and colon of 7- to 12-wk-old mice of the indicated genotypes was determined. Data for the Nhe2/Nhe3 mice are a subset of the data shown in Fig. 2 and are replotted to facilitate comparisons with gHKA/Nhe3 data. A: data for Nhe2/Nhe3 mice (wild type, 2+/+3+/+; Nhe2 knockout, 2-/-3+/+; Nhe3 knockout, 2+/+ 3-/-; Nhe2/Nhe3 double knockout, 2-/-3-/-). B: data for gHKA/Nhe3 mice (wild type, G+/+3+/+; gHKA knockout with 1 or 2 wild-type Nhe3 genes, G-/-3+/±; Nhe3 knockout with 1 or 2 wild-type gHKA genes, G+/±3-/-; gHKA/Nhe3 double knockout, G-/-3-/-). Values are means ± SE; n = 5 for each group. * P < 0.05, dagger  P < 0.01, significantly different from wild type.



View larger version (42K):
[in this window]
[in a new window]
 
Fig. 4.   pH of the intestinal contents of Nhe2/Nhe3 and gHKA/Nhe3 mice. The pH of the contents of the small intestine, cecum, and colon of 7- to 12-wk-old mice of the indicated genotypes was determined. Data for the Nhe2/Nhe3 mice are a subset of the data shown in Fig. 2 and are replotted to facilitate comparisons with the gHKA/Nhe3 data. A: data for Nhe2/Nhe3 mice. B: data for gHKA/Nhe3 mice. Note that loss of either the Nhe2 or gHKA genes caused acidification of the intestinal contents. Values are means ± SE; n = 5 for each group. * P <=  0.05, dagger  P < 0.01, significantly different from wild type.

When compared with wild-type mice, the loss of both copies of the Nhe2 (Fig. 3A) or gHKA genes (Fig. 3B) in mice carrying at least one functional copy of the Nhe3 gene led to only slight changes in the weight of the contents of each segment. When the quantities of the intestinal contents for the two double-knockout mice were compared directly, there was a significant difference for the small intestine (gHKA-/-Nhe3-/-, 0.84 ± 0.09 g; Nhe2-/-Nhe3-/-, 0.48 ± 0.08 g; P < 0.05), but not for the cecum (gHKA-/-Nhe3-/-, 1.77 ± 0.24 g; Nhe2-/-Nhe3-/-, 1.73 ± 0.30 g) or colon (gHKA-/- Nhe3-/-, 0.48 ± 0.07 g; Nhe2-/-Nhe3-/-, 0.67 ± 0.17 g).

Like that of Nhe2-/- mice, the pH of the intestinal contents of gHKA-/- mice was more acidic than those of wild-type mice (Fig. 4), consistent with an impairment of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> secretion secondary to achlorhydria. When the gHKA and Nhe3 null mutations were combined, the pH of the intestinal contents increased to levels similar to those observed in Nhe2/Nhe3 double knockouts. The pH of the contents of the small intestine was higher in gHKA-/-Nhe3-/- mice (7.75 ± 0.05) than in Nhe2-/-Nhe3-/- mice (7.54 ± 0.10; P < 0.05). The pH of the contents of the cecum (gHKA-/-Nhe3-/-, 7.81 ± 0.11; Nhe2-/-Nhe3-/-, 7.90 ± 0.09) and colon (gHKA-/-Nhe3-/-, 7.81 ± 0.11; Nhe2-/-Nhe3-/-, 7.61 ± 0.12) was not significantly different.

Northern blot analysis of NHE2 and NHE3 mRNA levels in intestinal tract of NHE-deficient mice. As an initial test of the relative responses of the Nhe2 and Nhe3 genes to the diarrheal state of NHE3-deficient mice, we compared the expression of the 4.4-kb NHE2 mRNA and both the 5.9-kb wild-type and the 3.8- and 2.1-kb mutant NHE3 mRNAs in the small intestine, cecum, proximal colon, and distal colon of adult Nhe3+/+, Nhe3+/-, and Nhe3-/- mice. The levels of NHE2 mRNA in each intestinal segment were approximately the same in all three genotypes (Fig. 5). In contrast, the sharp increase in hybridization intensities of the mutant Nhe3 mRNAs in the small intestine, cecum, and proximal colon of Nhe3-/- mice, relative to those of Nhe3+/- mice, suggests that expression of the Nhe3 gene (when corrected for gene copy number) is upregulated approximately threefold in the small intestine and approximately twofold in the cecum and proximal colon of Nhe3-/- mice. There was no apparent induction of mutant NHE3 mRNAs in the distal colon.


