Decreased renal Na-K-2Cl cotransporter abundance in mice with
heterozygous disruption of the
Gs
gene
Carolyn A.
Ecelbarger1,
Shuhua
Yu2,
Alanna J.
Lee1,
Lee S.
Weinstein2, and
Mark A.
Knepper1
1 Laboratory of Kidney and
Electrolyte Metabolism, National Heart, Lung and Blood Institute;
and 2 Metabolic Diseases Branch,
National Institute of Diabetes, Digestive, and Kidney Diseases,
Bethesda, Maryland 20892
 |
ABSTRACT |
Transport
processes along the nephron are regulated in part by hormone
stimulation of adenylyl cyclases mediated by the heterotrimeric G
protein Gs. To assess the role of
this pathway in the regulation of Na-K-2Cl cotransporter abundance in
the renal thick ascending limb (TAL), we studied mice with heterozygous
disruption of the Gnas gene, which
codes for the
-subunit of Gs.
Outer medullary Gs
protein
abundance (as assessed by semiquantitative immunoblotting) and
glucagon-stimulated cAMP production were significantly reduced in the
heterozygous Gs
knockout mice
(GSKO) relative to their wild-type (WT) littermates. Furthermore,
Na-K-2Cl cotransporter protein abundance in the outer medulla was
significantly reduced (band density, 48% of WT). In addition, GSKO
mice had a significantly reduced (72% of WT) urinary osmolality in
response to a single injection of
1-deamino-[8-D-arginine]vasopressin
(DDAVP), a vasopressin analog. In contrast, outer
medullary protein expression of the type 3 Na/H exchanger (NHE-3) or
Tamm-Horsfall protein did not differ between the GSKO mice and their WT
littermates. However, abundance of type VI adenylyl cyclase was
markedly decreased in the outer medullas of GSKO mice, suggesting a
novel feed-forward regulatory mechanism. We conclude that
expression of the Na-K-2Cl cotransporter of the TAL is dependent on
Gs
-mediated hormone stimulation, most likely due to long-term changes in cellular cAMP levels.
vasopressin; urinary concentrating mechanism; adenosine
3',5'-cyclic monophosphate; sodium-potassium-adenosinetriphosphatase; aquaporins
 |
INTRODUCTION |
TRANSPORT FUNCTIONS are regulated throughout the length
of the nephron by hormone stimulation of adenylyl cyclases mediated by
the heterotrimeric G protein Gs.
For example, activation of this signaling pathway via vasopressin
binding to V2 receptors in rat renal collecting duct and the thick
ascending limb (TAL) (18, 19) is key in renal water conservation. In
the TAL, acute exposure to vasopressin has been shown to increase NaCl
transport in isolated perfused tubule experiments (12, 14, 27).
Furthermore, chronic administration of vasopressin to Brattleboro rats
has been reported to increase TAL chloride transport (3). An increase in NaCl transport by the TAL would enhance counter-current
multiplication and thus the ability to form a concentrated urine.
Increased TAL chloride transport could be due to increased expression
of the apical Na-K-2Cl cotransporter, perhaps as a result of increased cAMP production. The finding of a cAMP regulatory element (CRE) in the
5'-flanking region of the NKCC-2 gene (17), which codes for the
Na-K-2Cl cotransporter of the TAL, is consistent with the hypothesis
that the expression level of this cotransporter is regulated by
adenylyl cyclase activation. We recently showed an increase in outer
medullary expression of the apically located Na-K-2Cl cotransporter of
the TAL in rats by
1-deamino-[8-D-arginine]vasopressin (DDAVP) administration or water restriction (conditions which raise
intracellular cAMP levels) (20). Furthermore, the expression of
aquaporin-2, another vital protein in the urinary concentrating mechanism, is thought to be mediated by cAMP stimulation of cAMP regulatory element binding protein (CREB) phosphorylation (16, 26, 35).
Phosphorylated CREB has been proposed to activate aquaporin-2 gene
transcription by binding to a CRE and possibly other elements in the
5'-flanking region of the aquaporin-2 gene.
A new animal model, recently introduced, is useful in addressing the
role of cAMP in the regulation of the expression of the Na-K-2Cl
cotransporter, namely mice with heterozygous disruption of the
Gnas gene, which codes for the
heterotrimeric G protein subunit,
Gs
. These
Gs
knockout (GSKO) mice were
created by targeted disruption of Gnas
through insertion of a GK-Neo cassette into exon 2 (36). The
homozygotes do not develop beyond the embryonic stage. However, the
heterozygotes survive and manifest an interesting phenotype that is
dependent upon whether the mutant gene is derived from the mother
(m
/p+) or from the father (m+/p
). This phenotypic variability is due to genomic imprinting, i.e., epigenetic inactivation of one of the two alleles coding for
Gnas (36). Thus proximal tubules of
m+/p
mice express normal (wild-type, WT) levels of Gs
protein and manifest normal
cAMP responses to parathyroid hormone, whereas proximal tubules of
m
/p+ mice express low levels of
Gs
protein and manifest
depressed cAMP responses to parathyroid hormone.
The primary objective of the present study was to test the effect of
reduced Gs
protein expression
in the heterozygous GSKO mice on signaling and transporter
expression in the TAL of Henle's loop. Specifically, the goals were to
evaluate 1) whether
Gs
protein expression differs
between m
/p+ GSKO mice and m+/p
GSKO mice in a manner
consistent with imprinting of Gnas in
TAL cells, 2) whether the TALs of
GSKO mice manifest a reduction in Na-K-2Cl cotransporter expression or
a reduction in expression of other TAL proteins (NHE-3, Tamm-Horsfall
protein, or Na-K-ATPase subunits), 3) whether a reduction in
Gs
expression in the TALs of
GSKO mice is associated with reduced cellular cAMP production in TAL
cells, and 4) whether GSKO mice have
a defect in urinary concentrating capacity.
