Departments of 1 Molecular Genetics, Biochemistry, and Microbiology and 2 Molecular and Cellular Physiology, The University of Cincinnati College of Medicine, Cincinnati, Ohio 45267-0524; and 3 Department of Biological Sciences, Northern Kentucky University, Highland Heights, Kentucky 41099
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ABSTRACT |
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The degree to which loss of the
NHE3 Na+/H+ exchanger in the kidney contributes
to impaired Na+-fluid volume homeostasis in NHE3-deficient
(Nhe3/
) mice is unclear because of the
coexisting intestinal absorptive defect. To more accurately assess the
renal effects of NHE3 ablation, we developed a mouse with transgenic
expression of rat NHE3 in the intestine and crossed it with
Nhe3
/
mice. Transgenic
Nhe3
/
(tgNhe3
/
)
mice tolerated dietary NaCl depletion better than nontransgenic knockouts and showed no evidence of renal salt wasting. Unlike nontransgenic Nhe3
/
mice,
tgNhe3
/
mice tolerated a 5% NaCl diet. When
fed a 5% NaCl diet, tgNhe3
/
mice had lower
serum aldosterone than tgNhe3
/
mice on a 1%
NaCl diet, indicating improved extracellular fluid volume status.
Na+-loaded tgNhe3
/
mice had
sharply increased urinary Na+ excretion, reflective of
increased absorption of Na+ in the small intestine;
nevertheless, they remained hypotensive, and renal studies showed a
reduction in glomerular filtration rate (GFR) similar to that observed
in nontransgenic Nhe3
/
mice. These data show
that reduced GFR, rather than being secondary to systemic hypovolemia,
is a major renal compensatory mechanism for the loss of NHE3 and
indicate that loss of NHE3 in the kidney alters the set point for
Na+-fluid volume homeostasis.
sodium/hydrogen exchanger; diarrhea; Slc9a3; sodium absorption; sodium-fluid volume homeostasis
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INTRODUCTION |
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NA+/H+
exchanger isoform 3 (NHE3) is one of several Na+
transport proteins in renal epithelial cells that are involved in
maintaining Na+-fluid volume homeostasis. Localized to
apical membranes of the proximal tubule and to a lesser extent in the
thick ascending limb of Henle, NHE3 transports Na+ into the
cell in exchange for H+ and is responsible for absorbing
large quantities of NaCl and HCO/
) mice have severe absorptive defects
in both the kidney and intestine, and they exhibit characteristics of
chronic volume depletion, including low blood pressure, high levels of
renin mRNA in kidney, and high serum aldosterone (21). In
situ microperfusion and micropuncture studies showed that reabsorption
of HCO
/
mice (12, 24).
However, fluid delivery to the distal convoluted tubule was not
significantly different from that in wild-type mice, and this appeared
to be due to a regulated reduction in the glomerular filtration rate
(GFR) resulting from tubuloglomerular feedback (TGF) mechanisms
(12). These observations suggested that the reduction in
GFR might be a compensatory mechanism by which the kidneys of
Nhe3
/
mice conserve Na+ and
HCO
The observed normalization of fluid delivery to the distal convoluted
tubule of NHE3-deficient mice (12) raised the possibility that the proximal tubule absorptive defect itself might not lead to
significant renal salt wasting. In a subsequent study, when subjected
to dietary Na+ restriction,
Nhe3/
mice lost weight rapidly and did in
fact exhibit urinary salt wasting (8), although apparently
not as severe as in mice lacking transporters along some of the more
distal segments of the nephron, such as the ROMK K+ channel
(10) and Na+-K+-2Cl
cotransporter (23) of the thick ascending limb and the
epithelial Na+ channel (ENaC) of the connecting tubule and
collecting duct (2, 6, 14, 18, 23). By the third day of
dietary Na+ restriction, however, many of the
Nhe3
/
mice were undergoing hypovolemic renal
failure. This raises the possibility that systemic volume depletion,
and not just the loss of NHE3 in the kidney, might cause some degree of
renal dysfunction. Thus extracellular fluid volume depletion itself,
which is exacerbated by the diarrheal state during dietary
Na+ restriction, may have contributed to the mild
impairment in the ability of the NHE3-deficient kidney to retain
Na+. Similarly, it was unclear whether the observed
reduction in GFR in Nhe3
/
mice might have
been due, in part, to systemic hypovolemia and hypotension rather than
to an appropriate regulation of fluid delivery to the distal tubule via
TGF mechanisms. Attempts to improve the fluid volume status of
Nhe3
/
mice by feeding them a high-NaCl diet
resulted in swelling of the intestine, severe hypovolemia, and death,
further suggesting that the intestinal defect impaired extracellular
fluid-volume homeostasis.
