Low aquaporin-2 levels in polyuric DI +/+ severe mice with
constitutively high cAMP-phosphodiesterase activity
Jørgen
Frøkiaer1,2,
David
Marples2,3,
Heinz
Valtin4,
John F.
Morris5,
Mark A.
Knepper6, and
Søren
Nielsen2
1 Department of Clinical
Physiology, Aarhus University Hospital and Institute of
Experimental Clinical Research,
2 Department of Cell Biology,
Institute of Anatomy, University of Aarhus, DK-8000 Aarhus,
Denmark; 3 Department of
Physiology, University of Leeds, LS2 9NQ Leeds, United Kingdom;
4 Department of Physiology,
Dartmouth Medical School, Hanover, New Hampshire 03755-3836;
5 Department of Anatomy,
University of Oxford, Oxford OX1 3QX, United Kingdom; and
6 Laboratory of Kidney and
Electrolyte Metabolism, National Heart, Lung, and Blood Institute,
National Institutes of Health, Bethesda, Maryland
20892
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ABSTRACT |
In the renal collecting duct, vasopressin acutely activates cAMP
production, resulting in trafficking of aquaporin-2 water channels
(AQP2) to the apical plasma membrane, thereby increasing water
permeability. This acute response is modulated by long-term changes in
AQP2 expression. Recently, a cAMP-responsive element has been
identified in the AQP2 gene, raising the possibility that changes in
cAMP levels may control AQP2 expression. To investigate this
possibility, we determined AQP2 protein levels in a strain of mice, DI
+/+ severe (DI), which have genetically high levels of
cAMP-phosphodiesterase activity, and hence low cellular cAMP levels,
and severe polyuria. Semiquantitative immunoblotting of membrane
fractions prepared from whole kidneys revealed that AQP2 levels in DI
mice were only 26 ± 7% (±SE) of those in control mice
(n = 10, P < 0.01). In addition,
semiquantitative Northern blotting revealed a significantly lower AQP2
mRNA expression in kidneys from DI mice compared with control mice (43 ± 6% vs. 100 ± 10%; n = 6 in
each group, P < 0.05). AQP3 levels
were also reduced. The mice were polyuric and urine osmolalities were
accordingly substantially lower in the DI mice than in controls (496 ± 53 vs. 1,696 ± 105 mosmol/kgH2O, respectively).
Moreover, there was a linear correlation between urine osmolalities and
AQP2 levels (P < 0.05).
Immunoelectron microscopy confirmed the markedly lower expression of
AQP2 in collecting duct principal cells in kidneys of DI mice and,
furthermore, demonstrated that AQP2 was almost completely absent from
the apical plasma membrane. Thus expression of AQP2 and AQP2
trafficking were severely impaired in DI mice. These
results are consistent with the view that in vivo regulation of AQP2
expression by vasopressin is mediated by cAMP.
collecting duct; nephrogenic diabetes insipidus; water balance; vasopressin
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INTRODUCTION |
COLLECTING DUCT WATER permeability has been shown to be
regulated both by short-term and long-term mechanisms (16, 19). Acutely, vasopressin acts via vasopressin
V2 receptors linked to adenylyl
cyclase, causing a rise in cytosolic cAMP. Ultimately, this cascade
results in the transfer of aquaporin-2 (AQP2) (10), the
vasopressin-regulated water channel of the collecting duct (3, 4, 15,
24, 27), from intracellular vesicles to the apical plasma membrane,
thereby increasing the water permeability of the collecting duct.
Phosphorylation of serine-256 of the AQP2 protein by cAMP-dependent
protein kinase (PKA) appears to be involved in AQP2 trafficking in
AQP2-transfected LLC-PK1 cells (9,
14), implying a direct role of AQP2 phosphorylation.
This acute response is modulated by long-term changes in AQP2
expression in response to the state of hydration, thought to be at
least partly mediated by vasopressin. Indeed, chronic vasopressin infusion in rats markedly increases AQP2 expression levels (4, 5, 31).
This finding raises the possibility that cAMP may increase the
transcription of the AQP2 gene as well as the vesicular trafficking of
AQP2. In support of this hypothesis, recent studies have identified a
putative cAMP responsive element (CRE) in the 5'-untranslated
region of the AQP2 gene (12). On the other hand, it has recently been
shown that water loading causes severe downregulation of AQP2
expression despite high circulating DDAVP levels maintained by
continuous subcutaneous infusion via osmotic minipumps ("vasopressin escape" phenomenon) (5). This finding supports earlier evidence for
the existence of vasopressin-independent mechanisms for the regulation
of AQP2 expression (20).
Downregulation of AQP2 has been shown to be associated with the
development of severe polyuria in multiple forms of acquired nephrogenic diabetes insipidus (8, 20, 21). To investigate the role of
cAMP in the control of AQP2 transcription we used a strain
of mice, called "DI +/+ severe" (DI), which have genetically high
levels of rolipram-sensitive cAMP-phosphodiesterase (cAMP-PDE; type IV)
activity, low levels of cytosolic cAMP levels, and which are unable to
raise the cytosolic cAMP in response to vasopressin (32). These mice
have extensive polyuria and low urine osmolality compared with the
control mice (1, 2, 11, 32).
It can be hypothesized that the polyuria of DI mice might be a
consequence of low AQP2 expression levels, or reduced trafficking of
AQP2 to the apical plasma membrane, or a combination of both. The
purpose of the present study was to examine the levels of AQP2
expression and its subcellular distribution in collecting duct
principal cells to evaluate these possibilities. This information will
allow us to evaluate whether cAMP plays a significant role in the
intracellular signaling pathways controlling AQP2 expression. Furthermore, in the context of the evidence for both
vasopressin-dependent and vasopressin-independent signals for
regulation of AQP2 expression, it remains a possibility that cAMP may
play a role in both pathways or even act as a final common pathway.
The results show that polyuric DI mice have low total AQP2 expression
and, moreover, low levels of AQP2 in the apical plasma membrane of
collecting duct principal cells. Since mice of this strain have
constitutively high levels of cAMP-PDE activity, and hence low cellular
cAMP levels, these results strongly support the view that cAMP is a
critical component in the long-term regulation of AQP2 and AQP3 gene
transcription as well as mediates the acute shuttling of AQP2 to and
from its active site, namely the apical plasma membrane. The very low
levels of AQP2 expression in DI mice, in the order of 10-15% in
severely polyuric male DI mice, raise the possibility that cAMP acts as
a common final pathway in controlling AQP2 expression.