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MATERIALS AND METHODS |
Knockout mouse model. The GSKO mice
used in this study were originally described by Yu et al. (36).
Homozygous GSKO mice did not develop beyond the embryonic stage.
Consequently, heterozygous knockout mice were used in these studies.
Heterozygous GSKO mice were obtained by mating either a heterozygous
GSKO female mouse to a WT male (to generate m
/p+ mice) or by
mating a heterozygous male to a WT female (to generate m+/p
mice). Genotyping of offspring was performed by Southern hybridization
or duplex PCR (34) by using primers for the
-globin gene promoter
and "neo-specific" primers as described in the initial
publication of Yu et al. (36). In some tissues and cell
types, Gnas is subject to imprinting or inactivation of one parent's donated allele, presumably by methylation (2). Therefore, it is important to distinguish between the
two types of heterozygotes. In this report, we present and
statistically analyze the immunoblotting data from the two groups of
heterozygotes both separately and in a pooled format.
Experimental design. Four studies were
conducted using the GSKO mice. In the first, we examined expression of
TAL proteins in the two groups of heterozygous mice by immunoblotting.
Four m
/p+ mice and four m+/p
mice were compared with four
each of their respective WT littermates. The mice were killed by
cervical dislocation, the kidneys were removed, and samples were
prepared for immunoblotting as described below. In the second study,
m+/p
mice were injected intramuscularly with 1 ng DDAVP
(Peninsula Laboratories, Belmont, CA), and urine was collected 1 h
later to assess the ability to increase urinary osmolality (model
5100C; Wescor, Logan, UT). In a third study (for cAMP measurement), six additional untreated heterozygous GSKO mice were used (three
m
/p+ mice and three m+/p
mice) along with their
respective WT littermates. In these mice, after death, TAL and inner
medullary collecting duct (IMCD) tubule suspensions were
prepared from both right and left kidney outer or inner medullas,
respectively, as described below. In the fourth study, six m
/p+
mice and six littermates were deprived of water for 48 h prior to urine
collection to assess Na-K-2Cl cotransporter expression and final urine
concentration in the context of a maximal long-term antidiuretic
stimulus. After measurement of urinary osmolality, kidneys from these
water-deprived mice were harvested, and whole homogenates were prepared
from the outer medullas of the left kidneys for immunoblotting.
Antibodies. We used a rabbit
polyclonal anti-Na-K-2Cl cotransporter antibody (L224) (9) raised
against a synthetic peptide corresponding to amino acids 109-129
of the amino-terminal tail of the rat Na-K-2Cl cotransporter based on
the cloned sequence reported by Gamba et al. (11). The antibody is
highly specific for the TAL form of the Na-K-2Cl cotransporter and does
not recognize either the secretory form of the Na-K-2Cl cotransporter
or the thiazide-sensitive Na-Cl cotransporter. Similarly, rabbit
polyclonal antibodies were raised against
1) amino acids 809-831 of the
type 3 Na/H exchanger (NHE-3) (L546) (10),
2) against amino acids 267-292
of aquaporin-3 water channel (L178) (8), and
3) against amino acids 911-929
of UTA1, the vasopressin-regulated urea channel of the IMCD (L403)
(30), by immunizing rabbits with synthetic peptides corresponding to
these regions in the carboxy-terminal tail of each protein. A rabbit
polyclonal anti-aquaporin-2 antibody (L414), likewise, was raised
against a 23-amino acid synthetic peptide corresponding to the
carboxy-terminal tail of aquaporin-2. The immunizing peptide was
identical to that used for the previously described polyclonal antibody
(L127) (6), and the two anti-aquaporin-2 antibodies have been found to
give equivalent labeling on immunoblots (unpublished data). These
polyclonal antibodies were affinity purified on columns prepared with
the corresponding synthetic peptides. An affinity-purified polyclonal
anti-Gs
antibody (33) was
kindly provided by Dr. Paul Goldsmith (National Institute of Diabetes
and Digestive and Kidney Diseases). Rabbit anti-Na-K-ATPase (
1- and
-subunit) polyclonal antibodies were obtained from Upstate Biotechnology (Lake Placid, NY). A polyclonal anti-Tamm-Horsfall antibody (15) was kindly provided by Dr. John Hoyer (University of
Pennsylvania). A polyclonal adenylyl cyclase VI antibody was purchased
from Santa Cruz Biotechnology (Santa Cruz, CA). This antibody
recognizes adenylyl cyclase VI, which is expressed in the TAL of the
collecting duct (4), as well as adenylyl cyclase V (not expressed in
the TAL). For immunoblotting, antibodies were used at the following
concentrations: Na-K-2Cl cotransporter, 0.21 µg/ml; aquaporin-2, 0.29 µg/ml; Na-K-ATPase (
1-subunit), 0.10 µg/ml, Na-K-ATPase
(
-subunit), 0.10 µg/ml;
Gs
, 0.73 µg/ml; adenylyl
cyclase VI, 0.20 µg/ml, NHE-3, 0.40 µg/ml; UTA1, 0.21 µg/ml; and
aquaporin-3, 0.12 µg/ml.
Immunoblotting sample preparation.