View larger version (96K):
[in this window]
[in a new window]
 
Fig. 5.   Northern blot analysis of RNA from the small intestine, cecum, proximal colon (Prox. Col.), and distal colon (Dist. Col.) of Nhe3+/+, Nhe3+/-, and Nhe3-/- mice. Each RNA sample (10 µg of total RNA per lane) was extracted from the pooled tissues of 3 adult mice. The blot was first hybridized with an NHE2 probe and then stripped and hybridized with an NHE3 probe that identifies both the wild-type NHE3 mRNA and 2 mutant mRNAs that lack codons 320-825 (Ref. 34). A relatively short exposure time was used for the NHE3 probe to allow visualization of the upregulation of aberrant mRNAs in the small intestine of the knockout; at longer exposures, the wild-type mRNA is clearly visible in the wild-type and heterozygous samples. The blot was stripped again and hybridized with a ribosomal L32 protein probe as a loading control. PhosphorImager analysis showed 6.2-, 3.6-, and 4.1-fold increases in hybridization intensity of the mutant mRNAs in the small intestine, cecum, and proximal colon, respectively, of Nhe3-/- mice (with 2 copies of mutant gene), relative to those of Nhe3+/- mice (with 1 copy of mutant gene). In contrast, PhosphorImager analysis of NHE2 mRNA indicated slight reductions in hybridization intensity in Nhe3-/- samples relative to those of wild-type and heterozygous samples.

Northern blot analyses of tissues from multiple animals revealed no significant differences in NHE2 mRNA levels in either the small intestine or colon of wild-type and Nhe3-/- mice (Fig. 6), consistent with the results shown in Fig. 5. Finally, there were no significant differences in NHE3 mRNA levels of wild-type and Nhe2-/- mice in either the small intestine or colon (Fig. 7).


View larger version (48K):
[in this window]
[in a new window]
 
Fig. 6.   Northern blot analysis of NHE2 mRNA in small intestine and colon of Nhe3-/- and Nhe3+/+ mice. A: each RNA sample (10 µg of total RNA per lane) was extracted from an individual adult mouse. The blot was hybridized with an NHE2 probe and then stripped and hybridized with a probe for the ribosomal L32 protein. B: hybridization signals for NHE2 mRNA were quantitated by PhosphorImager analysis and normalized to the corresponding signal for L32. Values are means ± SE.



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 7.   Northern blot analysis of NHE3 mRNA in small intestine and colon of Nhe2-/- and Nhe2+/+ mice. A: each RNA sample (10 µg of total RNA per lane) was extracted from an individual adult mouse. The blot was hybridized with an NHE3 probe and then stripped and hybridized with a probe for the ribosomal L32 protein as a loading control. B: hybridization signals for NHE3 mRNA were quantitated by PhoshorImager analysis and normalized to the corresponding signal for L32. Values are means ± SE.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The major objective of this study was to identify in vivo correlates of a worsening of the absorptive defect resulting from the additional loss of NHE2 in NHE3-deficient mice. In vitro studies utilizing Na+/H+ exchange inhibitors and isolated vesicles suggested that both NHE2 and NHE3 could provide a significant absorptive capacity in the intestine (8, 9, 40); however, clear evidence of an absorptive role for NHE2 in the intestine in vivo is lacking. As an in vivo test of the hypothesis that NHE2 provides compensation for the intestinal absorptive defect of Nhe3-/- mice, we developed Nhe2/Nhe3 double-knockout mice and analyzed their phenotype. Because of the severe absorptive defects in both the intestine and kidney (22, 34, 38), Nhe3-/- mice are in a chronic volume-depleted state, as indicated by low blood pressure and high aldosterone. When subjected to a Na+-depleted diet for three days, Nhe3 null mice lose weight rapidly, become severely acidotic, and are susceptible to hypovolemic shock (19). This poor tolerance of a dietary perturbation of Na+-fluid volume homeostasis suggested that the genetic loss of a transport mechanism that provides significant compensation for the absorptive defect of the Nhe3-/- intestine, as proposed for NHE2, would cause a substantial worsening of the phenotype. Contrary to our expectations, the results support the alternative hypothesis that NHE3 alone, or in combination with a yet to be identified transporter(s), provides the Na+/H+ exchange activity involved in fluid and electrolyte absorption in the intestine.