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METHODS |
Experimental animals. Mice were kept
in the Animal Care Facility of the Anatomy Department, Oxford
University. Mice had free access to standard mouse diet and water. The
abbreviation "DI" was used to describe the DI +/+ severe mice.
For controls, normal mice were used and are described as
"controls."
Antibodies. The affinity-purified
antibody to AQP2 has previously been described (25). Briefly, peptides
corresponding to the 22 COOH-terminal amino acids of rat AQP2 were
synthesized with the addition of a cysteine residue at the carboxy
terminus. Peptides were conjugated to keyhole limpet hemocyanin and
used to immunize rabbits. Crude antisera were affinity purified using a
column on which 2 mg of the synthetic peptide was immobilized via a
sulfhydryl linkage to the
NH2-terminal cysteine
(Immunobilization kit no. 2; Pierce, Rockford, IL). Antibodies to AQP3
and AQP1 were prepared using the same procedures (6, 31), and both antibodies were affinity purified as previously described. All antibodies have been characterized for immunoblotting,
immunocytochemistry, and immunoelectron microscopy previously (4, 6,
25, 26, 31).
Membrane fractionation for
immunoblotting. The kidneys from male and female DI
mice and from male and female control mice were removed and frozen in
liquid nitrogen. After thawing on ice, the kidneys were minced finely,
and homogenized in dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH
7.2) with five strokes of a motor-driven Potter-Elvehjem homogenizer,
at 1,250 rpm. To increase yields, the pellet was rehomogenized with
three strokes, and the centrifugation was repeated. The supernatants
were pooled and centrifuged at 200,000 g for 1 h. The resultant pellet was resuspended in dissecting buffer, and assayed for protein concentration using the method of Lowry. Gel samples (in Laemmli sample buffer containing 2% SDS) were made from this membrane preparation.
Electrophoresis and immunoblotting.
Samples were loaded 10 µg/lane onto 12% polyacrylamide SDS-PAGE gels
and run on minigel systems. After transfer by electroelution to
nitrocellulose membranes, blots were blocked for 1 h with 5% skimmed
milk in PBS-T (80 mM Na2HPO4,
20 mM
NaH2PO4,
100 mM NaCl, and 0.1% Tween 20, pH 7.5), washed three times in PBS-T
over 25 min, and subsequently incubated with affinity-purified
anti-AQP2, anti-AQP3, or anti-AQP1 overnight. After a wash as above,
the blots were incubated at room temperature for 1 h with horseradish
peroxidase-conjugated secondary antibody (P448, diluted 1:3,000; Dako,
Glostrup, Denmark). After a final wash as above, antibody binding was
visualized using an enhanced chemiluminescence system (ECL; Amersham
International). Controls were made with exchange of primary antibody to
antibody preabsorbed with immunizing peptide (100 ng per 40 ng IgG), or
with preimmune serum (diluted 1:1,000). All controls were without
labeling (not shown). Identical gels were run in parallel and were
stained with Coomassie blue.
Semiquantitative Northern blot
analysis. Total RNA was prepared from inner medulla
using a RNeasy Mini Kit (Qiagen). Northern blot analysis was performed
using digoxigenin-labeled RNA probes [rat AQP2 and human
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. Total RNA was
separated on a 1% agarose, 2% formaldehyde gel followed by blotting
on a nylon membrane filter (Hybond-N; Amersham Life Science,
Buckinghamshire, UK). Prehybridization was performed at 55°C for 30 min in 5× SSC (with 50% formamide, 0.1% sarcosyl, and 0.02%
SDS) and 2% blocking solution (blocking reagent in maleic acid; Boehringer, Mannheim, Germany). After prehybridization, blots
were hybridized with a digoxigenin-labeled RNA probe at 55°C for 24 h. RNA probe labeling was performed by in vitro transcription (MAXIscript In Vitro Transcription kit, Ambien). After
hybridization, blots were washed in 2× SSC + 0.1% SDS at room
temperature for two 5-min periods followed by wash in 0.1× SSC + 0.1% SDS at 68°C for two 15-min periods. Blots were equilibrated
for 1 min in maleic acid containing 0.3% Tween 20 and blocked for 30 min. After incubation for 30 min with anti-digoxigenin-AP conjugate,
blots were washed for two 15-min periods in maleic acid containing
0.3% Tween 20 and equilibrated for 5 min in 0.1 M
Tris · HCl + 0.1 M NaCl. The bands were visualized
using a chemiluminescent substrate, CSPD (Boehringer).
Quantitation of AQP2, AQP1, and AQP3
expression. ECL films were scanned using a Umax
Vista-S8 scanner and Corel Photopaint Software, and both the 29-kDa and
the 35- to 50-kDa band (corresponding to nonglycosylated and
glycosylated AQP2; Ref. 30) were scanned. For AQP3, the
affinity-purified anti-AQP3 recognized both nonglycosylated and
glycosylated AQP3, corresponding to the 27-kDa and the 33- to 40-kDa
bands, respectively. Both bands were scanned as described previously (6). Similarly, for AQP1 both the 29-kDa and the 35- to
50-kDa bands were scanned. The labeling density was quantitated using
specially written software (available upon request). Bands from gels
made with serial dilutions of protein from inner medulla, processed as
above, were found to be linear over a wide range (20). For quantitation
of AQP2 expression, ECL exposures were chosen that gave bands in the
control samples that were close to the top of the linear range. Equal
loading among lanes was confirmed by scanning and densitometry of
identical SDS-PAGE gels run in parallel to the gels for immunoblots and
stained with Coomassie blue.
Densitometry of AQP2 mRNA levels. The
band of ~1.6 kb corresponding to AQP2 mRNA was scanned. Values were
corrected by densitometry of GAPDH visualized using a
digoxigenin-labeled probe for human GAPDH on separate identical blots.
