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J Biol Chem, Vol. 273, Issue 44, 29150-29155, October 30, 1998
From the Departments of Mutations in the gene encoding the
thiazide-sensitive Na+-Cl The thiazide-sensitive Na+-Cl Identification of mutations in NCC as the molecular basis of
Gitelman's syndrome was a major breakthrough in the elucidation of the
mechanisms underlying the metabolic abnormalities seen in affected
patients (6, 7). Given the established biochemical function of NCC, it
was apparent that the loss of NCC or a reduction in its activity would
impair NaCl reabsorption in the DCT, and that the metabolic
disturbances must be secondary to this defect. Despite this advance in
our understanding of Gitelman's syndrome, the role of NCC in the
maintenance of sodium and fluid volume homeostasis is unclear, and the
mechanisms by which NCC deficiency causes perturbations of acid-base
homeostasis and renal handling of K+, Na+,
Ca2+, and Mg2+ have not been determined. To
begin addressing these issues we have prepared and analyzed a mouse
model with a null mutation in the Ncc gene (locus
Slc12a3).
Generation of Mutant Mice--
The strain 129/SvJ phage library
from which Ncc genomic clones were isolated and the
targeting vector, MJK+KO, were described previously (11). A
2.5-kb PvuII genomic fragment, ending with codon 485 in exon
12, was subcloned into a cloning site 5' of the neo gene in the vector
and served as the 5' homologous arm. The 3' arm (1.8 kb) was PCR
amplified from a genomic clone using primers corresponding to codons
494-506 in exon 12 and codons 528-539 in exon 13. XhoI
sites were included in the primers to facilitate subcloning into the
vector. The 3' outside probe used for Southern blot analysis began at
codon 546 in exon 13 and included 3.7 kb of genomic sequence.
Electroporation of embryonic stem cells, selection of cells in the
presence of G418 and gancyclovir, Southern blot analysis of DNA samples
from cells and mice, and blastocyst-mediated transgenesis were carried
out as described previously (11).
PCR Genotyping--
DNA from tail biopsies was analyzed by PCR.
Forward (5'-AGGGTCAAGGGCACGGTTGGC-3') and reverse
(5'-GGTAAAGGGAGCGGGTCCGAGG-3') primers corresponding to intron
sequences flanking the disrupted exon amplified a 265-base pair product
from the wild-type gene. A reverse primer (5'-GCATGCTCCAGACTGCCTTG-3')
complementary to sequences in the phosphoglycerate kinase promoter,
which drives the neo gene, and the forward primer described above
amplified a 188-base pair product from the targeted allele.
Northern Blot Analysis--
Total RNA was isolated from the
kidneys of adult mice using Tri-Reagent (Molecular Research Center,
Inc., Cincinnati, OH) according to the supplier's protocol. Blots were
prepared and hybridized with GAPDH, rat renin, and rat NCC cDNA
probes as described previosly (11). mRNA expression was quantified
by PhosphorImager analysis.
Analysis of Blood--
Blood (~50 µl) was collected into
heparin-treated capillary tubes from the femoral artery of anesthetized
mice or from the tail vein of awake mice and immediately analyzed for
gases, electrolytes, and pH using a Chiron diagnostics model 348 pH/blood gas analyzer (Chiron, Norwood, MA). Serum Mg2+ and
Ca2+ concentrations were determined colorimetrically
(calmagite and arsenazo III assays, Sigma). An 125I
radioimmunoassay kit (Diagnostic Products Corp., Los Angeles, CA) was
used to measure serum aldosterone concentrations.
Na+ Depletion Protocols and Analysis of
Urine--
Custom research diets were purchased from Harlan-Teklad
(Madison, WI). Adult Ncc+/+ and
Ncc Blood Pressure--
Mean arterial blood pressure of mice that
were maintained on a 1% NaCl diet or fed a 0.01% NaCl diet for 2 weeks was measured using a femoral artery catheter (13). The mice were
anesthetized with intraperitoneal injections of inactin (100 µg/g
body weight) and ketamine (50 µg/g body weight).
Microscopy and Morphometric Analyses of DCT Cells--
Kidney
sections from 10-15-week-old Ncc+/+ and
Ncc Statistical Analysis--
Values represent the mean ± S.E.