Viability of Nhe2/Nhe3 mutants. In an earlier study of the Nhe3 knockout (34), in which the mice were of a mixed 129/SvJ and Black Swiss background, the survival of null mutants was indistinguishable from that of wild-type mice. However, in later generations, some breeding pairs tended to yield null mutants with poor survival rates, and when we placed the mutation on a C57Bl6 background, null mutants did not survive beyond weaning (G. Shull, unpublished data). Those observations suggested that inbreeding leads to impaired viability, which we now minimize by frequent backcrossing with mice derived from a mating of 129/SvJ and Black Swiss mice. Because the observed morbidity was associated with abdominal bloating due to swelling of the intestinal tract, which is greater than that observed in normal knockout mice, it seemed likely that the diarrheal state is a major factor leading to death.

The more severe phenotype observed after inbreeding is apparently due to the existence of a recessive locus (or loci), other than Nhe2, that modifies the phenotype. If the Nhe2 gene were also an important modifier locus for the diarrheal state of NHE3-deficient mice, as should be the case if it made a major contribution to NaCl or NaHCO3 absorption, then the survival rate of Nhe2-/-Nhe3-/- mice should be sharply reduced relative to that of Nhe3-/- mice. Although the frequency of death was slightly higher among double knockouts, the association was weak. For one breeding pair, in which there were 10 Nhe3 null mutants, four of seven Nhe3-/- mice with at least one functional copy of the Nhe2 gene died, whereas the three double-knockout mice survived. These viability data do not support the hypothesis that NHE2 provides important compensation for the intestinal absorptive defect of NHE3-deficient mice.

Because the absence of NHE2 causes a progressive loss of gastric parietal cells and acid secretion, culminating with achlorhydria in adult mice (33), Nhe2-/- Nhe3-/- mice have both a stomach secretory defect and an intestinal absorptive defect. Thus the slight reductions in survival rate and body weight of Nhe2-/- Nhe3-/- mice, although not statistically significant when compared with the Nhe2+/+Nhe3-/- mice, may have been due to a nutritional deficit resulting from the combined effects of the stomach and intestinal phenotypes rather than a worsening of the intestinal absorptive defect.

Systemic acid-base and Na+-fluid volume status of Nhe2/Nhe3 mutants. NHE2 is expressed on apical membranes in both the kidney (5) and intestine (1, 14). If it provided significant compensation for the loss of NHE3 in either organ, then an impairment of acid-base or Na+-fluid volume homeostasis in Nhe2-/-Nhe3-/- mice, relative to that in Nhe3-/- mice carrying a functional copy of the Nhe2 gene, would have been expected. However, this did not occur.

An important renal function for NHE3 in the maintenance of systemic acid-base homeostasis has been well established. In situ microperfusion studies using wild-type and NHE3-deficient mice showed that NHE3 is responsible for 55-60% of the HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the proximal tubule (34, 38). Despite the severe absorptive defect, Nhe3-/- mice maintained on a normal diet exhibit only a mild acidosis (34, 38), suggesting that the deficit is largely overcome by compensatory mechanisms. In contrast, when Nhe3-/- mice were fed a Na+-restricted diet, which quickly leads to severe hypovolemia, the acidosis was severe (19). This latter observation suggested that a further impairment of Na+-fluid volume homeostasis in Nhe3-/- mice (as would be expected with the loss of NHE2 if this isoform played a compensatory role in Na+ absorption) would cause a worsening of the acid-base disorder. Some of the renal compensatory mechanisms for the proximal tubule absorptive defect have been identified. The glomerular filtration rate is reduced by 30-35% in Nhe3-/- mice (4, 22), thereby limiting the amount of HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> that must be reabsorbed, and HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> reabsorption in the collecting duct via the H+-ATPase and anion exchanger 1 Cl-/HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> exchanger is upregulated (28). Whether additional mechanisms, such as NHE2, contribute to compensation for the acid-base disorder of NHE3-deficient mice is unclear.

