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1 Department of Molecular Genetics, Biochemistry, and Microbiology and 2 Department of Environmental Health, College of Medicine, University of Cincinnati, Cincinnati, Ohio 45267
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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
sodium absorption; diarrhea; Slc9a2; Slc9a3
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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ABSORPTION
of NaCl, HCO
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
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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 
-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.
-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 
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.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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, HCONhe3+/).
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).
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/ 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/ 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).
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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.
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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.
/ 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).
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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.
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/ 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-subunit (gHKA) genes were prepared and analyzed. We reasoned that these mice should exhibit the same reductions in fluid
and HCO/ 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.
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/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/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.
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/
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).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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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.
/ 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/HCO/ 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/ 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 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.
/ mice, a likely explanation for the
acidity of the intestinal tract was a reduction in
HCO/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.
/ 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/ 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.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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0.05, 
P < 0.01, significantly different from wild type.