Left kidneys were removed from the mice and were dissected with small,
curved scissors into inner medulla, outer medulla, and
cortex. Outer medulla as dissected consisted primarily of inner stripe
and thus did not contain a significant proportion of proximal straight
tubule. Each region was homogenized separately using a tissue
homogenizer (Omni 2000 fitted with a 7 or 10 mm micro-sawtooth
generator) in chilled membrane-isolation solution (1 ml for outer or
inner medulla, 5 ml cortex) containing 250 mM sucrose, 10 mM
triethanolamine (Calbiochem, La Jolla, CA), 1 µg/ml leupeptin
(Bachem, Torrance, CA) and 0.1 µg/ml phenylmethylsulfonyl fluoride
(US Biochemical, Toledo, OH) adjusted to pH 7.6. The homogenates were
then centrifuged in a Beckman L8-M ultracentrifuge fitted with a type
80TI rotor at 200,000 g for 1 h to
obtain a total membrane pellet. This pellet was resuspended in a small
amount of isolation solution, and the protein concentration was
measured (BCA protein assay reagent kit; Pierce, Rockford, IL). All
samples were then diluted with isolation solution to a final protein
concentration of 1 µg/µl and solubilized at 60°C for 15 min in
Laemmli sample buffer. Samples were stored at
80°C until
ready to run on gels.
Electrophoresis and blotting of
membranes. SDS-PAGE was done on precast minigels of
7.5, 10, or 12% polyacrylamide (Bio-Rad, Hercules, CA). The proteins
were transferred from the gels electrophoretically to nitrocellulose
membranes. After a 30-min 5% milk block, membranes were probed
overnight at 4°C with the appropriate primary antibody. For this,
the antibodies were diluted into a solution containing 150 mM NaCl, 50 mM sodium phosphate, 10 mg/dl sodium azide, 50 mg/dl Tween 20, and 0.1 g/dl bovine serum albumin (pH 7.5). The secondary antibody was donkey
anti-rabbit IgG conjugated to horseradish peroxidase (Pierce no. 31458)
used at a concentration of 0.16 µg/ml. Sites of antibody-antigen
reaction were visualized using luminol-based enhanced chemiluminescence
(LumiGLO; Kirkegaard and Perry Laboratories, Gaithersburg, MD) before
exposure to X-ray film (no. 165-1579 Scientific Imaging Film;
Kodak, Rochester, NY). Immunoblot band densities were quantitated by
laser densitometry (Molecular Dynamics, San Jose, CA).
cAMP measurements in tubule
suspensions. Immediately after death, both kidneys were
rapidly removed from the mouse. The outer medulla (inner stripe) and
inner medulla were dissected out of both kidneys and separately minced
into small cubes (<1 mm) with a razor blade, while the tissue was
kept moist. Pieces of outer medulla (or inner medulla) were incubated
in a 50-ml centrifuge tube at 37°C in 10 ml collagenase digestion
solution which contained 50 mg collagenase B (Worthington type;
Boehringer, Mannheim, Germany), 50 mg bovine serum albumin (fraction V;
Sigma, St. Louis, MO), and 5 mg DNase I (DN-25, Sigma) dissolved in 50 ml of solution 1, which was composed
of DMEM-F12 (Sigma) with added 4 mM glycine (Sigma, no. G6388) and 1 mM
heptanoate (Sigma, no. H9278). A light stream of oxygen was blown
across the surface of the solution during the incubation, which was
done in a heated shaking water bath. After 8 min, the tube was removed,
and the partially digested outer (or inner) medullary tissue was
pipetted up and down (15 times) into a 10-ml disposable pipette to
facilitate digestion. Chunks were allowed to settle out in the pipette
by gravity. Large chunks were then redigested for 8 min more. The
remaining digested material (primarily short segments of TALs and
collecting ducts for the outer medullary suspensions and collecting
ducts and thin limbs for inner medullary suspensions) was added to 25 ml of ice-cold solution 1 (which
diluted out enzymes). After three total rounds of digestion, the
digested tubules were centrifuged at 100 g for 5 min to softly pellet tubules
and concentrate the TALs (for outer medullary suspensions) or
collecting ducts (for inner medullary suspensions). In this
preparation, for the outer medullary suspensions, thin limbs and
free-floating collecting duct cells generally stayed in supernatant, as
assayed by microscopic analysis of the fractions. After the gentle
centrifugation, the outer medullary suspension consisted of
~80-90% TALs compared with other tubule components. This was
based on microscopic analysis of several preparations and counting of
the tubules present of each type under the microscope. For the inner
medullary suspensions, this protocol has been previously shown to
result in relative enrichment of IMCD vs. other tubule components, such
as thin limbs (21).
The outer (or inner) medullary tubules were resuspended in 0.5 ml fresh
solution 1 immediately before
beginning the incubation. Incubations for cAMP production were
performed as previously described (7). After an initial 10-min
incubation with 0.5 mM IBMX to inhibit phosphodiesterases, 50-µl
aliquots of the suspension were incubated for 5 min with one level of
hormone or vehicle. For the TAL suspensions, treatments were as
follows: vehicle, 0.1, 1, 10, or 100 nM glucagon. The IMCD reportedly
expresses one receptor subtype coupled to
Gs
, the vasopressin receptor
(28). Therefore, the IMCD suspensions were exposed to vehicle or 0.1 or
10 nM DDAVP. The reactions were terminated by the addition of 10%
trichloroacetic acid. After centrifugation at 2,300 g for 10 min, cAMP was measured in the
supernatant using an ELISA-based approach (enzyme immunoassay kit no.
581001; Cayman Chemical, Ann Arbor, MI). Protein content of the pellet
was measured by a Bradford analysis kit (Bio-Rad).
 |
RESULTS |
Thick Ascending Limb
Gs
expression.
The mice evaluated in these studies were heterozygous for the
-subunit of the heterotrimeric GTP-binding protein,
Gs, and were previously
characterized by Yu et al. (36). Heterozygotes were derived from the
mating of a heterozygous female to a WT male (m
/p+, where
"m" indicates the maternally derived allele and "p"
indicates the paternally derived allele) or from the mating of a
heterozygous male to a WT female (m+/p
). Imprinting (epigenetic inactivation of one allele) of the gene that codes for
Gs
(Gnas) has been demonstrated in the
kidney proximal tubule, but not in IMCD, in this mouse model (36).