Given the previous observation of a mild acidosis in anesthetized Nhe3-/- mice (34, 38), we were surprised to see that there was little evidence of acidosis in the awake Nhe3 knockout mice, regardless of the number of functional Nhe2 genes. There were no trends in blood pH that would suggest that the loss of NHE2 contributes to a mild acidosis, which could occur either by a direct reduction in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption or indirectly via reduced Na+ absorption and subsequent impairment of Na+-fluid volume homeostasis. However, the groups lacking NHE3 did exhibit a suggestive trend (Table 2, row 1 vs. rows 4 and 7, row 2 vs. rows 5 and 8, and row 3 vs. rows 6 and 9). When the Nhe2 genotype of the awake mice used in the current study was ignored, thereby raising the number of mice being considered for statistical analysis, and the values for Nhe3-/- mice (Table 2, rows 1-3, n = 21) were compared with those of Nhe3+/- and Nhe3+/+ mice (rows 4-9, n = 42), a mild but statistically significant reduction in blood pH was apparent in Nhe3-/- mice (pH, 7.41 ± 0.01 for Nhe3-/- and 7.45 ± 0.01 for Nhe3+/- and Nhe3+/+ combined, P < 0.01). Although the differences are small, they are only slightly less than the decrease of 0.06 pH units we (34) observed previously in anesthetized mice. The milder acidosis observed in awake Nhe3-/- mice, relative to that observed in anesthetized mice (34, 38), may be due to the absence of a respiratory component to the acidosis, which has been attributed to the effects of anesthesia (38). The addition of a mild respiratory acidosis during anesthesia may stress the renal compensatory mechanisms and result in an enhancement of the small differences between wild-type and Nhe3-/- mice. The observation that Nhe2Nhe3 double-knockout mice did not exhibit a significant acidosis when compared with Nhe3-/- mice argues against an important role for NHE2 in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> absorption in either the kidney or intestine.

As an indication of the Na+-fluid volume status of the mutants, we measured the levels of serum aldosterone, which is known to increase in response to volume depletion or a deficit in NaCl absorption. Nhe3+/- mice exhibit decreased fluid absorption in the renal proximal tubule (22) and increased expression of colonic H+-K+-ATPase in the colon (34). Thus the loss of one copy of the Nhe3 gene stresses the absorptive capacity of both the kidney and colon, indicating that the Nhe3+/- background is suitable for testing the effects of the Nhe2 null mutation. Serum aldosterone was mildly elevated in Nhe3+/- mice and sharply elevated in Nhe3-/- mice, consistent with a deficit in Na+-fluid volume homeostasis caused by NHE3 deficiency. However, the loss of NHE2 in Nhe3+/- or Nhe3-/- mice caused no further increase in serum aldosterone, suggesting that its absence does not cause a worsening of the absorptive defects in either the kidney or intestine. Consistent with this observation, in a separate study designed to assess the absorptive functions of NHE2 and NHE3 in the kidney, we observed no renal or fecal salt wasting in Nhe2-/- mice maintained on a Na+-restricted diet, even when their Na+ retention capability was further stressed by placing the Nhe2 mutation on an Nhe3+/- background (19). The lack of an effect of the Nhe2 null mutation on serum aldosterone (Fig. 1) or NaCl retention during dietary Na+ restriction in Nhe3+/- mice (19) argues against a major role for NHE2 in NaCl absorption.

Intestinal phenotype of Nhe2/Nhe3 double knockout mice. When we observed the animals in their cages, the diarrhea of Nhe2-/-Nhe3-/- mice did not appear to be any more severe than that of Nhe3-/- mice. To further assess the diarrheal state of the various mutants, we measured the pH and weight of the contents of the small intestine, cecum, and colon. This method is a variation of the enteropooling assay (32), which is commonly used for quantitative assessment of induced diarrhea in rodents for up to 6 h following treatment. Although it has not been used previously to assess the severity of congenital diarrhea, our observation that mice exhibiting signs of morbidity have more severe swelling of all intestinal segments suggests that increased pooling of fluid occurs in the intestinal tracts of the more severely affected mice. The data must be interpreted with caution, however, as it is possible that these measurements lack the sensitivity required to detect a mild worsening of the diarrheal state. When Nhe2-/-Nhe3-/- mice were compared with Nhe2+/+ Nhe3-/- mice, the quantity of the luminal contents was not increased in any segment and, surprisingly, the quantity of the small intestinal contents was reduced. There was a slight increase in the pH of the contents of the cecum, but not of the other segments. These results do not support the hypothesis that NHE2 plays an important compensatory role in the diarrheal state of NHE3-deficient mice. However, interpretations of the data were complicated by the fact that the intestinal contents of Nhe2-/- mice carrying the wild-type Nhe3 gene were quite acidic when compared with those of wild-type mice.