A mixed factorial analysis of variance with repeated measures was used
to assess statistical significance, and post hoc analysis of
individual means was accomplished by contrast analysis.
Generation of NCC-deficient Mice--
Embryonic stem cells
carrying an Ncc null allele were generated by inserting the
neo gene into exon 12 (Fig. 1,
A and B) and used to establish a null mutant
mouse line. PCR analysis of tail DNAs from offspring of heterozygous
mating pairs (Fig. 1C) showed that mice of all three
genotypes were born at the expected Mendelian ratios (37 +/+, 83 +/ Acid-Base Status and Plasma Electrolytes in Mice Maintained on
Normal and Na+-depleted Diets--
Blood gases, blood pH,
and plasma electrolytes were measured in both awake and anesthetized
mice that were maintained on either a normal diet or on a
Na+-depleted diet for 14 or 20 days. As shown in Table
I, there were no significant differences
in blood gases, blood pH, or HCO3 Urinary Na+ and K+ Excretion in Mice
Maintained on Normal and Na+-depleted
Diets--
Ncc+/+ and
Ncc Intra-arterial Blood Pressure Measurements--
When the mice were
fed a normal diet, mean arterial pressures, measured using a femoral
artery catheter, were slightly lower in Ncc Renin mRNA in Kidney and Serum Aldosterone--
To assess
whether the renin-angiotensin-aldosterone axis was activated in
Ncc Urinary Ca2+ Excretion and Serum Mg2+
Levels--
Because hypocalciuria and hypomagnesemia are commonly
observed in Gitelman's syndrome, we analyzed urinary Ca2+
and serum Mg2+ concentrations in the
Ncc Morphometric Analyses and Ultrastructure of Renal Distal Convoluted
Tubules--
In response to treatment with diuretics or altered
dietary Na+, DCT cells undergo ultrastructural changes
(16-20), which are thought to be due to alterations in Na+
transport activity. Because Na+ transport is likely to be
severely reduced in NCC-deficient DCT cells, we examined kidneys of
Ncc In order to examine the role of NCC in sodium and fluid volume
homeostasis and in the pathogenesis of Gitelman's syndrome we
generated an NCC-deficient mouse line. Insertion of the neo gene into
exon 12 of the Ncc gene clearly produced a null mutation, as
Ncc mRNA could not be detected in the kidneys of
homozygous mutant mice. Ncc Although hypokalemic alkalosis is recognized as a major feature of
Gitelman's syndrome in humans, analysis of blood samples from
Ncc Serious impairments of sodium and fluid volume homeostasis are observed
in humans and/or mice with null mutations in the epithelial sodium
channel of the collecting duct (21, 22), the apical Na+-K+-2Cl Like patients with Gitelman's syndrome, NCC-deficient mice had low
levels of both urinary Ca2+ and serum Mg2+,
which are thought to be due to increased Ca2+ and decreased
Mg2+ reabsorption in the DCT (27, 28). The mechanisms by
which NCC dysfunction perturbs renal Ca2+ and
Mg2+ handling in Gitelman's patients are not well
understood. It has been suggested that a reduction in intracellular
Na+ levels as a result of the loss of NCC activity could
increase the rate of Ca2+ efflux via basolateral
Na+/Ca2+ exchange (27). Also, inhibition of
NaCl uptake by chlorothiazide has been shown to decrease intracellular
Cl We observed significant histological changes in the distal tubule of
NCC-deficient mice, but saw no evidence of apoptosis in this segment,
as observed following acute treatment with high doses of thiazides
(19). In Ncc In summary, our studies show that Ncc *
This work was supported by National Institutes of Health
Grants HL41496, DK50594, and ES06096.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.
¶
Present address: INSERM U367, 17 Rue du Fer a Moulin, 75005 Paris, France.
**
To whom correspondence should be addressed: Dept. of Molecular
Genetics, Biochemistry and Microbiology, University of Cincinnati, College of Medicine, 231 Bethesda Ave., Cincinnati, OH 45267-0524. Tel.: 513-558-0056; Fax: 513-558-1885; E-mail:
shullge{at}ucmail.uc.edu.