Because of the potential for imprinting in the TAL, we first evaluated
outer medullary tissue (primarily inner stripe, see
MATERIALS AND METHODS) from both types of GSKO mice separately. Figure 1
shows Gs
immunoblots prepared from gels loaded with outer medullary membrane samples. Each
lane was loaded with 5 µg of total protein prepared from a different
mouse. Figure 1A shows an immunoblot
prepared with crude membrane samples prepared from outer medullas of
m
/p+ mice with respective WT littermates. Figure
1B shows an immunoblot loaded with
membrane samples prepared from m+/p
mice and their respective
wild-type WT littermates. Two bands were present at ~50 and 45 kDa (33), secondary to alternative splicing of
Gnas transcript.
Gs
expression was approximately
equally reduced in the outer medullas of both groups of heterozygotes
(Table 1). Because there was an
approximately equal decrease (but not elimination) of
Gs
expression in both
groups of GSKO mice (Table 1), we conclude that imprinting
of Gnas does not occur in the TAL,
whose cells make up most of the cellular volume of the outer medulla
(22).

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Fig. 1.
Immunoblots of total outer medullary membranes (200,000 g pellet) of wild-type (WT) and
Gs knockout (GSKO) mice probed
with anti-Gs antibody.
A: m /p+ mice vs. WT
littermates. B: m+/p vs. WT
littermates. For both blots, each lane was loaded with 5 µg protein
from a different animal.
Gs -antibody was affinity
purified and used at an IgG concentration of 0.73 µg/ml. Preliminary
12% polyacrylamide gels were run in each case and were stained with
Coomassie blue to confirm equality of loading (not shown).
Gs abundance was significantly
reduced in the GSKO mice in both groups (see Table 1).
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Reduced expression of the Na-K-2Cl
cotransporter. Figure
2A shows
an immunoblot of outer medullary membranes (same samples as in Fig.
1A) from GSKO mice (m
/p+)
and their WT littermates. This immunoblot demonstrates a decrease in
Na-K-2Cl cotransporter expression in GSKO mice relative to WT mice from
the same litter. [In membrane fractions, commonly there are two
broad bands associated with the Na-K-2Cl cotransporter centered at
~161 and at 320 kDa. We have recently shown by cross-linking studies
in rat (25) that the upper band (320 kDa) is most likely due to the
natural existence of a homodimer of the Na-K-2Cl cotransporter in the plasma membrane. The dimer tends to be stabilized under low ionic strength conditions such as exists in the solution used for membrane isolation in the present studies.] Figure
2B shows normalized densitometry data
comparing pooled band densities from m
/p+ and m+/p
GSKO
mice (n = 8) and the pooled
band densities of their respective WT controls
(n = 8). This pooled analysis
confirmed the decrease in band density for the Na-K-2Cl cotransporter
in the GSKO mice (band density for GSKO mice: 49 ± 6% of WT
controls, means ± SE). Before running immunoblots,
Coomassie-stained "loading gels" were run on all samples and
evaluated by laser densitometry to confirm equality of loading on the
immunoblots (data not shown).

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Fig. 2.
A: Na-K-2Cl cotransporter immunoblot
of total outer medullary membranes (200,000 g pellet; 1 µg/lane) from 4 WT and 4 m /p+ GSKO heterozygotes. Each lane was loaded with a sample from
a different animal. Na-K-2Cl antibody was affinity purified and used at
an IgG concentration of 0.21 µg/ml. Preliminary 12% polyacrylamide
gels were run in each case and were stained with Coomassie blue to
confirm equality of loading (not shown).
B: densitometry of the Na-K-2Cl
cotransporter immunoblot bands from GSKO mice plotted as a percentage
of their WT littermates. Data were pooled from both groups of mice.
Na-K-2Cl abundance was significantly reduced in the GSKO mice in both
m /p+ and m+/p groups (see also Table 1).
* P < 0.05.
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Na-K-2Cl cotransporter expression also was reduced in the cortex. When
data were pooled from both groups of GSKO heterozygotes, Na-K-2Cl
cotransporter band density was significantly reduced (73 ± 11% of
WT littermates, means ± SE, P < 0.05).
Lack of effect on other TAL apical
proteins. We also measured the abundance of other
apically expressed proteins in the outer medullary TAL by
semiquantitative immunoblotting. Figure
3A shows an immunoblot for NHE-3 (the type 3 Na/H exchanger) in the outer medulla using m
/p+ GSKO heterozygotes and their WT littermates. The single band we observe for NHE-3 in mouse runs at ~84 kDa, similar to the band seen in rat kidney (1). Densitometric analysis revealed that the expression level of NHE-3 did not differ
significantly between the GSKO mice and their WT littermates. Table 1
shows that similar results were obtained in the m+/p
GSKO mice.

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Fig. 3.
Immunoblots of total outer medullary membranes (200,000 g pellet) from 4 WT and 4 m /p+
heterozygous Gs -knockout mice.
A: blot probed with anti-NHE-3
antibody, 0.40 mg/ml, with 10 µg protein from a different rat loaded
in each lane. B: blot probed with
anti-Tamm-Horsfall antibody, 1.4 µg/ml IgG, with 5 µg protein from
a different rat loaded in each lane. Preliminary 12% polyacrylamide
gels were run and were stained with Coomassie blue to confirm equality
of loading (not shown). Neither NHE-3 nor Tamm-Horsfall protein
abundance was significantly affected by
Gs knockout (see Table 1).
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Figure 3B shows an immunoblot for
Tamm-Horsfall protein in the outer medullas of m
/p+ GSKO mice
compared with WT littermates. Band density was not different between
the GSKO mice and the WT controls. Likewise, no difference in
expression of Tamm-Horsfall protein was seen in outer medullas of
m+/p
GSKO mice vs. their WT littermates (Table 1).