Thus the coexisting intestinal absorptive defect and chronic diarrhea
in Nhe3/
mice represent a major confounding
factor in determining the specific effects of the loss of NHE3 in the
kidney on renal Na+ conservation, GFR, and extracellular
fluid-volume homeostasis. To assess these issues, we developed a
transgenic mouse in which NHE3 is expressed in the small intestine via
the intestinal fatty acid binding protein (IFABP) promoter and
crossed it with Nhe3
/
mice. Transgenic
Nhe3
/
(tgNhe3
/
)
mice were then subjected to dietary Na+ restriction and
Na+ loading, and renal function was analyzed. Both dietary
Na+ restriction and Na+ loading were better
tolerated in tgNhe3
/
mice than in
nontransgenic Nhe3
/
mice. Salt loading led
to a substantial reduction of aldosterone levels in
tgNhe3
/
mice, indicating a partial
correction of the extracellular fluid-volume deficit. However,
tgNhe3
/
mice remained mildly hypotensive and
had reduced GFR compared with Nhe3+/+ mice also
harboring the IFABP-Nhe3 transgene
(tgNhe3+/+).
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METHODS |
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Production and genotyping of mutant and transgenic mice.
The rat NHE3 cDNA was cloned into an expression plasmid containing the
small intestine-specific IFABP promoter (nucleotides 1178 to +28) and
a t-intron polyadenylation cassette [provided by J. A. Whitsett
(27)] (Fig. 1A).
The IFABP/NHE3 construct was microinjected into fertilized oocytes from
Institute of Cancer Research (ICR) mice, and injected oocytes were
implanted into the uterus of pseudopregnant mice to produce transgenic
animals by the University of Cincinnati transgenic core facility. Mice carrying the transgene integrated into their genome were identified by
PCR analysis. The 5'-oligonucleotide primer sequence was from the IFABP
promoter sequence (5'-CTGCCAGGTTATCTCTTGAAC-3'), and the 3' reverse
primer sequence was from the NHE3 cDNA sequence (5'-CTGTTCGGTTCCTCCTCAATG-3'). PCR conditions were 94°C for 3 min,
then 35 cycles at 94°C for 30 s, 60°C for 30 s, and
72°C for 30 s, followed by 72°C for 10 min. ICR transgenic
mice were backcrossed for two to three generations with
Nhe3+/
mice of a mixed 129SvJ and Black Swiss
background (21) to produce Nhe3+/+
and Nhe3
/
mice carrying the IFABP/NHE3
transgene (tgNhe3+/+ and
tgNhe3
/
). Thus the genetic background of the
mice used in these experiments was 12.5-25% ICR, with the
remainder being an equal mix of 129SVJ and Black Swiss.
Nhe3+/
mice were generated and maintained as
previously described (21). PCR genotyping was performed
using the following primers: a forward primer corresponding to a
sequence in exon 6 (5'-CTTTTGCGGCATCTGCTGTCAG-3'), a reverse primer
corresponding to a sequence in intron 6 (5'-ACTACTAAGAGTGCTCCTAGCTCTCACC-3'), and a reverse primer
corresponding to a sequence in the neomycin resistance gene
(5'-GCATGCTCCAGACTGCCTTG-3'). PCR conditions were 94°C for 3 min,
then 40 cycles at 94°C for 30 s, 62°C for 30 s, and
72°C for 30 s. The experiments described below were performed in
accordance with the guidelines established by the Institutional Animal
Care and Use Committee at the University of Cincinnati College of
Medicine. All experimental pairs of tgNhe3
/
and tgNhe3+/+ mice were 8-12 wk old and
were littermates matched by both age and sex to ensure that no strain,
age, or sex biases contributed to physiological outputs.