The abbreviations used are:
NCC, Na+-Cl
Phenotype Resembling Gitelman's Syndrome in Mice Lacking the
Apical Na+-Cl
Cotransporter of the Distal
Convoluted Tubule*
,¶,,,,,
, and**
Molecular Genetics, Environmental Health, the University of Cincinnati College of
Medicine, Cincinnati, Ohio 45267-0524
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
cotransporter
(NCC) of the distal convoluted tubule cause Gitelman's syndrome, an
inherited hypokalemic alkalosis with hypomagnesemia and hypocalciuria.
These metabolic abnormalities are secondary to the deficit in NaCl
reabsorption, but the underlying mechanisms are unclear. To gain a
better understanding of the role of NCC in sodium and fluid volume
homeostasis and in the pathogenesis of Gitelman's syndrome, we used
gene targeting to prepare an NCC-deficient mouse. Null mutant
(Ncc/) mice appear healthy and are normal
with respect to acid-base balance, plasma electrolyte concentrations,
serum aldosterone levels, and blood pressure.
Ncc/ mice retain Na+ as well as
wild-type mice when fed a Na+-depleted diet; however, after
2 weeks of Na+ depletion the mean arterial blood pressure
of Ncc/ mice was significantly lower than
that of wild-type mice. In addition, Ncc/
mice exhibited increased renin mRNA levels in kidney,
hypomagnesemia and hypocalciuria, and morphological changes in the
distal convoluted tubule. These data indicate that the loss of NCC
activity in the mouse causes only subtle perturbations of sodium and
fluid volume homeostasis, but renal handling of Mg2+ and
Ca2+ are altered, as observed in Gitelman's syndrome.
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
cotransporter (NCC)1 is
expressed on the apical membranes of distal convoluted tubule
epithelial cells (1, 2) and is closely related to the apical
Na+-K+-2Cl cotransporter of the
thick ascending limb (3, 4). Both transporters play major roles in the
reabsorption of NaCl in the kidney (5). In humans, mutations in the
genes encoding NCC and the apical
Na+-K+-2Cl cotransporter have
been shown to cause Gitelman's (6, 7) and Bartter's (8) syndromes,
respectively. Hypokalemic alkalosis is a common feature of both
diseases but there are a number of significant differences. Patients
with Bartter's syndrome present clinically at an early age, have
severe salt wasting and volume depletion (9, 10) and exhibit increased
urinary Ca2+ and relatively normal serum Mg2+
levels. In contrast, patients with Gitelman's syndrome are frequently asymptomatic at younger ages, display only limited evidence of salt
wasting or hypovolemia, and exhibit hypocalciuria and hypomagnesemia (9, 10).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/ mice were housed in metabolic cages as
described previously (12). The mice were given deionized water ad
libitum and fed either Na+-replete (TD 90229, 1%
NaCl) or Na+-depleted diets (TD 94268, 0.1% NaCl; TD
90228, 0.01% NaCl). The volume of urine collected during each 24-h
period was measured and Na+ and K+ content was
assayed by flame photometry (Corning model 480). Urinary
Ca2+ concentrations were determined colorimetrically
(arsenazo III assay, Sigma).
/ mice (n = 5 for each
genotype) were prepared for light and transmission electron microscopy
as described previously (11). Both objective and subjective criteria
were used in the analysis of DCT and other distal tubule cells, and
morphometric analyses were performed without prior knowledge of the
genotype. Features used to distinguish DCT cells from other cortical
cell types (14, 15) included the absence of a brush border, apically
placed nuclei, epithelial thickness, and the shape and placement of
mitochondria relative to invaginations of the basolateral membrane. The
transition point between DCT and connecting tubule cannot be clearly
delineated in some species (15), and there is evidence that the
morphology of DCT cells is dependent on the transport activity of the
cell (16-18). Therefore, objective measurements were used to
distinguish DCT and connecting tubule cells, and to identify
alterations in the DCT. Measurements were made on
Ncc+/+ and Ncc/ DCT
cells which had been classified based on both appearance and, as an
objective criterion, the position of their nuclei. At × 1250 magnification, the distances from the basement membrane to the inferior
pole of the nucleus and the apical membrane were measured. Distal
tubule cells containing apically (within the upper two-thirds of the
cell) and basally (within or penetrating into the lower one-third of
the cell) positioned nuclei were classified as DCT and "other"
distal tubule cells, respectively. While this method cannot be used to
show definitively that DCT cells are altered in number, rather than
having an altered morphology that more closely resembles connecting
tubule cells, it does indicate that the features used to classify them
as DCT have changed. To determine the volume density of the DCT a
32-point grid was projected onto sequentially encountered fields of
kidney cortex at × 1250 magnification, with 25 fields surveyed
for kidneys of each mouse. The height of DCT and other distal tubule
cells was obtained by digitizing a line that extended from the basement
membrane to the tubule lumen.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
,
and 48 /). Northern blot analysis of kidney RNA (Fig.