Na-K-ATPase abundance in outer medullas of GSKO
mice. Figure 4 shows
immunoblots of outer medullary membranes from m
/p+ GSKO mice and
their WT controls probed with antibodies to the
1-subunit (Fig.
4A) and
-subunit (Fig.
4B) of Na-K-ATPase. As shown in Fig.
4 and summarized in Table 1, the expression level of the
-subunit
was significantly reduced in the GSKO mice (equally so in both groups
of heterozygotes). Although the band density for the
1-subunit also
appeared to be lower in the GSKO mice relative to the WT mice
(especially in the m
/p+ group), a significant decrease was not
detected in the pooled groups (Table 1).

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Fig. 4.
Immunoblots of total outer medullary membranes (200,000 g pellet) from 4 WT and 4 m /p+
heterozygous Gs -knockouts
(GSKO). A: blot probed with
anti- 1-subunit Na-K-ATPase antibody, 0.10 µg/ml, with 10 µg
protein from a different rat loaded in each lane.
B: blot probed with anti- -subunit
Na-K-ATPase antibody, 0.10 mg/ml, with 10 µg protein from a different
rat loaded in each lane. Preliminary 12% polyacrylamide gels were run
and were stained with Coomassie blue to confirm equality of loading
(not shown). The 1-subunit of Na-K-ATPase was not significantly
changed in either group of GSKO heterozygotes, whereas -subunit of
Na-K-ATPase was significantly reduced in both groups of GSKO
heterozygotes (Table 1).
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Decreased cAMP production in TAL
suspensions. To assess a potential mechanism for the
decrease in Na-K-2Cl cotransporter expression in these mice, we first
tested whether the observed reduction in
Gs
protein was associated with
a corresponding reduction in cAMP production by the TAL cells. In the
TAL, Gs is coupled to four
different receptors: glucagon, vasopressin V2, calcitonin, and
parathyroid hormone (28). In the present studies, glucagon was used.
Figure 5 shows a dose response of outer
medullary suspensions to one of four levels of glucagon (0.1, 1, 10 or
100 nM) or vehicle. The cAMP production rate in outer medullary TAL
suspensions from the GSKO mice was reduced significantly (by 50% or
more) relative to the WT mice, regardless of whether they were exposed
to glucagon or vehicle alone.

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Fig. 5.
cAMP production in 50-µl aliquots of outer medullary suspensions
prepared from 6 WT mice (black bars) and 6 GSKO heterozygotes (striped
bars) (3 m /p+ and 3 m+/p GSKO mice were used with their
respective littermates). Samples were incubated for 5 min with the
agonist or vehicle in presence of the phosphodiesterase inhibitor, IBMX
(0.25 mM). Each reaction was terminated by the addition of
trichloroacetic acid. Aliquots were incubated with glucagon (0.1, 1, 10 or 100 nM) or vehicle. cAMP accumulation was significantly reduced in
the GSKO mice throughout the dose profile.
* P < 0.05.
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Decreased adenylyl cyclase abundance in the
TAL. Figure 6 shows
immunoblots of the outer medullary membranes probed with an antibody to
type VI adenylyl cyclase, the adenylyl cyclase isoform expressed in the
TAL (4). The m
/p+ group is shown in Fig. 6A, and the m+/p
group is shown
in Fig. 6B. The abundance of type VI
adenylyl cyclase was significantly reduced in the outer medullas from
both groups of GSKO mice relative to their WT littermates (Table 1).

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Fig. 6.
Immunoblots of total outer medullary membranes (200,000 g pellet) from WT and heterozygous
(GSKO) mice probed with anti-adenylyl cyclase (V, VI) antibody, 0.20 mg/ml. A: m /p+ mice.
B: m+/p mice. For both blots,
individual lanes were loaded with 10 µg protein from a different
animal. Preliminary 12% polyacrylamide gels were run in each case and
were stained with Coomassie blue to confirm equality of loading (not
shown). Expression of adenylyl cyclase (VI) was significantly reduced
in both m /p+ and m+/p knockouts (Table 1).
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Decreased urinary concentrating
capacity. A decrease in Na-K-2Cl cotransporter
abundance in the TAL would predict a reduction in countercurrent
multiplication by the loop of Henle and decreased medullary solute
concentrations. Therefore, one would predict a reduction in maximum
urinary osmolality achieved in response to acute stimulation by
vasopressin. To assess this possibility, six m+/p
GSKO mice and
six WT littermates were injected intramuscularly with DDAVP, a V2
receptor-selective vasopressin agonist. After 1 h, urine was collected
from all 12 mice for measurement of osmolality. Figure
7 is a plot of individual measurements
obtained from the urine of each mouse. Average urinary osmolality was
significantly lower in the GSKO mice relative to their WT littermates
(2,449 ± 211 vs. 3,399 ± 60 mosmol/kgH2O, respectively;
P
0.005).

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Fig. 7.
Final urine osmolality
(mosmol/kgH2O) 1 h after injection
of 1 ng of
1-deamino-[8-D-arginine]vasopressin
(DDAVP, im) to 6 m+/p Gs
knockout (GSKO) mice and 6 WT littermates. Each symbol represents a
different litter. All mice were in the basal (untreated) state before
injection of DDAVP. Urinary osmolality of the GSKO mice was
significantly lower than their WT littermates,
P < 0.01.