Preparation of tissue for
immunocytochemistry. Immunocytochemistry was performed
as previously described (20). Kidneys were perfusion fixed with 4%
paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. The kidneys
were postfixed for 2 days in 2% paraformaldehyde. Tissue blocks were
cut from the kidney inner medulla (close to the junction between inner
and outer medulla) and infiltrated for 30 min with 2.3 M sucrose + 2%
paraformaldehyde. Blocks were subsequently mounted on holders and
rapidly frozen in liquid nitrogen. The blocks were used either directly
for cryosectioning or for cryosubstitution and Lowicryl HM20 plastic
resin embedding.
Semithin (0.85 µm) cryosections cut on a Reichert Ultracut FCS were
preincubated with PBS containing 1% skimmed milk and 0.05 M glycine,
then incubated with affinity-purified antibodies against AQP2
(100-600 ng IgG/ml) in PBS containing 1% skimmed milk and 0.3%
Triton X-100. The labeling was visualized as previously described (20,
21) using horseradish peroxidase-conjugated secondary antibody (P448,
1:100, Dako), and sections were counterstained with Meier counter- stain.
The frozen samples were freeze substituted (28) in a Reichert AFS
(Reichert, Vienna, Austria). Samples were sequentially equilibrated
over 3 days in 0.5% uranyl acetate in methanol at temperatures
gradually increasing from
80°C to
70°C, and then rinsed in pure methanol for 24 h at
70°C to
45°C
and infiltrated at
45°C with Lowicryl HM20 and methanol at
1:1, then 2:1, and finally pure HM20 (1 day in each
solution) before ultraviolet polymerization in pure HM20 for 2 days at
45°C and 2 days at 0°C.
Ultrathin sections (Reichert Ultracut FCS) placed on nickel grids (with
or without Formvar coating) were quickly dipped in absolute ethanol
containing saturated NaOH, washed extensively in water, and then
incubated in TBST (0.05 M Tris, 0.1% Triton X-100, 0.1% sodium
borohydride, and 50 mM glycine) for 10 min and in TBST solution
containing 2% BSA for 10 min. The grids were then incubated with
affinity-purified anti-AQP2, and the labeling was visualized using
goat-anti-rabbit-gold (GAR 10, 10-nm colloidal gold particles; BioCell
Research Laboratories, Cardiff, UK), and grids were stained with uranyl
acetate. Immunolabeling controls were performed using nonimmune IgG and
revealed absence of labeling. Semiquantitation of the immunogold
labeling was performed on kidneys from two controls and two DI
mice. Ultrathin sections were prepared from
cryosubstituted and Lowicryl HM20-embedded tissue. Sections were
obtained from proximal parts of the kidney inner medulla. The sections
were labeled with affinity-purified anti-AQP2 in parallel, and very low
antibody concentrations were used to assure extremely low background
labeling (this is essential due to the very low labeling density in the
DI mice). Electron micrographs were obtained at a primary
magnification of ×15,500 or ×21,000, and the gold particle
distribution was determined. Light microscopy was performed using a
Leica Laborlux S microscope, and electron microscopy was performed
using Philips CM100 and Philips EM208 electron microscopes.
Statistics. For densitometry of
immunoblots, samples from kidneys of DI mice of each gender were run
together with samples from the corresponding controls. AQP2, AQP1, and
AQP3 labeling in the samples from the DI mice was calculated as a
fraction of the mean value of the samples from the controls for that
gel. Parallel Coomassie-stained gels were subjected to densitometry and
used for correction of potential minor differences in loading. Values
are presented in the text as means ± SE. Comparisons between groups
were made by unpaired t-tests.
P < 0.05 was considered significant.
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RESULTS |
Reduced expression of AQP2 and AQP2 mRNA levels in
male DI mouse kidneys. To test whether DI mice manifest
a decrease in AQP2 expression, semiquantitative immunoblotting of
kidney membranes was performed using anti-AQP2 antibodies.
Immunoblotting using membrane fractions prepared from total mouse
kidney revealed that the affinity-purified anti-AQP2 antibody
exclusively recognized 29-kDa and 35- to 50-kDa bands (Fig.
1), corresponding to nonglycosylated and
glycosylated forms of AQP2, respectively (30). The AQP2 bands were
ablated by use of antibody previously absorbed with immunizing peptide
(not shown), consistent with previous evidence in rat tissue (8, 20,
21).

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Fig. 1.
A: representative immunoblot comparing
aquaporin-2 (AQP2) expression in kidneys from 5 male DI +/+ severe mice
(DI-Male) and from 5 male control mice (control). Membranes were
prepared from total kidneys and loaded at 10 µg/lane. Antibody labels
29-kDa and 35- to 50-kDa bands corresponding, respectively, to
nonglycosylated and glycosylated forms of AQP2 (see
METHODS). Also shown are
densitometry of AQP2 expression (B)
and urine osmolality (C) from male
DI mice (n = 5) and male control mice
(control, n = 9). Values were obtained
by densitometry of immunoblots, and expression is presented as fraction
of expression in controls (control).
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Immunoblotting of membrane fractions prepared from kidneys of male DI
mice and control mice revealed markedly lower AQP2 expression levels in
DI male mice (Fig. 1A). Both AQP2
bands (29 and 35-50 kDa) were decreased. Densitometry (Fig.
1B) revealed that in male DI mice
(n = 5) AQP2 expression levels were
reduced to 13 ± 4% of that in male controls (100 ± 17%,
n = 5). Consistent with this, male DI
mice were severely polyuric, and consequently, the urine osmolalities
were low, averaging 347 ± 42 compared with 1,740 ± 80 mosmol/kgH2O in urine from control
mice (Fig. 1C). Thus there is a
marked downregulation of AQP2 expression in kidneys of polyuric male DI mice.
Total RNA purified from kidney inner medulla of male DI mice and male
control mice was analyzed by semiquantitative Northern blotting. As
shown in Fig. 2, a band of ~1.6 kb was
detected, consistent with the predicted size of AQP2 mRNA (10).
Compared with control animals, semiquantitative Northern blotting
revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice, i.e., 43 ± 6% (n = 6) vs.
100 ± 10% (n = 6;
P < 0.05).

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Fig. 2.
A: Northern blot showing AQP2 mRNA in
whole kidney from control mice and male DI mice. Digoxigenin-labeled
human AQP2 mRNA probe labels a band of 1.6 kb consistent with the
predicted size of AQP2 mRNA (10). B: a
parallel blot identical to the blot shown in
A was hybridized with a cDNA probe of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control for
loading of kidney mRNA levels. C:
semiquantitative densitometric analysis revealed a significantly lower
AQP2 mRNA expression in kidneys from DI male mice compared with control
mice of 43 ± 6% vs. 100 ± 10%.