1D) showed that Ncc mRNA expression was
reduced in Ncc+/ mice and absent in
Ncc/ mice, demonstrating that the
Ncc gene was inactivated. Ncc/
mice grew normally, were indistinguishable from
Ncc+/+ littermates in appearance and behavior,
and were fertile.
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Fig. 1.
Generation of Ncc null mice.
A, gene-targeting strategy. Top, region of the
gene containing exons 12 and 13; middle, targeting construct
with the neo gene disrupting exon 12; bottom, targeted
allele. 8- and 9.6-kb HindIII restriction fragments unique
to the wild-type and targeted alleles, respectively, are indicated.
H, HindIII; P, PvuII;
X, XhoI; tk, herpes simplex
virus-thymidine kinase gene used for negative selection of targeted
cells; NotI, site used to linearize construct. B,
Southern blot analysis of DNA from wild-type and targeted embryonic
stem cell lines. DNA was digested with HindIII and
hybridized with the 3'-outside probe (hashed bar in
panel A). C, PCR genotyping of DNA from tail
biopsies. D, Northern blot analysis of total kidney RNA (10 µg/lane) from adult Ncc+/+,
Ncc+/ , and Ncc/
mice. The blot was hybridized with 5' and 3' Ncc cDNA
probes that span codons 2-475 and 475-930, respectively, and a GAPDH
cDNA probe as a loading control.
concentrations between Ncc+/+ and
Ncc/ mice when maintained on either diet,
nor were there significant differences between the two genotypes in
plasma Na+, Cl, K+, or
Ca2+ concentrations. Interestingly, serum Ca2+
concentrations of both Ncc+/+ and
Ncc/ mice maintained on the
Na+-depleted diet were significantly reduced when
compared with the levels observed under Na+-replete
conditions.
Blood gases, acid-base status, and plasma electrolytes
/ mice were fed a Na+-replete
diet (1% NaCl) for 9 days followed by a Na+-depleted diet
(0.01% NaCl) for 3 weeks, and the amounts of Na+ and
K+ excreted in the urine were measured (Fig.
2, A and B).
Urinary Na+ excretion was similar in
Ncc+/+ (0.50 ± 0.02 mmol/day) and
Ncc/ (0.46 ± 0.03 mmol/day) mice
during the Na+-replete period, and after only 2 days of
Na+ restriction (Fig. 2A) was reduced to very
low levels (~0.5% of the excretion rate during the
Na+-replete period) in mice of both genotypes. During the
Na+ depletion period, Na+ excretion averaged
2.6 ± 0.45 and 1.9 ± 0.39 µmol/day in
Ncc+/+ and Ncc/ mice,
respectively; the differences were not statistically significant. No
significant difference in daily urinary K+ excretion was
observed between Ncc+/+ and
Ncc/ mice during the Na+-replete
(0.63 ± 0.03 mmol/day in Ncc+/+ mice;
0.57 ± 0.03 mmol/day in Ncc/ mice) or
Na+ depletion (0.50 ± 0.02 mmol/day in
Ncc+/+, 0.51 ± 0.02 mmol/day in
Ncc/) periods (Fig. 2B).
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Fig. 2.