One of the confounding problems in gene knockout studies is that the loss of a transporter in one tissue can affect the phenotype in another tissue. Because the loss of NHE2 in the stomach causes reduced viability of the gastric parietal cell and achlorhydria in adult Nhe2-/- mice, a likely explanation for the acidity of the intestinal tract was a reduction in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and fluid secretion from the pancreas, which normally occurs when acidic contents from the stomach are emptied into the duodenum (18). It was important to control for this variable, because the reduction in HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> and fluid secretion from the pancreas, as well as electrolyte and fluid secretion from the stomach, would be expected to reduce the need for absorptive Na+/H+ exchange activity in the intestine. If this were the case, then the apparent lack of an enhancement of the diarrheal state in Nhe2-/-Nhe3-/- mice (relative to that of Nhe3-/- mice with a functional copy of Nhe2) might be the result of a decrease in secretion from both the stomach and pancreas (secondary to achlorhydria) balancing a decrease in absorption resulting from the loss of NHE2. Mice lacking the gastric H+-K+-ATPase are achlorhydric (35) and, like Nhe2-/- mice, the luminal contents of all intestinal segments were acidic (Fig. 4), indicating that the achlorhydria resulting from the gHKA null mutation would provide a suitable control for the achlorhydric phenotype of Nhe2 null mutants. However, achlorhydric gHKA-/-Nhe3-/- mice with functional NHE2 in the intestine had essentially the same intestinal phenotype as achlorhydric Nhe2-/-Nhe3-/- mice lacking NHE2 in the intestine.

As an additional indication of whether NHE2 might provide compensation for the diarrheal state of NHE3-deficient mice, we examined the expression of NHE2 and NHE3 mRNAs in intestinal segments from wild-type, Nhe3+/-, and Nhe3-/- mice. The diarrheal state did not lead to the induction of NHE2 mRNA in the Nhe3-/- intestine, suggesting that NHE2 does not provide major compensation for the intestinal absorptive defect of Nhe3-/- mice. In contrast, analysis of the same RNA samples showed that mutant transcripts from the Nhe3 gene were sharply upregulated in the small intestine, cecum, and proximal colon of Nhe3-/- mice when compared with the corresponding segments of Nhe3+/- mice. Finally, the wild-type NHE3 mRNA was not upregulated in the Nhe2-/- intestine, which provides suggestive evidence that the absence of NHE2 does not affect the absorptive capacity of the intestine.

Concluding remarks. When we initiated this study, we favored the hypothesis that NHE2 provided an important reserve absorptive capacity that would blunt the severity of the diarrheal state in Nhe3-/- mice. However, our studies failed to reveal any in vivo correlates of a worsening of the absorptive defect, such as decreased viability, a more severe acid-base disorder, an increase in serum aldosterone, or an increase in the volume or pH of the intestinal contents, which would be expected if NHE2-mediated Na+/H+ exchange were an important compensatory mechanism.

Although our data suggest that NHE2 provides little if any compensation for the diarrheal state of Nhe3-/- mice, the results must be interpreted with caution and do not negate the possibility that NHE2 plays a role in NaCl absorption under other circumstances. Through eliminating the activity of NHE3, we induced a diarrheal state in which aldosterone levels were sharply elevated. The response to hyperaldosteronism includes an increase in the expression and activity of NHE3 in the proximal colon (6). Northern analysis shows that the expression of mutant transcripts of the Nhe3 gene increases in the small intestine, cecum, and proximal colon in Nhe3-/- mice, indicating that the Nhe3 gene responds to the diarrheal state. Other studies (2, 16, 27) have shown that NHE2 and NHE3 are regulated differently and in some cases reciprocally. Thus it is conceivable that NHE2 might normally be activated under pathophysiological conditions in which NHE3 is downregulated and inactivated under conditions in which NHE3 is upregulated, as would occur when serum aldosterone is elevated. If this were the case, then the conditions resulting from the Nhe3 null mutation might activate signaling mechanisms that would downregulate the activity of NHE2. Also, it is possible that a worsening of the absorptive defect in the small intestine could be fully compensated by electrogenic Na+ absorption in the distal colon, thereby preventing a further loss of viability or worsening of acid-base or Na+-fluid volume homeostasis. In response to aldosterone, Na+ absorption in the colon switches from predominately electroneutral to electrogenic absorption via the epithelial Na+ channel (12, 31). We (34) showed previously that the colonic H+-K+-ATPase and epithelial Na+ channel, which in concert with an apical K+ channel mediate Na+/H+ exchange, are sharply upregulated in the Nhe3-/- colon. This might be sufficient to overcome a more severe absorptive defect in the small intestine and proximal colon. Although the reduction in the contents of the small intestine of Nhe2-/-Nhe3-/- mice relative to that of Nhe3-/- mice with functional NHE2 argues against this interpretation, our measurements of intestinal contents may not be an adequate reflection of the diarrheal state. On the basis of our data, however, it is clear that NHE3 is the predominant absorptive exchanger and that any compensation that might be provided by NHE2 is not essential.