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Differential AQP2 protein expression levels in male
vs. female DI mice. Although both male and female DI
mice are known to be polyuric (2, 11), previous studies have indicated
a significant difference in the concentrating ability of males and
females, with males displaying a more severe polyuria (32). To examine whether this difference correlates with changes in AQP2 expression, a
differential analysis of AQP2 levels in male and female DI mice was
undertaken. As shown in Fig.
3A, the
decrease in AQP2 levels in females relative to female control mice was
not as extensive as seen in males. The AQP2 expression in female DI
mice was 39 ± 12% (n = 5, Fig. 3A) of that of female
controls (100 ± 17%, n = 4). In males (n = 5), AQP2
expression was reduced to 13 ± 4% of levels in male controls (Fig.
1, A and
B). Combining data from females and
males confirmed a significant decrease both in AQP2 expression and
urine osmolality in DI mice (Fig. 4). A
direct comparison of the expression levels between male and female DI mice confirmed a significantly lower AQP2 expression in male DI mice
(Fig.
5A).
Densitometry (Fig. 5B) of the
immunoblot revealed that AQP2 levels in kidneys of male DI mice
constituted only 36 ± 11% of the levels in female DI mice (100 ± 13%). Consistent with the lower AQP2 expression in male compared
with female DI mice, urine osmolalities were significantly lower in
male compared with female DI mice (347 ± 42 vs. 615 ± 31 mosmol/kgH2O in males and females,
respectively) (Figs. 1C and
3B).

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Fig. 3.
Densitometry of AQP2 expression (A)
and urine osmolality (B) from female
DI mice (n = 5) and female control
mice (control, n = 4). Values are
obtained by densitometry of immunoblots, and expression is presented as
fraction of expression in controls (control).
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Fig. 4.
Densitometry of AQP2 expression (A)
and urine osmolality (B) from DI
mice (n = 10) and control mice
(control, n = 9). Both genders are
included. Values are obtained by densitometry of immunoblots, and
expression is presented as fraction of expression in controls.
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Fig. 5.
A: immunoblot comparing AQP2
expression in kidneys from 5 male DI mice (DI-Male) and 5 female DI
mice (DI-Female). Membranes were prepared from total kidneys and loaded
at 10 µg/lane. Antibody labels 29-kDa and 35- to 50-kDa bands
corresponding, respectively, to nonglycosylated and glycosylated forms
of AQP2. B: densitometry of
AQP2 expression. Values are obtained by densitometry of immunoblot, and
expression is presented as fraction of expression in controls. In
males, AQP2 expression is 36 ± 11% of expression in female (100 ± 13%) DI mice.
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Urine osmolality correlates with AQP2 protein
expression in DI mice. As demonstrated in Figs.
1C and
3B, the urine osmolality is markedly
lower in both male and female DI mice compared with control mice. In
Fig. 6, the relationship between urine
osmolality and relative AQP2 expression in both male and female DI as
well as male and female control mice is depicted. The analysis revealed a significant correlation
(R2 = 0.6). Thus
the results are consistent with the view that low AQP2 levels and hence
low levels in the apical plasma membrane (see below) are a significant
factor determining the high urine output and low urine osmolality
observed in DI mice.

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Fig. 6.
Urine osmolality in DI (n = 10) and
control (n = 9) mice plotted as
function of AQP2 expression. Both genders are included. Linear
regression analysis reveals a significant correlation
(R2 = 0.6). This
increase in AQP2 expression and increase in urine osmolality is similar
to the parallel increase in AQP2 and urine osmolality previously shown
in Brattleboro rats in response to long-term (5 days) vasopressin
treatment. Long-term treatment with vasopressin results in a 3- to 4-fold increase in AQP2 expression and a 4- to 5-fold increase
in urine osmolality (to ~1,100
mosmol/kgH2O) (4).
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Immunocytochemistry and immunoelectron microscopical
analysis of AQP2 distribution. cAMP plays a pivotal
role in the acute hydrosmotic response to vasopressin by inducing the
transfer of AQP2 from intracellular vesicles to its active site in the
apical plasma membrane (22, 25, 29, 35). It would be expected that this
process might be disturbed in the kidneys of the DI mice having low
cytosolic cAMP levels. To investigate whether there are major
differences in the subcellular distribution of AQP2 in the kidney
collecting duct of DI mice, immunocytochemistry and immunoelectron
microscopy were performed. Immunocytochemistry (Fig.
7) confirmed the markedly lower AQP2
expression in the inner medullary collecting ducts of kidneys from DI
mice (Fig. 7B), compared with that
in control mice (Fig. 7A). The
sparse labeling in DI mice was primarily intracellular. Immunolabeling
controls revealed absence of labeling (Fig.
7C). Immunoelectron microscopy was
used to analyze the subcellular distribution of AQP2 in kidneys from
control mice (Fig. 8) and DI mice (Fig.
9). Qualitative analysis of multiple inner
medullary collecting duct cells in kidneys of several DI mice uniformly
revealed a significant reduction in overall labeling (confirming the
light microscopical observations). In addition, a very marked reduction
in immunogold labeling of the apical plasma membrane of collecting duct
principal cells was observed (Fig. 9). This was consistently seen in
all collecting duct principal cells in the different DI mice. The
remaining labeling (albeit markedly reduced in abundance) was present
in vesicles (Fig. 9). In contrast, significant AQP2 labeling was
consistently found in the apical plasma membrane of collecting duct
principal cells in kidneys from control mice (Fig. 8). Semiquantitation of the immunogold labeling (703 gold particles) revealed that in
controls ~43% of the labeling was associated with the apical plasma
membrane compared with 4% in the DI mice. Importantly, the AQP2
labeling density was extremely low in DI mice compared with the
controls. Thus there was a marked reduction in labeling of the apical
plasma membrane (being the "active" site of AQP2 to allow water
to be reabsorbed from the collecting duct lumen) in DI mice. The
immunocytochemistry and immunoelectron microscopy confirmed the low
AQP2 protein levels and demonstrated that there was indeed impaired
delivery of AQP2 to the apical plasma membrane in DI mice. This
supports the view that both the short- and long-term regulation of AQP2
are severely disturbed in DI mice with low cytosolic levels of cAMP in
kidney collecting duct cells.