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Obliteration of effect on Na-K-2Cl cotransporter
expression by water restriction. Finally, to test
whether the impairment of outer medullary Na-K-2Cl cotransporter
expression in GSKO mice could be overcome by a chronic increase in
circulating vasopressin, five m
/p+ GSKO mice and five WT
littermates were thirsted for 48 h. Analysis of urine samples collected
just prior to death revealed that urinary osmolality was not
significantly different between the two groups of mice. Final urine
osmolalities for the WT mice and their GSKO littermates were 3,518 ± 82 and 3,465 ± 96 mosmol/kgH2O, respectively, as
previously reported (36). Immunoblots of outer medullary membrane
samples from these mice (Fig. 8) revealed
that after the water deprivation, Na-K-2Cl cotransporter abundance was
no longer lower in outer medullas of the GSKO mice than in
their WT littermates (band density for GSKO was 140 ± 58% of WT
littermates, mean ± SE; not significant).

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Fig. 8.
Immunoblot (probed with anti-Na-K-2Cl cotransporter antibody, 0.21 µg/ml) of outer medullary homogenates from 6 WT and 6 heterozygous
(m /p+) Gs -knockouts
(GSKO) after a 48-h water deprivation period. Each lane was loaded with
1 µg total protein from a different mouse. Each letter
(A-E)
designates a different litter. (Note: absence of observable 320-dimer
band is most likely due to the fact that these were whole homogenates
rather than membrane fractions, as were used in Fig. 2.) Preliminary
12% polyacrylamide gels were run and were stained with Coomassie blue
to confirm equality of loading (not shown). Expression of the Na-K-2Cl
cotransporter was not significantly different between the two groups.
|
|
Collecting Duct
Aquaporin-2 expression is reduced in renal cortex, but
not medulla. Because the urinary concentrating defect
we observed in the GSKO mice could be partly due to a defect in
collecting duct water and/or solute transport, we also assessed the
expression level of several important collecting duct transporters.
Figure 9A
shows an immunoblot of cortical membranes from m
/p+ GSKO mice and their WT littermates probed with anti-aquaporin-2 antibody demonstrating a decrease in aquaporin-2 expression in the GSKO mice. As
shown in Table 1, the expression level of aquaporin-2 protein was
significantly lower in cortical membranes from the GSKO mice
relative to their WT littermates in both groups (Table 1). When data were pooled from both groups, aquaporin-2 band density of
GSKO mice was 55 ± 6% of WT littermates (means ± SE, P < 0.05). Figure
9B shows an immunoblot of the outer
medullary membranes (m
/p+ vs. WT controls) probed with
anti-aquaporin-2 antibody. In contrast to the cortex, in the outer
medulla, no significant difference was observed between the GSKO mice
and their WT littermates when data were analyzed separately or when data from both groups were pooled (Fig.
9B, Table 1). Similarly, there was no
significant difference in aquaporin-2 expression in the inner medulla
between the GSKO mice and their WT littermates (data not shown).

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Fig. 9.
A: aquaporin-2 immunoblot of renal
cortical membranes (200,000 g pellet;
10 µg protein/lane) from 4 WT and 4 m /p+ heterozygous
Gs -knockout mice (GSKO).
B: aquaporin-2 immunoblot of renal
outer medullary membranes (200,000 g
pellet; 3 µg protein/lane) from 4 WT and 4 m /p+ heterozygous
Gs -knockout (GSKO) mice. Both
blots were probed with anti-aquaporin-2 antibody, 0.29 mg/ml. In each
case, preliminary 12% polyacrylamide gels were run and were stained
with Coomassie blue to confirm equality of loading (not
shown).
|
|
Aquaporin-3 expression modestly decreased while urea
transporter expression was unchanged. Aquaporin-3 is a
major basolateral water transporter in kidney collecting duct.
Interestingly, aquaporin-3 protein was significantly reduced in the
m
/p+ mice (40 ± 14% of WT mean; mean ± SE) outer medulla, but not in the m+/p
mice, or
when data were pooled. (see Table 1). Finally, immunoblotting of the
inner medulla revealed no significant differences in basal expression
levels of the vasopressin-regulated urea transporter (UTA1), the apical
urea transporter of the IMCD (Table 1). Thus the decreased urinary
concentrating capacity is not likely to be due to decreased urea
transporter abundance in the collecting duct.
No differences in IMCD cAMP
production. IMCD suspensions prepared from three
m
/p+ and three m+/p
mice were exposed to vehicle or one
of two doses of DDAVP (0.1 and 10 nM). Although cAMP production tended
to be lower in the GSKO mice at both doses, there were no significant
differences between the two groups (Fig.
10). Thus this is consistent with our
finding of no differences in aquaporin-2 protein expression (see Fig.
9, Table 1).

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Fig. 10.
cAMP production in 50-µl aliquots of inner medullary suspensions
prepared from 6 WT mice (solid bars) and 6 GSKO heterozygotes (hatched
bars) (3 m /p+ and 3 m+/p GSKO mice were used with their
respective littermates). Samples were incubated for 5 min with DDAVP
(0.1 or 10 nM) or vehicle in presence of the phosphodiesterase
inhibitor, IBMX (0.25 mM). Each reaction was terminated by the addition
of trichloroacetic acid. cAMP production was not significantly
different between the groups at any concentration.
|
|
No change in adenylyl cyclase abundance in the inner
medulla. Figure 11 shows immunoblots of the outer
medullary membranes probed with an antibody to type VI adenylyl
cyclase, the adenylyl cyclase isoform expressed in the collecting duct
(4). The m
/p+ group is shown in Fig.
11A,
and the m+/p
group is shown in Fig. 11B. The abundance of type VI adenylyl
cyclase in GSKO was not significantly different from the abundance in
their WT littermates.

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Fig. 11.