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RNA isolation and Northern blot analysis.
Total RNA was extracted from tissues using Tri-Reagent (Molecular
Research Center). Total RNA (10 µg/sample) was mixed with Glyoxal
Sample Buffer (BioWhittaker Molecular Applications, Rockland, ME),
separated by electrophoresis in 1% agarose, and transferred to
Hybond-N+ nylon membrane (Amersham Pharmacia Biotech, Piscataway, NJ).
Northern blots were screened using [32P]-labeled cDNA
probes specific for NHE3, renin, and the L32 ribosomal protein.
Quantitation of renin mRNA levels was determined by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA) using ImageQuant software
(Molecular Dynamics). Renin expression levels were normalized using L32
ribosomal protein mRNA as a loading control and reported as mean volume
integrated values for four pairs of tgNhe3/
and tgNhe3+/+ mice on 1 or 5% NaCl diets.
Total membrane preparations and Western blot analysis. Small intestines and kidneys were homogenized in homogenization buffer (0.25 M sucrose, 30 mM imidizole, 1 mM EDTA) to which a protease inhibitor cocktail without metal chelating reagents (Sigma, St. Louis, MO) was added. Homogenized suspensions were centrifuged at 6,000 g for 15 min at 4°C. The supernatant was saved, and the pellet was homogenized and centrifuged again. The combined supernatants were centrifuged at 200,000 g for 1 h at 4°C, and the pellet containing total membranes was resuspended in homogenization buffer. A BCA protein assay kit (Pierce, Rockford, IL) was used to quantitate protein concentrations. Total membrane preparations were analyzed by Western blot analyses using 1 µg/ml of the rabbit NHE3 polyclonal antibody (Chemicon, Temecula, CA), as described previously (26). Signal detection was accomplished using the SuperSignal West Pico Chemiluminescent Substrate (Pierce).
Analysis of size and contents of intestinal segments. Mice were anesthetized with intraperitoneal injections of 2.5% avertin (0.02 ml/g body wt) and euthanized by cervical dislocation. The contents of each segment were removed, and the weights of the tissue and contents were recorded. Contents were mixed with 1 ml sterile saline and centrifuged, and the pH of the supernatant was recorded.
Balance studies using varying Na+-content diets. Mice were housed in metabolic cages and provided with drinking water and food ad libitum, as described previously (22). Food (Harlan Teklad, Madison, WI) contained normal (1%), low (0.01%), or high (5%) levels of NaCl. Body weights and the amount of food and water consumed were recorded every day. Urine samples were collected daily and were processed and analyzed for Na+ and K+ concentrations as described previously (8).
Serum aldosterone levels.
Mice were anesthetized, and blood was drawn by cardiac puncture. Serum
was separated from blood cells and stored at 20°C. Serum samples
were diluted 1:4 in sterile PBS, and aldosterone concentrations were
determined using an RIA kit (Diagnostic Products, Los Angeles, CA).
Renal hemodynamic measurements.