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Fig. 7.
Immunocytochemical localization of AQP2 in kidney inner medulla.
A: kidney from a control mouse (male).
Collecting duct principal cells are heavily labeled (arrows), whereas
intercalated cells (arrowhead), thin limbs, and vascular structures are
unlabeled. Within collecting duct principal cells, AQP2 labeling is
localized to the apical plasma membrane domains and subapical
intracellular vesicles. B: in male DI
mice, AQP2 labeling is very sparse. Labeling is localized in the
subapical region. C: immunolabeling
controls using nonimmune IgG reveal no labeling. Magnification,
×1,000.
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Fig. 8.
Immunoelectron microscopical localization of AQP2 in kidney inner
medullary collecting duct principal cells from male control mouse.
Abundant AQP2 labeling of the apical plasma membrane (arrows) and
intracellular vesicles (arrowheads) is seen.
Inset: immunolabeling controls using
nonimmune IgG reveal no labeling. Magnification, ×71,000.
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Fig. 9.
Immunoelectron microscopical localization of AQP2 in kidney inner
medullary collecting duct principal cells from male DI mouse. Only
sparse AQP2 labeling of intracellular vesicles (arrowheads) is seen,
with virtually no labeling of the apical plasma membrane.
Inset: immunolabeling controls using
nonimmune IgG reveal no labeling. Magnification, ×71,000.
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Dysregulation of other water channels.
Several transporters have been shown to be regulated by vasopressin via
V2 receptors and, hence, cAMP. It
has been shown that AQP3 expression can be induced by continuous
infusion of vasopressin via osmotic minipumps, whereas the expression
of the proximal tubule/descending thin limb water channel AQP1 remains
unchanged (31). The expression pattern of AQP1 and AQP3 was therefore
examined. As demonstrated in Fig. 10,
there was no change in AQP1 expression in immunoblots of membrane
fractions from kidneys of male DI mice compared with male controls
(Fig. 10A). In contrast, there was a
lower AQP3 expression in kidneys of male DI mice compared with controls
(Fig. 10B). Immunolabeling controls
using peptide absorption of the affinity-purified anti-AQP3 antibody
revealed a complete absence of labeling (not shown). Densitometry
revealed that AQP3 expression was 26 ± 7% of levels in kidneys
from control mice (100 ± 16%; Fig.
10C). Thus, in addition to the
marked downregulation of the apical collecting duct water channel AQP2,
there is also a significant reduction in the basolateral collecting
duct water channel AQP3. These results strongly suggest that the
expression of both channels is regulated by cAMP and that dysregulation
of both channels may play a critical role in the severe polyuria and
concentrating defect associated with the increased cAMP-PDE activity in
this strain.

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Fig. 10.
A: immunoblot comparing AQP1
expression in kidneys from 5 male DI mice (DI-Male) and from 5 male
control mice. Membranes were prepared from total kidneys and loaded at
~10 µg/lane. B: immunoblot
comparing AQP3 expression in kidneys from 5 male DI mice (DI-Male) and
from 5 male control mice. Membranes were prepared from total kidneys
and loaded at ~10 µg/lane. A prominent band was observed at
~26-28 kDa, and higher molecular weight bands were also seen,
which are likely to represent glycosylated and/or multimeric
AQP3. C: densitometry of AQP3
expression in male DI mice (n = 5) and
control mice (n = 5). Values are
obtained by densitometry of immunoblots, and expression is presented as
fraction of expression in controls.
|
|
 |
DISCUSSION |
It has previously been demonstrated that the renal collecting ducts of
DI mice have markedly decreased intracellular levels of cAMP and a
failure to increase intracellular cAMP in response to vasopressin (1,
2, 11). The present study demonstrated that these mice manifest a
marked suppression of AQP2 protein abundance and AQP2 mRNA levels. The
low AQP2 protein levels correlated closely with low urinary osmolality
in these mice. Furthermore, AQP2 levels were lower in male than in
female DI mice in concordance with the lower urine osmolalities in male
compared with female DI animals. These results support the view that
cAMP plays an important role in the regulation of AQP2 expression. The
very low AQP2 protein and AQP2 mRNA levels, especially in male DI mice, strongly support the view that cAMP may represent a final common pathway for modulating AQP2 expression, presumably through regulation of gene transcription. Furthermore, immunoelectron microscopy revealed
low labeling of the apical plasma membrane of collecting duct principal
cells in kidneys of DI mice, providing direct evidence that delivery of
AQP2 to the apical plasma membrane is also severely impaired in the DI
mice. In addition, the expression level of AQP3, the basolateral water
channel of the collecting duct prin-cipal cell, was decreased in
the DI mice. Thus the present study provides evidence that
intracellular cAMP levels regulate
1) AQP2 trafficking, 2) AQP2 protein and AQP2 mRNA
expression, and 3) AQP3 expression.
Reduced expression of AQP2 protein and AQP2 mRNA in DI
mice. As determined both by semiquantitative
immunoblotting and by immunocytochemistry, the expression of AQP2
protein was significantly reduced in kidneys from DI mice compared with
control mice. In addition, semiquantitative Northern blotting revealed
a significantly lower AQP2 mRNA expression in kidneys from DI mice
compared with control mice. These findings are consistent with the fact
that the DI mice have high nucleotide PDE activity and consequently low
levels of cytosolic cAMP (2, 11). Thus the finding of low AQP2 levels
in the kidneys of DI mice suggests that cAMP is important for
regulation of AQP2 expression, most likely via cAMP-dependent regulation of AQP2 gene transcription. This possibility is discussed in
more detail below. The mechanisms responsible for the difference in
AQP2 expression and urine production between male and female DI mice
remain unknown. It may be speculated that the defects in these mice may
render them more sensitive to a potential effect of sex hormones.
Although a potential effect of such hormones on the regulation of AQP2
and urine production has not been directly examined, a recent study
indicated that there was a difference in urine excretion of AQP2
between male and female individuals (7), raising this as a possibility.
Involvement of PDEs in vasopressin regulation of water
transport. Vasopressin binds to
V2 receptors present in the
basolateral membrane of collecting duct principal cells. Acting through
the GTP-binding protein Gs, the
interaction of vasopressin with the V2 receptor activates adenylyl
cyclase, which accelerates the production of cAMP from ATP (16).