Urinary Na+ and K+
excretion in response to Na+-replete and
Na+-depleted diets. Mice were fed a diet containing
1% NaCl for nine days followed by a Na+-depleted diet
(0.01% NaCl) for 3 weeks (shaded area). The amount of
Na+ (A) and K+ (B)
excreted in urine of Ncc+/+ (filled
circles) and Ncc / (open
squares) mice was determined by flame photometry. Results are
expressed as mmoles of Na+ or K+ excreted per
day. No significant difference in Na+ or K+
excretion was observed between Ncc+/+
(n = 5) and Ncc/
(n = 5) mice during the Na+-replete or
Na+ depletion periods. Average
Ncc+/+ and Ncc/ body
weights at the end of the Na+-replete (34.8 ± 1.4 g in Ncc+/+, 31.3 ± 2.0 g in
Ncc/) and Na+ depletion
(35.2 ± 2.2 g in Ncc+/+, 34.2 ± 2.4 g in Ncc/) periods were not
significantly different.
/
(84.7 ± 5.6 mm Hg) than in Ncc+/+
(87.4 ± 6.6 mm Hg) mice (Fig. 3),
but the difference was not statistically significant. However, after
both groups of mice were fed a Na+-depleted diet for 2 weeks (Fig. 3), the mean arterial pressure of
Ncc/ mice (72.3 ± 5.0 mm Hg) was
significantly lower than that of wild-type mice (86.1 ± 3.9 mm
Hg).
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Fig. 3.
Intra-arterial blood pressure
measurements. Mean arterial blood pressure was measured in
anesthetized Ncc+/+ and
Ncc / mice maintained on a
Na+-replete diet and after being fed a
Na+-depleted diet (0.01% NaCl) for 2 weeks. The number of
animals analyzed for each group is shown in parentheses.
*Significantly different from wild-type as determined by single factor
analysis of variance (p = 0.05). Values for all
analyses are means ± S.E.
/ mice, renin mRNA in kidney and
serum aldosterone levels were measured in mice that were fed a
Na+-replete diet. In an initial experiment we analyzed
renin mRNA in pooled kidney samples (two mice of each genotype) and
found that renin mRNA, when normalized against GAPDH mRNA, was
1.9-fold higher in kidneys of Ncc/ mice
(data not shown). An additional experiment was performed in which
kidney RNA from three mice of each genotype was analyzed. As shown in
Fig. 4A, renin mRNA levels
were higher in Ncc/ kidneys than in
Ncc+/+ kidneys (averaging 1.7-fold when
normalized against GAPDH mRNA). Serum aldosterone levels (Fig.
4B) were similar in Ncc+/+ (0.67 ± 0.16 ng/ml) and Ncc/ (0.65 ± 0.13 ng/ml) mice that were fed a normal diet (1% NaCl). After 2 weeks of
Na+ restriction, aldosterone levels were higher in
Ncc/ mice (2.73 ± 0.32 ng/ml) than in
Ncc+/+ mice (2.18 ± 0.27 ng/ml), but the
difference was not statistically significant.
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Fig. 4.
Renin mRNA and serum aldosterone.
A, Northern blot analysis of total kidney RNA (10 µg)
isolated from individual adult Ncc+/+,
Ncc+/ , and Ncc/
mice maintained on a Na+-replete diet. The blot was
hybridized with a renin cDNA probe and then stripped and hybridized
with a GAPDH probe. After autoradiography, the blots were analyzed by
PhosphorImager analysis and the renin/GAPDH signal intensity ratios
(1.00 ± 0.02 for Ncc/ mice; 0.58 ± 0.01 for Ncc+/+ mice; p = 0.05) were calculated. B, serum aldosterone levels in adult
Ncc+/+ and Ncc/ mice
after being fed a Na+-replete diet and then a
Na+-depleted diet (0.01% NaCl) for 2 weeks. The number of
animals analyzed for each group is shown in parentheses.
Serum aldosterone increased in response to Na+ depletion in
mice of both genotypes (p 
0.0005) but there were no
significant differences between Ncc+/+ and
Ncc/ mice maintained on the same diet.
/ and Ncc+/+ mice
that were used in the Na+ depletion study. Urinary
Ca2+ excretion was significantly lower in
Ncc/ (0.045 ± 0.004 mg/day) than in
Ncc+/+ (0.208 ± 0.014 mg/day) mice when
they were fed a Na+-replete diet (Fig.