The results of the current study are consistent with recent studies (5, 19, 20, 23, 30) of the physiological functions of NHE2 in other tissues, none of which have revealed an absorptive function. The loss of NHE2 caused no apparent perturbation of the ability of the kidney to retain Na+ (19), suggesting that its activity in apical membranes of the thick ascending limb and distal convoluted tubule (5) is not essential for maximum recovery of filtered Na+. Although NHE2 is expressed on apical membranes of pancreatic duct cells, the loss of NHE2 had no effect on absorption (20). In parotid glands, where it is expressed on apical membranes of both acinar and duct cells, the loss of NHE2 had no effect on absorption but, paradoxically, caused a significant decrease in secretion (30). NHE2 is expressed in apical membranes of submandibular gland duct cells and, on the basis of sensitivity to amiloride analogs, was proposed to play an absorptive role (21). However, a more recent study (23) revealed that the activity that had been attributed to NHE2 was also present in the submandibular ducts of Nhe2-/- and Nhe2-/-Nhe3-/- mice and is most likely due to NaHCO3 cotransport. Interestingly, the activity remaining in Nhe2-/-Nhe3-/- ducts was inhibited by 50 µM HOE-694, the same concentration used to selectively inhibit NHE2 in intestinal brush-border membranes in the studies (8, 9, 40) that attributed an absorptive function to NHE2.

If NHE2 does not play a role in NaCl, HCO<UP><SUB>3</SUB><SUP>−</SUP></UP>, and fluid absorption, what might its function be? One possibility is that it provides a H+ recycling mechanism that maintains the appropriate pH microenvironments on the extracellular and cytoplasmic sides of the plasma membrane that are critical for H+-coupled transport processes. A study (32) using Caco-2 intestinal epithelial cells, in which both NHE2 and NHE3 were expressed, showed that apical Na+/H+ exchange is activated by uptake of dipeptides and amino acids across the apical membrane via H+-coupled symport. Similarly, apical Na+/H+ exchange in cultured HT-29 cells, attributed to NHE2, has been shown (13) to be activated by the drop in pH of the apical cytoplasm following entry of short-chain fatty acids across the enterocyte apical membrane, thereby recycling H+ and maintaining the driving force for short-chain fatty acid uptake. Other possibilities are that NHE2 functions as follows: in the development or repair of the gut epithelium, as suggested by its upregulation in response to serum (24); in the regulation of anion channel activity via control of intracellular pH in apical membrane microdomains, as suggested by the decreased secretion from Nhe2-/- salivary acinar cells (30); or in the regulation of intracellular pH and cell volume. Additional studies in vivo and analysis of isolated tissues in vitro will be required to gain a detailed understanding of the physiological functions of NHE2.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-50594 and National Institute of Environmental Health Sciences Grant ES-06096.


    FOOTNOTES

Address for reprint requests and other correspondence: G. E. Shull, Dept. of Molecular Genetics, Biochemistry and Microbiology, College of Medicine, Univ. of Cincinnati, 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 21 June 2001; accepted in final form 14 August 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Bookstein, C, DePaoli AM, Xie Y, Niu P, Musch MW, Rao MC, and Chang EB. Na+/H+ exchangers, NHE-1 and NHE-3, of rat intestine. Expression and localization. J Clin Invest 93: 106-113, 1994[ISI][Medline].

2.   Bookstein, C, Musch MW, Xie Y, Rao MC, and Chang EB. Regulation of intestinal epithelial brush border Na+/H+ exchanger isoforms, NHE2 and NHE3, in C2bbe cells. J Membr Biol 171: 87-95, 1999[ISI][Medline].

3.   Bookstein, C, Xie Y, Rabenau K, Musch MW, McSwine RL, Rao MC, and Chang EB. Tissue distribution of Na+/H+ exchanger isoforms NHE2 and NHE4 in rat intestine and kidney. Am J Physiol Cell Physiol 273: C1496-C1505, 1997[ISI][Medline].

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.   Cho, JH, Musch MW, Bookstein CM, McSwine RL, Rabeneau K, and Chang EB. Aldosterone stimulates intestinal sodium absorption in rats by increasing NHE3 expression of the proximal colon. Am J Physiol Cell Physiol 274: C586-C594, 1998[Abstract/Free Full Text].

7.   Chow, CW, Khurana S, Woodside M, Grinstein S, and Orlowski J. The epithelial Na+/H+ exchanger, NHE3, is internalized through a clathrin-mediated pathway. J Biol Chem 274: 37551-37558, 1999[Abstract/Free Full Text].