Subsequently, cAMP binds to the regulatory subunit of PKA, resulting in
dissociation of the regulatory subunit from the catalytic subunit. This
activates the catalytic subunit, which phosphorylates various proteins
including AQP2. AQP2 is then translocated from intracellular vesicles
to the apical plasma membrane, thereby increasing the water
permeability of the apical plasma membrane. cAMP-dependent PDE
inactivates cAMP by hydrolyzing it to 5'-AMP, to be recycled into
a cellular pool of adenylyl nucleotides. In a series of studies, Dousa
and Valtin (2, 11, 13) and their associates analyzed the role of PDEs
in DI mice, which are completely unresponsive to high levels of
arginine vasopressin (AVP) to increase cytosolic levels of cAMP. When measured in microdissected segments of outer medullary (13) and inner medullary (11) collecting duct, the cAMP-PDE activity
was significantly higher in DI mice than in normal mice. Rolipram, a
selective inhibitor of the isozyme PDE IV, fully restored the
AVP-stimulated cAMP accumulation (11). In addition, the authors
demonstrated a parallel increase in the density of intramembranous particle clusters (thought to be an index of water channels present in
the plasma membrane) in freeze-fracture replicas of the apical plasma
membrane and demonstrated that the antidiuretic response to AVP was
fully restored (2). The activity of PDE IV was found to be fourfold
higher in the DI mice (2, 33). The use of cilostamide, a selective
inhibitor of the isozyme PDE III, suggested a role also for PDE III in
the defect (11). The critical role of PDEs was further substantiated in
subsequent studies using renal cells in vitro by Dousa and colleagues
(17, 33, 34).
In the DI mouse, both the activation of adenylyl cyclase and the
content of cAMP substrate ATP are normal, but there is a fourfold
increase in PDE types III and IV, and hence low baseline levels of
cAMP, and a lack of any rise in cAMP levels in response to vasopressin
even at high concentrations (11). Our results demonstrating low AQP2
expression in DI mice strongly suggest that cAMP plays a pivotal role
in AQP2 gene transcription. Recently, it was demonstrated that a CRE is
present in the 5'-untranslated flanking region of the AQP2 gene,
suggesting that changes in intracellular levels of cAMP may represent
an important pathway regulating AQP2 expression (12, 23, 36). Thus the
lower expression of AQP2 in DI mice is likely to reflect an impaired
synthesis of AQP2, although other mechanisms cannot be ruled out. For
example, it appears possible that the absence of, or reduction in,
phosphorylation of AQP2 may also induce trafficking of AQP2 into a
degradative pathway.
Role of cAMP in AQP2 trafficking.
Immunocytochemistry revealed a marked reduction in AQP2 labeling in
collecting ducts from kidneys of DI mice compared with kidneys from
control animals. This is consistent with the marked downregulation of
AQP2 determined by semiquantitative immunoblotting and is consistent
with the parallel decrease in urine osmolality (Figs.
1C and
3B). Moreover, immunocytochemistry
and immunoelectron microscopy demonstrated markedly reduced AQP2
labeling of the apical plasma membrane (as well as intracellular
vesicles) consistent with impaired targeting and delivery of
AQP2 to the plasma membrane in response to vasopressin and with the
observed increase in urine production and reduced urine
osmolality. This strongly suggests that both AQP2
expression levels and AQP2 targeting (already well established from
previous investigations) to the apical plasma membrane are critically
dependent on intracellular cAMP levels. This is consistent with recent
studies concerning the role of cAMP in PKA-mediated phosphorylation of AQP2 itself for trafficking. It was shown that the serine residue at
position 256 (S256) in the cytoplasmic COOH terminus of AQP2 is the
consensus site for PKA phosphorylation (9, 18). By introducing a
serine-to-alanine mutation into AQP2 by PCR-directed mutagenesis in
LLC-PK1 cells, it was recently
shown that PKA failed to phosphorylate AQP2 and prevented vasopressin-
or forskolin-stimulated translocation of AQP2 from the intracellular
site to the cell surface (9, 14). This indicates that
vasopressin-induced exocytosis of AQP2 is dependent on the presence of
the S256 PKA phosphorylation site (9). The present studies extend this
by demonstrating in vivo that kidneys with low cAMP levels in the collecting ducts (kidneys of DI mice) have a marked
reduction in AQP2 in the apical plasma membrane in vivo.
Reduced AQP3 expression in kidneys of DI
mice. Semiquantitative immunoblotting also revealed a
decrease in AQP3 protein expression in kidneys of male DI mice to
one-third of control levels (Fig. 10). Thus there was a decrease in the
two collecting duct principal cell water channels AQP2 and AQP3. The
results indicate that intracellular cAMP is likely to be an important
factor in the regulation of AQP3 expression and raise the possibility
that AQP3 gene transcription may also be, at least in part, dependent
on cytosolic levels of cAMP. However, a CRE element has not been
demonstrated for AQP3. Thus it is at present unclear whether a
potential role of cAMP in AQP3 expression is a primary or a secondary
effect. The parallel decrease of both AQP2 and AQP3 protein levels is
consistent with previous studies showing that long-term treatment of
rats with exogenous vasopressin in osmotic minipumps is associated with increased AQP3 as well as AQP2 expression, indicating that the expression of both channels can be regulated by vasopressin (31). Furthermore, prolonged dehydration is associated with a parallel increase in both AQP2 and AQP3 (6). In contrast AQP1 expression levels
are not changed by vasopressin treatment or dehydration (31). The lack
of altered AQP1 expression in DI mice compared with controls is
consistent with this.