Baseline renal function was determined in two groups of seven pairs of
tgNhe3+/+ and tgNhe3/
mice maintained on either a 1 or a 5% NaCl diet for 5 days. Mice were
anesthetized with ketamine (50 µg/g body wt) and inactin (100 µg/g
body wt) and surgically instrumented for renal measurements as
described previously (12). Immediately after surgery, a
bolus (3 µl/g body wt) of 1% FITC-inulin and 3% PAH in isotonic
saline was administered. This was followed by a maintenance infusion of
the same solution at 0.15 µl · min
1 · g
body wt
1. After a 30-min equilibration period, baseline
renal function was determined through two 30-min urine samples
collected through a catheter in the bladder (12). At the
midpoint of each baseline collection, an arterial blood sample (60 µl) was obtained for determination of plasma FITC-inulin
(11) and PAH (25) concentrations, and donor
blood was administered to replace the lost volume after each sample was
obtained. At the end of the second baseline collection, another blood
sample was acquired and plasma electrolyte levels were measured using a
pH/blood-gas analyzer (Bayer, Medfield, MA). Urinary Na+
and K+ concentrations were determined using a Corning 480 Flame Photometer (Bayer). GFR was calculated from inulin clearance, and
effective renal plasma flow (ERPF) was calculated from PAH clearance.
Statistics. Statistical analysis was performed by either Student's t-test or analysis of variance. When analysis of variance was applied, either a single-factor design or a mixed-factorial design with repeated measures on the second factor was used, and individual contrasts were used to compare individual group means when needed. Data are presented as means ± SE, and statistical significance was regarded as P < 0.05.
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RESULTS |
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Generation of transgenic mice expressing NHE3 in the small
intestine.
No promoters are available that would allow transgenic expression of
NHE3 throughout the intestinal tract. To partially rescue the
intestinal absorptive defect of Nhe3/
mice,
we used the rat IFABP promoter to drive expression of a rat NHE3
transgene (Fig. 1A) in the small intestine. Genomic
integration of the transgene was detected by PCR analysis (Fig.
1B). Northern blot analysis of kidney, small intestine,
cecum, and colon tissue from tgNhe3+/+ mice
revealed expression of a 3.5-kb mRNA corresponding to the transgene
only in small intestine, whereas the 5.6-kb mouse NHE3 mRNA was
expressed in all tissues (Fig. 1C). The transgenic mice were
then bred with Nhe3+/
mice. Western blot
analysis demonstrated that NHE3 protein was expressed in kidneys (data
not shown) and small intestines (Fig. 1D) of both transgenic
and nontransgenic wild-type mice but not in the kidneys (data not
shown) of tgNhe3
/
mice. In
tgNhe3
/
mice, NHE3 protein was expressed in
the small intestine, although at a lower level than in wild-type
controls (Fig. 1D). In addition to a protein corresponding
to full-length rat NHE3, a smaller band of unknown identity was also
detected in tgNhe3
/
small intestine.
Analysis of intestinal weight and intestinal contents.
The loss of NHE3 in the intestinal tract causes a severe absorptive
defect, resulting in chronic diarrhea. All segments of the intestine
are enlarged in Nhe3/
mice, and the volume
and pH of the luminal contents are increased (21).
Expression of functional NHE3 in the Nhe3
/
small intestine (Fig. 1, C and D) would be
expected to at least partially alleviate these defects in this segment.
Gross examination of the tgNhe3
/
intestinal
tract revealed a less bloated small intestine, whereas the cecum and
colon appeared similar to those of Nhe3
/
mice (21). The weight of the small intestine was greater
in tgNhe3
/
mice than in
tgNhe3+/+ mice; however, the weight and pH of
the small intestinal contents in tgNhe3
/
mice were not significantly different from those in
tgNhe3+/+ mice (Fig.
2). On the other hand, the weights and
luminal contents of the tgNhe3
/
cecum and
colon, where the transgene was not expressed, were significantly
greater than in tgNhe3+/+ mice, and the pH of
the luminal contents was significantly more alkaline (Fig. 2). These
data suggest that the observed levels of expression of the NHE3
transgene partially restored the absorptive capabilities of the small
intestine.
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tgNhe3/
mice exhibit increased tolerance for a
Na+-restricted diet.
When Nhe3
/
mice not carrying the transgene
were fed a Na+-restricted (0.01% NaCl) diet, they
exhibited severe weight loss and renal salt wasting (8).