8.   Collins, JF, Xu H, Kiela PR, Zeng J, and Ghishan FK. Functional and molecular characterization of NHE3 expression during ontogeny in rat jejunal epithelium. Am J Physiol Cell Physiol 273: C1937-C1946, 1997[Abstract/Free Full Text].

9.   Donowitz, M, De La Horra C, Calonge ML, Wood IS, Dyer J, Gribble SM, De Medina FS, Tse CM, Shirazi-Beechey SP, and Ilundain AA. In birds, NHE2 is major brush-border Na+/H+ exchanger in colon and is increased by a low-NaCl diet. Am J Physiol Regulatory Integrative Comp Physiol 274: R1659-R1669, 1998[Abstract/Free Full Text].

10.   D'Souza, S, Garcia-Cabado A, Yu F, Teter K, Lukacs G, Skorecki K, Moore HP, Orlowski J, and Grinstein S. The epithelial sodium-hydrogen antiporter Na+/H+ exchanger 3 accumulates and is functional in recycling endosomes. J Biol Chem 273: 2035-2043, 1998[Abstract/Free Full Text].

11.   Dudeja, PK, Rao DD, Syed I, Joshi V, Dahdal RY, Gardner C, Risk MC, Schmidt L, Bavishi D, Kim KE, Harig JM, Goldstein JL, Layden TJ, and Ramaswamy K. Intestinal distribution of human Na+/H+ exchanger isoforms NHE-1, NHE-2, and NHE-3 mRNA. Am J Physiol Gastrointest Liver Physiol 271: G483-G493, 1996[Abstract/Free Full Text].

12.   Foster, ES, Zimmerman TW, Hayslett JP, and Binder HJ. Corticosteroid alteration of active electrolyte transport in rat distal colon. Am J Physiol Gastrointest Liver Physiol 245: G668-G675, 1983[Abstract/Free Full Text].

13.   Gonda, T, Maouyo D, Rees SE, and Montrose MH. Regulation of intracellular pH gradients by identified Na/H exchanger isoforms and a short-chain fatty acid. Am J Physiol Gastrointest Liver Physiol 276: G259-G270, 1999[Abstract/Free Full Text].

14.   Hoogerwerf, WA, Tsao SC, Devuyst O, Levine SA, Yun CH, Yip JW, Cohen ME, Wilson PD, Lazenby AJ, Tse CM, and Donowitz M. NHE2 and NHE3 are human and rabbit intestinal brush-border proteins. Am J Physiol Gastrointest Liver Physiol 270: G29-G41, 1996[Abstract/Free Full Text].

15.   Janecki, AJ, Montrose MH, Zimniak P, Zweibaum A, Tse CM, Khurana S, and Donowitz M. Subcellular redistribution is involved in acute regulation of the brush border Na+/H+ exchanger isoform 3 in human colon adenocarcinoma cell line Caco-2. Protein kinase C-mediated inhibition of the exchanger. J Biol Chem 273: 8790-8798, 1998[Abstract/Free Full Text].

16.   Kandasamy, RA, Yu FH, Harris R, Boucher A, Hanrahan JW, and Orlowski J. Plasma membrane Na+/H+ exchanger isoforms (NHE-1, -2, and -3) are differentially responsive to second messenger agonists of the protein kinase A and C pathways. J Biol Chem 270: 29209-29216, 1995[Abstract/Free Full Text].

17.   Kaunitz, JD, Barrett KE, and McRoberts JA. Electrolyte secretion and absorption: small intestine and colon. In: Textbook of Gastroenterology (2nd ed), edited by Yamada T.. Philadelphia, PA: Lippincott, 1995, p. 326-361.

18.   Konturek, SJ, Krzyzek E, and Bilski J. The importance of gastric secretion in the feedback control of interdigestive and postprandial pancreatic secretion in rats. Regul Pept 36: 85-97, 1991[ISI][Medline].

19.   Ledoussal, C, Lorenz JN, Nieman ML, Soleimani M, Schultheis PJ, and Shull GE. Renal salt wasting in mice lacking NHE3 Na+/H+ exchanger but not in mice lacking NHE2. Am J Physiol Renal Physiol 281: F718-F727, 2001[Abstract/Free Full Text].

20.   Lee, MG, Ahn W, Choi JY, Luo X, Seo JT, Schultheis PJ, Shull GE, Kim KH, and Muallem S. Na+-dependent transporters mediate HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> salvage across the luminal membrane of the main pancreatic duct. J Clin Invest 105: 1651-1658, 2000[Abstract/Free Full Text].