Ecelbarger et al. (5) recently used a model where rats were implanted
with osmotic minipumps administrating DDAVP, and the rats were
subsequently water loaded for several days. In this model, there was a
parallel increase in both AQP2 and AQP3 protein expression in response
to DDAVP treatment alone prior to water loading (this is consistent
with the results in the present study showing a lower expression of
both AQP2 and AQP3 in DI mice). But after initiating water loading in
the continued presence of DDAVP treatment, a marked downregulation of
AQP2 but not of AQP3 was observed (5). This indicates that during
vasopressin escape, which presumably takes place via a
vasopressin-independent mechanism, the regulation of AQP2 and AQP3
differs. Although further studies are warranted to clarify this, it is
possible that cAMP is important for regulation of both AQP2 and AQP3
expression, whereas the induced downregulation of AQP2 alone during
vasopressin escape may be independent of cAMP.
 |
ACKNOWLEDGEMENTS |
We thank Mette Vistisen, Gitte Christensen, Annette Blak Rasmussen,
and Zhila Nikrozi for expert technical assistance.
 |
FOOTNOTES |
Support for this study was provided by the Karen Elise Jensen
Foundation, Novo Nordisk Foundation, Danish Medical
Research Council, University of Aarhus Research Foundation, the
University of Aarhus, the University of Leeds, and the intramural
budget of the National Heart, Lung, and Blood Institute, National
Institutes of Health.
J. Frøkiaer, D. Marples, and S. Nielsen contributed equally to
this study.
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: S. Nielsen, Dept. of Cell Biology,
Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus, Denmark.
Received 5 March 1998; accepted in final form 24 September 1998.
 |
REFERENCES |
1.
Brown, D.,
G. I. Shields,
H. Valtin,
J. F. Morris,
and
L. Orci.
Lack of intramembranous particle clusters in collecting ducts of mice with nephrogenic diabetes insipidus.
Am. J. Physiol.
249 (Renal Fluid Electrolyte Physiol. 18):
F582-F589,
1985[Medline].
2.
Coffey, A. K.,
D. J. O'Sullivan,
S. Homma,
T. P. Dousa,
and
H. Valtin.
Induction of intramembranous particle clusters in mice with nephrogenic diabetes insipidus.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F640-F646,
1991[Abstract/Free Full Text].
3.
Deen, P. M.,
M. A. Verdijk,
N. V. Knoers,
B. Wieringa,
L. A. Monnens,
C. H. van Os,
and
B. A. van Oost.
Requirement of human renal water channel aquaporin-2 for vasopressin-dependent concentration of urine.
Science
264:
92-95,
1994[Medline].
4.
DiGiovanni, S. R.,
S. Nielsen,
E. I. Christensen,
and
M. A. Knepper.
Regulation of collecting duct water channel expression by vasopressin in Brattleboro rat.
Proc. Natl. Acad. Sci. USA
91:
8984-8988,
1994[Abstract].
5.
Ecelbarger, C. A.,
S. Nielsen,
B. R. Olson,
T. Murase,
E. A. Baker,
M. A. Knepper,
and
J. G. Verbalis.
Role of renal aquaporins in escape from vasopressin-induced antidiuresis in rat.
J. Clin. Invest.
99:
1852-1863,
1997[Abstract/Free Full Text].
6.
Ecelbarger, C. A.,
J. Terris,
G. Frindt,
M. Echevarria,
D. Marples,
S. Nielsen,
and
M. A. Knepper.
Aquaporin-3 water channel localization and regulation in rat kidney.
Am. J. Physiol.
269 (Renal Fluid Electrolyte Physiol. 38):
F663-F672,
1995[Abstract/Free Full Text].
7.
Elliot, S.,
P. Goldsmith,
M. A. Knepper,
M. Haughey,
and
B. M. Olson.
Urinary excretion of aquaporin-2 in humans: a potential marker of collecting duct responsiveness to vasopressin.
J. Am. Soc. Nephrol.
7:
403-409,
1996[Abstract].
8.
Frøkiaer, J.,
D. Marples,
M. A. Knepper,
and
S. Nielsen.
Bilateral ureteral obstruction downregulates expression of vasopressin-sensitive AQP-2 water channel in rat kidney.
Am. J. Physiol.
270 (Renal Fluid Electrolyte Physiol. 39):
F657-F668,
1996[Abstract/Free Full Text].
9.
Fushimi, K.,
S. Sasaki,
and
F. Marumo.
Phosphorylation of serine 256 is required for cAMP-dependent regulatory exocytosis of the aquaporin-2 water channel.
J. Biol. Chem.
272:
14800-14804,
1997[Abstract/Free Full Text].
10.
Fushimi, K.,
S. Uchida,
Y. Hara,
Y. Hirata,
F. Marumo,
and
S. Sasaki.
Cloning and expression of apical membrane water channel of rat kidney collecting tubule.
Nature
361:
549-552,
1993[Medline].
11.
Homma, S.,
S. M. Gapstur,
A. Coffey,
H. Valtin,
and
T. P. Dousa.
Role of cAMP-phosphodiesterase isoenzymes in pathogenesis of murine nephrogenic diabetes insipidus.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F345-F353,
1991[Abstract/Free Full Text].
12.
Hozawa, S.,
E. J. Holtzman,
and
D. A. Ausiello.
cAMP motifs regulating transcription in the aquaporin 2 gene.
Am. J. Physiol.
270 (Cell Physiol. 39):
C1695-C1702,
1996[Abstract/Free Full Text].
13.
Jackson, B. A.,
R. M. Edwards,
H. Valtin,
and
T. P. Dousa.
Cellular action of vasopressin in medullary tubules of mice with hereditary nephrogenic diabetes insipidus.
J. Clin. Invest.
66:
110-122,
1980[Medline].
14.
Katsura, T.,
C. E. Gustafson,
D. A. Ausiello,
and
D. Brown.
Protein kinase A phosphorylation is involved in regulated exocytosis of aquaporin-2 in transfected LLC-PK1 cells.
Am. J. Physiol.
272 (Renal Physiol. 41):
F816-F822,
1997.
15.
Knepper, M. A.
Molecular physiology of urinary concentrating mechanism: regulation of aquaporin water channels by vasopressin.
Am. J. Physiol.
272 (Renal Physiol. 41):
F3-F12,
1997[Abstract/Free Full Text].
16.
Knepper, M. A.,
S. Nielsen,
C. L. Chou,
and
S. R. DiGiovanni.
Mechanism of vasopressin action in the renal collecting duct.
Semin. Nephrol.
14:
302-321,
1994[Medline].
17.
Kusano, E.,
N. Murayama,
J. L. Werness,
S. Christensen,
S. Homma,
A. N. Yusufi,
and
T. P. Dousa.
Effects of calcium on the vasopressin-sensitive cAMP metabolism in medullary tubules.
Am. J. Physiol.