To test the Na+-handling capabilities of
tgNhe3
/
mice,
tgNhe3+/+ and tgNhe3
/
mice were housed in metabolic cages and fed a 1% NaCl diet for 3 days,
followed by a 0.01% NaCl diet for 3 days. On the normal diet (1%
NaCl), tgNhe3
/
mice had lower urinary
Na+ excretion compared with
tgNhe3+/+ mice, consistent with only a partial
rescue of the intestinal phenotype. However, during dietary
Na+ restriction, tgNhe3
/
mice
lost only 6% of their body weight (Table
1), which was a major improvement over
the 17% average loss of body weight for Nhe3
/
mice subjected to the same protocol
(8). Furthermore, in contrast to nonrescued
Nhe3
/
mice that continued to excrete
substantial amounts of Na+ even after 3 days on low salt
(8), tgNhe3
/
mice lowered their
Na+ excretion after 3 days to very low levels that were not
significantly different from that for tgNhe3+/+
mice (Fig. 3A, Table 1).
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tgNhe3/
mice fed a 5% NaCl diet have increased urinary
Na+ excretion indicative of increased
intestinal NaCl absorption.
NaCl loading via continuous infusion of saline through a venous
catheter can restore extracellular fluid volume in
Nhe3
/
mice, as shown by increases in blood
pressure to a level similar to that for
Nhe3+/+ mice (Lorenz JN, unpublished
observations). This suggested that it might be possible to
restore extracellular fluid volume by dietary NaCl loading. However,
when Nhe3
/
mice not carrying the transgene
were fed a high-salt diet (5% NaCl), their small intestines became
severely swollen, probably due to an osmotic effect from the high
levels of NaCl in the gut, and they died within 48 h (Lorenz JN
and Shull GE, unpublished observations). In contrast, when
tgNhe3
/
mice were fed a normal-salt diet
(1% NaCl) for 3 days followed by a 5% NaCl diet for 4 days, they not
only tolerated the high-salt diet, but their urinary Na+
excretion increased to levels higher than that seen in
tgNhe3+/+ mice maintained on a normal diet (Fig.
3B, Table 2). These data demonstrate, importantly, that the intestinally rescued
NHE3-deficient mice can compensate for possible urinary NaCl losses
through increases in intestinal NaCl absorption, whereas their
nonrescued counterparts could not. There was no difference in weight
between tgNhe3
/
and
tgNhe3+/+ mice fed either diet, although body
weight decreased slightly in both genotypes when on the 5% NaCl diet
(Table 2). Again, tgNhe3
/
mice drank more
water than tgNhe3+/+ mice regardless of diet,
and both genotypes consumed more water and increased their urinary
output when fed the 5% NaCl diet (Table 2). Urinary K+
excretion was not different between the two genotypes fed either diet
(Table 2). These data indicate that dietary salt loading increased
intestinal Na+ absorption in
tgNhe3
/
mice.
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Serum aldosterone and renin mRNA levels in the kidney are decreased
in
tgNhe3/
mice fed a 5% NaCl diet.
The major hormonal mechanism for correction of a deficit in
extracellular fluid volume is an increase in serum aldosterone, which
stimulates the absorption of NaCl in the kidney and intestine. When fed
a diet containing 1% NaCl, tgNhe3
/
mice had
a serum aldosterone level that was similar to that observed previously
in Nhe3
/
mice (21) and was
11-fold greater than that in tgNhe3+/+ mice
(Fig. 4). These data indicate that
tgNhe3
/
mice fed a 1% NaCl diet have a
severe deficit in extracellular fluid volume. Serum aldosterone
was sharply decreased in tgNhe3
/
mice when
they were fed a 5% NaCl diet, although it was still higher than that
in tgNhe3+/+ mice (Fig. 4), indicating that the
deficit in extracellular fluid volume can be partially corrected by
dietary salt loading.
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Renal function in
tgNhe3/
mice fed a 1 or 5% NaCl diet.