21.   Lee, MG, Schultheis PJ, Shull GE, Chang E, Donowitz M, Park K, and Muallem S. Membrane limited expression and regulation of NHE isoforms by P2 receptors in the rat submandibular gland. J Physiol (Lond) 513: 341-357, 1998[Abstract/Free Full Text].

22.   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].

23.   Luo, X, Choi JY, Ko SBH, Pushkin A, Kurtz I, Ahn W, Lee MG, and Muallem S. HCO<UP><SUB>3</SUB><SUP>−</SUP></UP> salvage mechanisms in the submandibular gland acinar and duct cells. J Biol Chem 276: 9808-9816, 2001[Abstract/Free Full Text].

24.   Maher, MM, Gontarek JD, Bess RS, Donowitz M, and Yeo CJ. The Na+/H+ exchange isoform NHE3 regulates basal canine ileal sodium absorption in vivo. Gastroenterology 112: 174-183, 1997[ISI][Medline].

25.   Maher, MM, Gontarek JD, Jimenez RE, Donowitz M, and Yeo CJ. Role of brush border Na+/H+ exchange in canine ileal absorption. Dig Dis Sci 41: 651-659, 1996[ISI][Medline].

26.   Malakooti, J, Dahdal RY, Schmidt L, Layden TJ, Dudeja PK, and Ramaswamy K. Molecular cloning, tissue distribution, and functional expression of the human Na+/H+ exchanger NHE2. Am J Physiol Gastrointest Liver Physiol 277: G383-G390, 1999[Abstract/Free Full Text].

27.   McSwine, RL, Musch MW, Bookstein C, Xie Y, Rao M, and Chang EB. Regulation of apical membrane Na+/H+ exchangers NHE2 and NHE3 in intestinal epithelial cell line C2/bbe. Am J Physiol Cell Physiol 275: C693-C701, 1998[Abstract].

28.   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].

29.   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].

30.   Park, K, Evans RL, Watson GE, Nehrke K, Richardson L, Bell SM, Schultheis PJ, Hand AR, Shull GE, and Melvin JE. Defective fluid secretion and NaCl absorption in the parotid glands of Na+/H+ exchanger-deficient mice. J Biol Chem 276: 27042-27050, 2001[Abstract/Free Full Text].

31.   Perrone, RD, and Jenks SL. Suppression of coupled Na-Cl absorption by aldosterone and dexamethasone in rat distal colon in vitro. Am J Physiol Renal Fluid Electrolyte Physiol 246: F785-F793, 1984[ISI][Medline].

32.   Robert, A, Nezamis JE, Lancaster C, Hanchar AJ, and Klepper MS. Enteropooling assay: a test for diarrhea produced by prostaglandins. Prostaglandins 11: 809-828, 1976[Medline].

33.   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].

34.   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].

35.   Spicer, Z, Miller ML, Andringa A, Riddle TM, Duffy JJ, Doetschman T, and Shull GE. Stomachs of mice lacking the gastric H,K-ATPase alpha -subunit have achlorhydria, abnormal parietal cells, and ciliated metaplasia. J Biol Chem 275: 21555-21565, 2000[Abstract/Free Full Text].

36.   Thwaites, DT, Ford D, Glanville M, and Simmons NL. H+/solute-induced intracellular acidification leads to selective activation of apical Na+/H+ exchange in human intestinal epithelial cells. J Clin Invest 104: 629-635, 1999[Abstract/Free Full Text].

37.   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].

38.   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].

39.   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].

40.   Wormmeester, L, De Medina FS, Kokke F, Tse CM, Khurana S, Bowser J, Cohen ME, and Donowitz M. Quantitative contribution of NHE2 and NHE3 to rabbit ileal brush-border Na+/H+ exchange. Am J Physiol Cell Physiol 274: C1261-C1272, 1998[Abstract/Free Full Text].

41.   Yun, CH, Gurubhagavatula S, Levine SA, Montgomery JL, Brant SR, Cohen ME, Cragoe EJ, Jr, Pouyssegur J, Tse CM, and Donowitz M. Glucocorticoid stimulation of ileal Na+ absorptive cell brush border Na+/H+ exchange and association with an increase in message for NHE-3, an epithelial Na+/H+ exchanger isoform. J Biol Chem 268: 206-211, 1993[Abstract/Free Full Text].


Am J Physiol Gastrointest Liver Physiol 281(6):G1385-G1396
0193-1857/01 $5.00 Copyright © 2001 the American Physiological Society