Immunoblots of inner medullary homogenates from WT and heterozygous
(GSKO) mice probed with anti-adenylyl cyclase (V, VI) antibody, 0.20 mg/ml. A: WT vs. GSKO (m /p+)
mice. B: WT vs. GSKO (m+/p )
mice. For both blots, individual lanes were loaded with 5 µg protein
from a different animal. Preliminary 12% polyacrylamide gels were run
in each case and were stained with Coomassie blue to confirm equality
of loading (not shown).
|
|
 |
DISCUSSION |
The knockout mice used in these studies had a heterozygous disruption
of Gnas, the gene coding for the
-subunit of Gs. The model was
previously characterized by Yu et al. (36). Homozygous knockout mice do
not develop in utero and are not born. To obtain heterozygotes,
heterozygous females were mated with WT males, yielding m
/p+
mice, and WT females were mated with heterozygous males yielding
m+/p
mice. Because of imprinting in various tissues, the two
groups of heterozygotes have distinct phenotypes. For example,
m
/p+ mice are born larger than their WT littermates, and
m+/p
mice are born smaller than their WT littermates.
Furthermore, as discussed in the introduction, proximal tubules from
the two groups of heterozygotes manifest different levels of
Gs
expression and different
rates of cAMP production in response to parathyroid hormone (36).
Therefore, it was important initially to evaluate the two groups
separately with regard to Gs
expression in the TAL. We found that expression of
Gs
protein (as assessed by
immunoblotting band density) was ~50% reduced in the outer medullas
obtained from heterozygotes relative their WT littermates, regardless
of whether m
/p+ or m+/p
heterozygotes were used (Table
1). Thus there is no imprinting of
Gnas in the TAL of these mice, whose cells make up the greatest proportion of cell volume in the inner stripe of the outer medulla (22). Lack of
Gnas imprinting has also been reported
for inner medulla, where collecting ducts are the predominant structure
(36).
The chief new finding in this study was that the abundance of the
Na-K-2Cl cotransporter was reduced in the TALs of both groups of
heterozygotes (band density for GSKO mice 48% of WT) in association with a significant reduction in acute urinary concentrating capacity and decreased cAMP production by TAL cells. The most direct explanation for the decreased expression of the Na-K-2Cl cotransporter in the GSKO
mice is that cotransporter expression is regulated by cAMP and that
cAMP levels in the TAL cells are reduced as a result of reduced
Gs
expression. An unexpected
finding from these studies was that adenylyl cyclase type VI abundance
was dramatically reduced in the outer medulla of the GSKO mice (Fig. 6,
Table 1). Thus it appears possible that the decline in cAMP production
was a consequence of the decrease in adenylyl cyclase expression rather than the decrease in Gs
expression per se.
However, other explanations should be considered in this relatively
complex animal model, i.e., the
Gs
heterozygote, where many
systems and cells are variably affected (36). First, a reduction in
expression of the
-subunit of
Gs protein would hypothetically result in an increase in the amount of free
-
subunits. Free
-
(like free GTP-bound
) has well defined effects on several components of classic signaling pathways, e.g., phospholipase C (
2
and
3) and adenylyl cyclase (subtypes II, IV, VII, I)
(13).
Second, it would be predicted that a fall in
Gs
expression in the proximal
tubule would increase salt and water absorption (by decreasing cAMP
production) in this segment (23). This would reduce flow of both salt
and water to downstream segments of the tubule, which conceivably could
affect expression of various transporters. Increased NaCl load to the
TAL has been shown to increase sodium reabsorption by the loop of Henle
(as assessed by micropuncture) (24), and we have shown that increased
NaCl in the drinking water will increase the expression of the Na-K-2Cl
cotransporter in the outer medulla (9), possibly due to flow effects.
With this model, we can get some estimate of the importance of altered proximal tubule fluid reabsorption in affecting expression of the
Na-K-2Cl cotransporter, because of the existence of
Gnas imprinting in the proximal tubule
(36). The m
/p+ GSKO mice express very low levels of
Gs
in the proximal tubule,
whereas m+/p
GSKO mice have normal expression levels. Therefore,
if the decrease in Na-K-2Cl cotransporter expression in the TAL were
due entirely to reduced salt and water flow from the proximal tubule,
then we would have expected a relatively greater decrease in Na-K-2Cl
cotransporter expression in the m
/p+ mice. Instead, we see no
difference in the relative decrease in Na-K-2Cl cotransporter
expression between the m
/p+ mice (47% of WT) and the
m+/p
mice (51% of WT) (Table 1). However, it is possible that
factors other than Gs
expression in the proximal tubule might affect salt delivery to the TAL.
Third, it is possible that factors, such as aberrations in hormonal
status, autacoids, or sympathetic nervous activity in the GSKO mice
might directly or indirectly influence TAL protein expression. To date,
we have no evidence to suspect that any of these factors as influence
expression of the Na-K-2Cl cotransporter.
Thus it appears that the most plausible explanation for the decrease in
the expression of the Na-K-2Cl cotransporter in the GSKO mice is
decreased production of cAMP secondary to decreased Gs
expression and/or the
associated decrease in adenylyl cyclase expression. Several G
protein-coupled receptors, for example, vasopressin, glucagon, and
parathyroid hormone, are linked to adenylyl cyclase activation in the
kidney TAL (28). Because the TAL cells in this model had a markedly
diminished ability to produce cAMP when stimulated in vitro with
glucagon (Fig. 5), we suggest that chronically depressed cAMP levels in
the TAL in these GSKO mice may be responsible for the reduction in
outer medullary Na-K-2Cl cotransporter expression. Consistent with this mechanism, Igarashi et. al. (17) have reported the existence of a CRE
in the 5'-flanking region of the Na-K-2Cl cotransporter gene.
This motif potentially could have an important role in regulation of
cotransporter abundance. Furthermore, we have recently reported studies
in rats showing that DDAVP infusion and restriction of water intake to
amounts matching respiratory losses (conditions which increase cellular
cAMP production via the V2 receptor) both strongly upregulate Na-K-2Cl
cotransporter abundance (20).