After 5 days on either a 1 or a 5% NaCl diet,
tgNhe3+/+ and tgNhe3
/
mice were anesthetized and surgically prepared for analysis of renal
function, blood pressure and heart rate, and collection of blood
samples. Under anesthesia, mean arterial pressure (Fig. 6A) was lower in
tgNhe3
/
mice than in
tgNhe3+/+ mice regardless of diet, but
administration of the high-salt diet increased blood pressure in
tgNhe3
/
mice (88.4 ± 3.2 compared with
77.0 ± 4.6 mmHg when fed the 1% NaCl diet), whereas it had no
effect in tgNhe3+/+ mice. There were no
significant differences in heart rate between any of the groups (Fig.
6B). Also, in tgNhe3+/+ mice, the
hematocrit did not change when animals were placed on the high-salt
diet, but in tgNhe3
/
animals, the 5% NaCl
diet significantly reduced the hematocrit (Table
3). The effects of high-salt intake on
blood pressure and hematocrit are consistent with a partial correction
of the extracellular fluid volume deficit in NHE3-deficient mice
expressing the NHE3 transgene in the small intestine. None of the
groups differed with respect to plasma Na+ or arterial
blood HCO
/
mice regardless of diet (Table 3).
Plasma K+ increased significantly in
tgNhe3+/+ mice on the 5% NaCl diet compared
with the same genotype on the 1% NaCl diet, but this increase did not
occur in tgNhe3
/
mice (Table 3).
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DISCUSSION |
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Previous studies demonstrated that both Na+
reabsorption in the proximal tubule and systemic Na+-fluid
volume homeostasis are severely perturbed in
Nhe3/
mice (8, 12, 21, 24) and
that partial compensation for the renal absorptive defect occurs by a
reduction in GFR (4, 8, 12). However,
Nhe3
/
mice also have an intestinal
absorptive defect, which clearly contributes to volume depletion during
dietary Na+ restriction and might also play a role in
volume depletion under normal conditions. This makes it difficult to
accurately assess 1) the capacity of the NHE3-deficient
kidney to recover NaCl; 2) the degree to which the reduction
in GFR represents direct compensation for the proximal tubule
absorptive defect, via renal mechanisms such as TGF (12)
and the intrarenal renin-angiotensin system (15, 19); and
3) the specific contribution of the renal defect to chronic
extracellular fluid volume depletion.
To resolve these issues, we generated Nhe3/
mice expressing NHE3 in the small intestine with the expectation that
this would allow dietary salt loading, an approach that was not
successful with nontransgenic Nhe3
/
mice,
apparently because of the osmotic effects of high salt in the lumen of
the gut. It would have been preferable to express NHE3 in small
intestine, cecum, and colon; however, no promoters were available that
would allow this. Transgenic expression of rat NHE3 under the control
of the IFABP promoter yielded lower levels of expression than that of
the endogenous protein, and expression was limited to the small
intestine and therefore did not correct the absorptive defect in the
cecum or colon. In addition to an NHE3 protein corresponding in size to
wild-type NHE3, there was a diffuse product of lower molecular weight
in the tgNhe3
/
small intestine; the identity
of this smaller product is unclear. It is possible that glycosylation
or trafficking of NHE3, expressed from the transgene, is inefficient in
the Golgi complex of mouse small intestinal epithelial cells. An
alternative possibility is that the rat NHE3 mRNA derived from the
transgene, which lacks most of the wild-type 5'- and 3'-untranslated
sequences, may not be efficiently localized to endoplasmic
reticulum-bound ribosomes, as there is evidence that untranslated mRNA
sequences might be important in this process (16). Either
of these possibilities might explain the apparently smaller or
partially degraded NHE3 and the discrepancy between the amount of mRNA
expressed from the transgene and the relatively low amount of normal
NHE3 protein detected. Nevertheless, these low levels of NHE3
expression did lead to normalization of the pH of small intestinal
contents and, as discussed below, allowed a substantial amount of salt
loading when the mice were fed a 5% NaCl diet, thereby eliminating the diarrheal state as a major factor in extracellular fluid volume depletion.