5A). However, urinary
Ca2+ excretion in Ncc+/+ mice
dropped by ~60% after 24 h of Na+ depletion and
remained at this level (0.084 ± 0.008 mg/day) throughout the
3-week Na+ depletion period. In contrast, there was no
significant change in urinary Ca2+ excretion in
Ncc/ mice when they were fed the
Na+-depleted diet. Serum Mg2+ was significantly
lower in Ncc/ than in
Ncc+/+ mice when the animals were fed either
normal (0.51 ± 0.03 and 0.87 ± 0.03 mM,
respectively) or Na+-depleted (0.76 ± 0.02 and
1.17 ± 0.07 mM, respectively) diets (Fig.
5B). For both Ncc/ and
Ncc+/+ mice, serum Mg2+ was
significantly higher (p < 0.002) after being fed the
Na+-depleted diet for 3 weeks.
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Fig. 5.
Urinary Ca2+ excretion and serum
Mg2+. A, urine samples assayed for
Ca2+ content were the same as those analyzed in Fig. 2.
Results were calculated for the Na+-replete (days 1-9) and
Na+-depletion (days 11-30) periods and are expressed as
milligrams of Ca2+ excreted per day. B,
Mg2+ concentration in serum samples collected at the
beginning of the Na+-replete diet period and at the end of
the Na+ depletion period. Values for all analyses are
means ± S.E. *Significantly different from levels in wild-type
mice fed the same diet (p 0.0003).
/ and Ncc+/+ mice
by light and electron microscopy to determine whether there were
morphological correlates of the Na+ transport defect.
Readily identifiable DCT cells were 6.1 ± 1.2% of cortical
epithelial cells in Ncc+/+ kidneys and only
1.9 ± 0.7% in Ncc/ kidneys
(p = 0.012). In contrast, there was no significant
difference in the number of other distal tubule cell types (9.1 ± 1.4% and 10.5 ± 2.2% for Ncc+/+ and
Ncc/ kidneys, respectively). Cell height was
significantly decreased in Ncc/ DCT cells
(14.4 ± 0.5 µm in Ncc+/+, 12.7 ± 0.1 µm in Ncc/, p = 0.009)
but not in other distal tubule cells (11.9 ± 0.7 µm in
Ncc+/+, 11.6 ± 0.2 µm in
Ncc/). As evident in the representative
electron micrographs of mutant and wild-type cells shown in Fig.
6, mitochondria of
Ncc/ DCT cells were less elongated in shape
and the association of basolateral membrane folds with the mitochondria
was developed less well than in Ncc+/+
cells.
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Fig. 6.
Electron micrographs of
Ncc+/+ and Ncc /
DCT cells. Relative to the mitochondria in
wild-type cells (A), mitochondria in
Ncc/ cells (B) appear to be
reduced in number and less elongated in shape. Cells of both genotypes
have extensive basolateral membrane folds, but the membrane folds are
more closely associated with the mitochondria in
Ncc+/+ cells than in
Ncc/ cells. Scale bar, 5 µm.
DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
/ mice were born
at the expected Mendelian ratios, exhibited no overt disease phenotype,
and were fertile. Subsequent analyses provided evidence for a mild but
largely compensated perturbation of sodium and fluid volume
homeostasis, revealed morphological changes in the DCT, and
demonstrated that Ncc/ mice have
hypocalciuria and hypomagnesemia, which are characteristic of
Gitelman's syndrome.
/ mice revealed no apparent disturbances
of potassium or acid-base homeostasis. The absence of hypokalemic
alkalosis in Ncc/ mice may indicate a major
species difference in the effects of NCC dysfunction; however,
Gitelman's syndrome is often quite mild and many patients are not
identified until adulthood. In many of the case histories that have
been reported, as well as in the studies demonstrating that mutations
in the NCC gene cause Gitelman's syndrome (6, 7), hypokalemic
alkalosis was among the criteria used in selecting patients to be
studied. Thus, it is possible that the patients that have been reported
in the literature represent a more strongly affected subset of
individuals with mutations in the NCC gene, and that the physiological
consequences of such mutations differ depending on genetic background
and environmental factors.
cotransporter of the
thick ascending limb (8), and the NHE3 Na+/H+
exchanger of the proximal tubule (23). Given the role of NCC in NaCl
reabsorption in the distal tubule, one might anticipate that NCC null
mutations would also perturb sodium and fluid volume homeostasis.