249 (Renal Fluid Electrolyte Physiol. 18):
F956-F966,
1985[Medline].
18.
Kuwahara, M.,
K. Fushimi,
Y. Terada,
L. Bai,
F. Marumo,
and
S. Sasaki.
cAMP-dependent phosphorylation stimulates water permeability of aquaporin-collecting duct water channel protein expressed in Xenopus oocytes.
J. Biol. Chem.
270:
10384-10387,
1995[Abstract/Free Full Text].
19.
Lankford, S. P.,
C. L. Chou,
Y. Terada,
S. M. Wall,
J. B. Wade,
and
M. A. Knepper.
Regulation of collecting duct water permeability independent of cAMP-mediated AVP response.
Am. J. Physiol.
261 (Renal Fluid Electrolyte Physiol. 30):
F554-F566,
1991[Abstract/Free Full Text].
20.
Marples, D.,
S. Christensen,
E. I. Christensen,
P. D. Ottosen,
and
S. Nielsen.
Lithium-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla.
J. Clin. Invest.
95:
1838-1845,
1995[Medline].
21.
Marples, D.,
J. Frøkiaer,
J. Dorup,
M. A. Knepper,
and
S. Nielsen.
Hypokalemia-induced downregulation of aquaporin-2 water channel expression in rat kidney medulla and cortex.
J. Clin. Invest.
97:
1960-1968,
1996[Abstract/Free Full Text].
22.
Marples, D.,
M. A. Knepper,
E. I. Christensen,
and
S. Nielsen.
Redistribution of aquaporin-2 water channels induced by vasopressin in rat kidney inner medullary collecting duct.
Am. J. Physiol.
269 (Cell Physiol. 38):
C655-C664,
1995[Abstract].
23.
Matsumura, Y.,
S. Uchida,
T. Rai,
S. Sasaki,
and
F. Marumo.
Transcriptional regulation of aquaporin-2 water channel gene by cAMP.
J. Am. Soc. Nephrol.
8:
861-867,
1997[Abstract].
24.
Nielsen, S.,
and
P. Agre.
The aquaporin family of water channels in kidney.
Kidney Int.
48:
1057-1068,
1995[Medline].
25.
Nielsen, S.,
C. L. Chou,
D. Marples,
E. I. Christensen,
B. K. Kishore,
and
M. A. Knepper.
Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma membrane.
Proc. Natl. Acad. Sci. USA
92:
1013-1017,
1995[Abstract].
26.
Nielsen, S.,
S. R. DiGiovanni,
E. I. Christensen,
M. A. Knepper,
and
H. W. Harris.
Cellular and subcellular immunolocalization of vasopressin-regulated water channel in rat kidney.
Proc. Natl. Acad. Sci. USA
90:
11663-11667,
1993[Abstract].
27.
Nielsen, S.,
D. Marples,
J. Frøkiaer,
M. Knepper,
and
P. Agre.
The aquaporin family of water channels in kidney: an update on physiology and pathophysiology of aquaporin-2.
Kidney Int.
49:
1718-1723,
1996[Medline].
28.
Nielsen, S.,
T. Pallone,
B. L. Smith,
E. I. Christensen,
P. Agre,
and
A. B. Maunsbach.
Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney.
Am. J. Physiol.
268 (Renal Fluid Electrolyte Physiol. 37):
F1023-F1037,
1995[Abstract/Free Full Text].
29.
Saboli
, I.,
T. Katsura,
J. M. Verbavatz,
and
D. Brown.
The AQP2 water channel: effect of vasopressin treatment, microtubule disruption, and distribution in neonatal rats.
J. Membr. Biol.
143:
165-175,
1995[Medline]. [Corrigenda. J. Membr. Biol. 145: 107-108, 1995]
30.
Sasaki, S.,
K. Fushimi,
H. Saito,
F. Saito,
S. Uchida,
K. Ishibashi,
M. Kuwahara,
T. Ikeuchi,
K. Inui,
K. Nakajima,
T. X. Watanabe,
and
F. Marumo.
Cloning, characterization, and chromosomal mapping of human aquaporin of collecting duct.
J. Clin. Invest.
93:
1250-1256,
1994[Medline]. [Corrigenda. J. Clin. Invest. 94: following p. 216, 1994]
31.
Terris, J.,
C. A. Ecelbarger,
S. Nielsen,
and
M. A. Knepper.
Long-term regulation of four renal aquaporins in rats.
Am. J. Physiol.
271 (Renal Fluid Electrolyte Physiol. 40):
F414-F422,
1996[Abstract/Free Full Text].
32.
Valtin, H.
Genetic models of diabetes insipidus.
In: Handbook of Physiology. Renal Physiology. Bethesda, MD: Am. Physiol. Soc., 1992, sect. 8, vol. 2, chapt. 28, p. 1281-1315.
33.
Yamaki, M.,
S. McIntyre,
J. M. Murphy,
J. V. Swinnen,
M. Conti,
and
T. P. Dousa.
ADH resistance of LLC-PK1 cells caused by overexpression of cAMP-phosphodiesterase type-IV.
Kidney Int.
43:
1286-1297,
1993[Medline].
34.
Yamaki, M.,
S. J. McIntyre,
and
T. P. Dousa.
Dexamethasone (DEX) down-regulates Ca2+ calmodulin (CaM)-dependent cAMP-phosphodiesterase isozyme (PDE-I) in rat inner medulla (Abstract).
J. Am. Soc. Nephrol.
2:
467,
1991.
35.
Yamamoto, T.,
S. Sasaki,
K. Fushimi,
K. Ishibashi,
E. Yaoita,
K. Kawasaki,
F. Marumo,
and
I. Kihara.
Vasopressin increases AQP-CD water channel in apical membrane of collecting duct cells in Brattleboro rats.
Am. J. Physiol.
268 (Cell Physiol. 37):
C1546-C1551,
1995[Abstract/Free Full Text].
36.
Yasui, M.,
S. M. Zelenin,
G. Celsi,
and
A. Aperia.
Adenylate cyclase-coupled vasopressin receptor activates AQP2 promoter via a dual effect on CRE and AP1 elements.
Am. J. Physiol.
272 (Renal Physiol. 41):
F443-F450,
1997[Abstract/Free Full Text].
Am J Physiol Renal Physiol 276(2):F179-F190
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