When the mice were maintained on a 1% NaCl diet, transgenic expression
of NHE3 in the small intestine of Nhe3/
mice
did not appear to improve extracellular fluid volume status, as they
had elevated levels of serum aldosterone, highly induced renin mRNA in
kidney, and low blood pressure similar to that seen in nontransgenic
Nhe3
/
mice (21). The net
intestinal absorption and urinary excretion of NaCl (which must be
equivalent when the mice are in balance) corresponded to ~0.22 and
~2.1 ml of isotonic fluid/day for tgNhe3
/
and tgNhe3+/+ mice, respectively, consistent
with the possibility that poor absorption of NaCl from the intestinal
tract was a major factor in systemic volume depletion. Surprisingly, in
response to dietary Na+ restriction,
tgNhe3
/
mice exhibited little evidence of
urinary salt wasting. The relatively small reduction in body weight,
relative to that observed earlier for Na+-restricted
Nhe3
/
mice (8), is probably due
primarily to intestinal losses of NaCl because urinary losses on
days 2 and 3 of Na+ restriction were
only equivalent to ~0.015 ml of isotonic fluid/day. These results
indicate that the NHE3-deficient kidney has a substantial ability to
recover NaCl and suggest that the hypovolemic renal failure observed in
our previous study after 3 days of dietary Na+ restriction
(8), which was a likely factor in the observed renal salt
wasting, was brought on largely by the continuing intestinal losses of
salt and water.
When the mice were maintained on a 5% NaCl diet, urinary
Na+ excretion in tgNhe3/
mice
increased to a level ~18 times that in
tgNhe3
/
mice on a normal 1% NaCl diet and
~2.5 times that in tgNhe3+/+ mice on a 1%
NaCl diet. The net urinary excretion (and intestinal absorption) of
NaCl per day in tgNhe3
/
mice was equivalent
to that in ~4.3 ml of extracellular fluid and was far in excess of
that excreted by the NHE3-deficient kidney when the mice were fed
either a normal or a Na+-restricted diet (Tables 1 and 2).
This indicates that the effects of the intestinal absorptive defect on
extracellular fluid volume homeostasis can be overcome by dietary salt
loading in these animals. As shown by the sharply reduced serum
aldosterone levels, the decrease in the level of induction of renin
mRNA in the kidney, and the increase in mean arterial pressure, the
extracellular fluid volume status of tgNhe3
/
mice was substantially improved when they were fed the 5% NaCl diet.
Nevertheless, serum aldosterone and kidney renin mRNA were still
elevated and blood pressure was still reduced relative to that for
tgNhe3+/+ mice. These data suggest that the
absence of NHE3 in the kidney, even when the mice are subjected to
dietary NaCl loading, results in a certain degree of chronic volume
depletion. Thus these data are consistent with the hypothesis that the
activity of NHE3 in the kidney is required for maintenance of the
normal set point for Na+-fluid volume balance.
The genetic background of the mice used in this study differed slightly
from that of our previous studies (8, 9, 12, 21), in which
the mice were an equal mix of 129SVJ and Black Swiss strains. The
transgenic mice were prepared on an ICR background and then backcrossed
with Nhe3+/ mice for two to three generations
before breeding pairs were established to generate the animals used in
these experiments. Half of the mice used in the dietary NaCl-loading
experiments were derived from pairs that had been backcrossed for three
generations and would have had a genetic background of only ~12.5%
ICR; the remaining mice were ~25% ICR. It is conceivable that the
addition of some ICR genetic background onto the already mixed 129SVJ
and Black Swiss background might have made the mice hardier, thereby contributing to their improved ability (via a reduction in both the
degree of volume depletion and the consequent hypovolemic renal
failure) to tolerate a low-salt diet. However, this would probably
require the presence of numerous modifier loci because the majority of
the mice would have lacked any given ICR locus. It seems highly
unlikely that the differences in genetic background between the mice in
this study and in our previous studies could be a significant factor in
the ability of the tgNhe3
/
mice to tolerate
a high-salt diet, which clearly involves a substantial increase in
Na+ absorption from the gut.