The effect of the Gs
knockout
on Na-K-2Cl cotransporter abundance appeared to be selective and not
due to general atrophy of the TAL because the expression levels of
other apical TAL proteins, such as NHE-3 and Tamm-Horsfall protein,
were not similarly reduced (Fig. 3). However, the expression level of
the
-subunit of Na-K-ATPase was markedly decreased in outer medullas
of GSKO mice, whereas the
1-subunit was much less profoundly
affected. TAL segments are thought to account for at least 90% of the
total Na-K-ATPase in the outer medulla. Therefore, measurements of
Na-K-ATPase subunit abundance in the outer medulla is tantamount to
measurement of the abundance in the TAL. Consequently, we conclude that
GSKO mice had reduced amounts of Na-K-ATPase in their TALs. The
activity of Na-K-ATPase has been reported to be increased acutely by
vasopressin in the TAL (5). However, we are unaware of studies
investigating the long-term effect of vasopressin on Na-K-ATPase
activity in the TAL. The mechanism by which a reduction in
Gs
expression leads to
decreased Na-K-ATPase
-subunit expression has not been defined by
the present studies. Na-K-ATPase activity has been reported to be
influenced by intracellular sodium concentration (31, 32). Conceivably,
the reduction in the
-subunit expression could be secondary to the
reduced expression of the Na-K-2Cl cotransporter and the resulting fall
in apical sodium entry.
An unexpected finding from these studies was that adenylyl cyclase type
VI abundance was dramatically reduced in the outer medulla of the GSKO
mice (Fig. 6 and Table 1). Adenylyl cyclase VI is expressed in both the
TALs and the collecting ducts of the outer medulla. Little is known
about long-term regulation of adenylyl cyclase abundance. Possibly, the
half-life of the type VI adenylyl cyclase is influenced by its state of
activation by Gs
or by concomitant changes in cellular signaling such as changes in cAMP level
or protein kinase A activation. The reduction in expression of adenylyl
cyclase in the GSKO mice might also be responsible, and certainly would
be expected to contribute, to the decrease in cAMP production measured
in the TAL suspensions (Fig. 5).
The expression of aquaporin-2 protein of the collecting duct would be
predicted to be decreased in the GSKO mice, because, like the Na-K-2Cl
cotransporter, the abundance of aquaporin-2 is thought to be regulated
through a CRE in the 5'-flanking region of the gene. As we
predicted, aquaporin-2 abundance was significantly reduced in the renal
cortex regardless of whether data were pooled (band density of GSKO on
average 55% of WT) or whether the two groups of heterozygotes were
analyzed separately (Table 1). However, in the inner and outer medulla,
aquaporin-2 protein expression was not reduced in either group of
heterozygotes or when data were pooled (Table 1). Consistent with the
lack of change in aquaporin-2 expression, we also observed no
significant differences in cAMP production by the IMCD suspensions from
the GSKO mice compared with their WT littermates. Therefore, it appears
that decreased Gs
expression
does not necessarily result in decreased cAMP production and thus
Gs
is not rate-limiting for
cAMP production in the IMCD. It is interesting that, unlike the TAL,
the IMCD did not show any decrease in adenylyl cyclase VI expression.
Thus it seems likely that the decrease in Na-K-2Cl cotransporter
expression in the outer medulla was critically dependent on the
observed fall in adenylyl cyclase VI abundance. These observations
point to the regulation of the abundance of adenylyl cyclase as a
possible control site for cAMP-mediated cell signaling and call for
additional studies to investigate the mechanisms involved.
Finally, in the basal (untreated state), the ability of the GSKO mice
to concentrate their urine after a short-term (1 h) DDAVP challenge was
significantly compromised (Fig. 7). This may be attributable to the
downregulation of the Na-K-2Cl cotransporter in the TAL, which would
substantially impair countercurrent multiplication. However, it is also
possible that decreased collecting duct water or solute permeability
could have contributed to the concentrating defect. Indeed, the
expression level of aquaporin-3 was significantly decreased in the
m
/p+ group in the outer medulla, although significant changes in
aquaporin-2 or UTA1 expression were not seen.
After a 48-h thirsting period, there were no differences in final urine
osmolality between the heterozygotes and their WT littermates.
Furthermore, after the 48-h thirst, the whole kidney expression of the
Na-K-2Cl cotransporter (Fig. 8) or aquaporin-2 (data not shown) was not
significantly different between the two groups. Presumably, prolonged
stimulation with high levels of vasopressin can overcome the limiting
effects of decreased Gs
expression on cotransporter expression. These findings therefore provide additional evidence for the importance of expression level of
the Na-K-2Cl cotransporter in the TAL of the outer medulla of rodents
as a determinant of urine concentrating capacity, although decreased
expression of aquaporin-2 protein in the cortical collecting duct and
aquaporin-3 in the outer medullary collecting duct might also play a role.
Human patients with heterozygous inactivating mutations of the
Gs
gene
(pseudohypoparathyroidism type Ia) did not manifest evidence for a
decrease in concentrating ability after overnight fluid restriction or
in response to exogenous vasopressin (29), suggesting that impairment
of countercurrent multiplication may be less severe in these human
patients than in mice with analogous mutations.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Paul Goldsmith for the
anti-Gs
antibody and for
assistance with the ELISA measurements, Dr. Brian Doctor for advice
with regard to preparation of tubule suspensions, and Dr. K. Spring for
careful reading of the manuscript.
 |
FOOTNOTES |
These studies were supported by the intramural research budgets of the
National Heart, Lung, and Blood Institute and the National Institute of
Diabetes and Digestive and Kidney Diseases.
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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. A. Knepper,
National Institutes of Health, 10 Center Dr., MSC 1603, Bldg. 10, Rm.
6N260, Bethesda, MD 20892-1603 (E-mail:
knep{at}helix.nih.gov).
Received 4 September 1998; accepted in final form 26 April 1999.
 |
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