However, when cases of Gitelman's syndrome were distinguished from
Bartter's syndrome by relatively rigorous diagnostic criteria, Gitelman's syndrome patients were typically normotensive and exhibited little direct evidence of hypovolemia (9). Nevertheless, elevated serum
aldosterone levels (9) indicated that some of the mechanisms that
compensate for a perturbation of sodium and fluid volume homeostasis
were activated in these patients. The results of the current study
suggest that NCC-deficient mice also have a mild, compensated
perturbation of sodium and fluid volume homeostasis. Serum aldosterone
was not elevated in Ncc/ mice, indicating
that the systemic renin-angiotensin-aldosterone axis was not activated.
However, the increased renin mRNA levels in
Ncc/ kidneys is consistent with the
possibility that compensation might be mediated by the intra-renal
renin-angiotensin system (24-26). There was no significant difference
in mean arterial blood pressure between Ncc/
and Ncc+/+ mice when maintained on a
Na+-replete diet, but anesthetized
Ncc/ mice exhibited a lower blood pressure
than wild-type mice when fed a Na+-depleted diet for 2 weeks. This was surprising because Ncc/ mice
were able to retain Na+ as well as wild-type mice during
the Na+-depletion period. A possible explanation for this
result is that Ncc/ mice are mildly
hypovolemic, and that gradual Na+ loss during
Na+ restriction reduces intravascular volume to a point
where the mechanisms that regulate blood pressure can no longer fully
compensate. If this interpretation is correct, it implies that
Ncc/ mice have an altered set point for
Na+-fluid volume balance in which the reduction in
intravascular volume due to the loss of Na+ reabsorption
via NCC is largely, but not completely, corrected by compensatory
mechanisms.
activity, causing hyperpolarization that in turn
causes increased Ca2+ uptake via apical Ca2+
channels (29). With regard to Mg2+ handling, there are data
indicating that the hypomagnesemia might be secondary to hypokalemia
(30). Our results tend to negate this hypothesis as
Ncc/ mice develop hypomagnesemia in the
absence of hypokalemia. Also, clinical studies of patients with
hypomagnesemia and hypokalemia show that correction of hypomagnesemia
by dietary Mg2+ supplements often leads to correction of
the hypokalemia (31-33), thereby suggesting that the hypokalemia is
secondary to hypomagnesemia.
/ mice, there was a sharp
reduction in the number of readily identifiable DCT cells, the height
of DCT cells was decreased, their mitochondria were less elongated, and
their basolateral membrane folds were not as closely associated with
the mitochondria. These changes are the opposite of those observed in
rats treated with furosemide (16) or maintained on a high salt diet
(17), in which Na+ reabsorption in the DCT is increased.
Thus, it seems likely that the alterations in the DCT of
Ncc/ mice are due to the reduced
transcellular Na+ transport capacity. These data support
the view (16-18, 20) that the structure of DCT cells is regulated by
the level of Na+ reabsorption, and suggest that the
alterations in renal Ca2+ and Mg2+ handling
could be due, at least in part, to the altered structure of the
DCT.
/ mice
have hypocalciuria and hypomagnesemia, but not hypokalemic alkalosis.
This suggests that the renal defects in Ca2+ and
Mg2+ reabsorption are a consequence of functional and/or
structural alterations in the DCT caused by the loss of NCC activity
rather than being secondary to systemic metabolic disturbances such as hypokalemia or alkalosis. Our results also indicate that NCC plays a
relatively subtle role in the maintenance of sodium and fluid volume
homeostasis, compared with that of the NHE3
Na+/H+ exchanger (23), the apical
Na+-K+-2Cl cotransporter (8), and
the epithelial Na+ channel (21, 22). In future studies the
NCC-deficient mouse should serve as a valuable model for deciphering
the mechanisms by which NCC deficiency alters renal Ca2+
and Mg2+ handling and for further investigations of the
role of NCC in electrolyte, fluid volume, and acid-base
homeostasis.
FOOTNOTES
cotransporter; Ncc, mouse
gene encoding NCC; Ncc+/+, Ncc+/, and Ncc/ are
wild-type, heterozygous, and homozygous mutant mice, respectively; DCT, distal convoluted tubule; neo, neomycin resistance; PCR, polymerase
chain reaction; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; kb, kilobase pair(s).
REFERENCES
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Abstract
Introduction
Procedures
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Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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