Previous studies showed that both single-nephron GFR (12)
and whole kidney GFR (4, 8) are reduced in
Nhe3/
mice and that NHE3-deficient kidneys
have intact TGF mechanisms (12). Analysis of renal
function in the salt-loaded tgNhe3
/
mice
revealed that ERPF and GFR were also significantly reduced relative to
that for tgNhe3+/+ mice and that both were
essentially the same in tgNhe3
/
mice fed
either a 1 or a 5% NaCl diet. If reduced perfusion pressure resulting
from the hypovolemic state were a major factor in the reduced GFR, then
dietary salt loading would have been expected to increase GFR. Although
it is clear from the results of a previous study (8) that
severe hypovolemia in nontransgenic Nhe3
/
mice during dietary Na+ restriction leads to a further
reduction in GFR and hypovolemic renal failure, the present results
support the view that the reduced GFR in NHE3-deficient mice occurs as
a direct compensation for the absorptive defect in the proximal tubule
and is due to renal mechanisms such as TGF (12) and a
reduction in renal plasma flow.
Although the intestinal function of NHE2 was not a subject of this investigation, it is interesting that a low level of NHE3 in the small intestine was sufficient to absorb large quantities of NaCl when the mice were salt loaded, whereas wild-type levels of NHE2 present throughout the intestinal tract provide little, if any, capacity for salt loading. NHE2-deficient mice do not have diarrhea and exhibit no alterations in aldosterone levels or blood pressure, suggesting that its absence does not impair intestinal or renal Na+ absorption (8, 9, 20). Studies of the intestinal phenotype of NHE3 and NHE2/NHE3 double-knockout mice revealed no evidence that NHE2 compensates for the loss of NHE3 (5, 9). Using the NHE2-deficient mouse, other investigators also have been unable to identify an absorptive function for NHE2 (13, 17). In parotid glands, where NHE2 is expressed on apical membranes, targeted ablation of NHE2 impaired secretion (17), a result opposite to what would be expected if NHE2 served an absorptive function. The results of the present study further support the view that NHE3 is the critical absorptive Na+/H+ exchanger in the intestine and that NHE2 has little, if any, role in Na+ absorption.
In summary, we used a transgenic approach to partially rescue the
intestinal absorptive defect of Nhe3/
mice.
When subjected to dietary Na+ restriction,
tgNhe3
/
mice were able to reduce urinary
Na+ excretion to very low levels, consistent with the view
that normalization of fluid delivery to the distal convoluted tubule
via a reduction in GFR (4, 12) largely prevents the
overloading of more distal mechanisms for Na+ reabsorption
and consequent salt wasting. After dietary salt loading, to partially
alleviate the extracellular fluid volume deficit, GFR remained lower in
tgNhe3
/
mice than in wild-type controls,
suggesting that reduced perfusion pressure resulting from systemic
hypovolemia is probably not a major factor in the reduced GFR.
Therefore, the reduction is more likely the result of intrarenal
homeostatic mechanisms involving TGF and reduced renal plasma flow.
Finally, after dietary salt loading that far exceeded the levels
occurring in wild-type controls on a normal diet,
tgNhe3
/
mice were still in a chronic
volume-depleted state, indicating that NHE3 in the kidney affects the
set point for Na+-fluid volume homeostasis.
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ACKNOWLEDGEMENTS |
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We thank Maureen Luehrmann and Angel Whitaker for expert animal husbandry.
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FOOTNOTES |
---|
This work was supported by National Institutes of Health Grants DK-50594, DK-57552, HL-61974, and T32-DK-07727.
Address for reprint requests and other correspondence: G. E. Shull; Dept. of Molecular Genetics, Biochemistry, and Microbiology; Univ. of Cincinnati College of Medicine, 231 Albert Sabin Way, 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.
First published February 11, 2003;10.1152/ajprenal.00418.2002
Received 3 December 2002; accepted in final form 5 February 2003.
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