Vol. 277, Issue 2, R427-R433, August 1999
Lack of vasopressin-independent upregulation of AQP-2 gene
expression in homozygous Brattleboro rats
Takako
Saito1,
San-E
Ishikawa1,
Sei
Sasaki2,
Minori
Higashiyama1,
Shoichiro
Nagasaka1,
Nobuya
Fujita1,
Kiyohide
Fushimi2,
Fumiaki
Marumo2, and
Toshikazu
Saito1
1 Division of Endocrinology and
Metabolism, Department of Medicine, Jichi Medical School, Tochigi
329-0498; and 2 Second
Department of Internal Medicine, Tokyo Medical and Dental
University, Tokyo 113-0034, Japan
 |
ABSTRACT |
Arginine
vasopressin (AVP) plays an important role in the expression of
aquaporin (AQP-2) in the collecting duct. The present study was
undertaken to determine whether there is an AVP-independent regulation
of AQP-2 gene expression in homozygous Brattleboro rats in which
endogenous AVP is absent. Exogenous administration of
1-deamino-8-D-AVP produced an
antidiuresis and expressed AQP-2 mRNA and AQP-2 protein in the renal
medulla of the homozygous Brattleboro rats. Twelve hours of water
deprivation produced severe dehydration in the homozygous Brattleboro
rats, such that urinary osmolality increased from 200 to 649 mosmol/kgH2O. However, no increase
in AQP-2 mRNA expression was observed after this dehydration, and the
medullary tissue content and urinary excretion of AQP-2 also remained
unchanged. Increases in AQP-2 mRNA expression and AQP-2 protein were
evident in Long-Evans rats after 64 h of water deprivation, with a
severity of dehydration almost equal to the 12-h dehydrated, homozygous
Brattleboro rats. These results indicate the lack of an AVP-independent
mechanism for upregulating AQP-2 mRNA expression in renal collecting
duct cells.
arginine vasopressin; aquaporin-2
| |
INTRODUCTION |
ARGININE VASOPRESSIN (AVP) produces the water
permeability of the renal collecting duct, and its action is mediated
by cAMP (12, 15). We recently cloned cDNA of the apical
collecting duct water channel, aquaporin-2 (AQP-2), from rat and human
kidney cDNA libraries (8, 24). The expression of AQP-2 mRNA is
increased by exogenous and endogenous AVP in various pathophysiological conditions (1, 7, 18, 30). The enhanced expression of AQP-2 mRNA is
abolished by the presence of an antidiuretic AVP antagonist (7, 9, 30)
or an acute water load that causes a reduction in plasma AVP levels
(22). Also, the 5'-flanking region of the AQP-2 gene contains
cAMP-responsive element (27). AVP is known to be the important
regulator of the transcription rates of the AQP-2 gene (10, 17).
However, other factors that might be involved in the regulation of
AQP-2 gene expression could not be ruled out (5, 16). Homozygous
Brattleboro rats are suitable for such a study, because endogenous AVP
is absent (28). There are a few reports in homozygous Brattleboro rats
showing that there is no change in AQP-2 protein in response to water deprivation and that
1-deamino-8-D-arginine
vasopressin (dDAVP) treatment increased its expression (4, 20, 26).
In the present study, we further examined whether there is an
AVP-independent regulation of AQP-2 mRNA expression by using the
homozygous Brattleboro rats.
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METHODS |
Animals and experimental protocols.
Male homozygous Brattleboro rats, weighing 250-280 g, in which
endogenous AVP was absent due to an inherited defect in the AVP gene
(25, 28) were used in the present experiments to examine the expression
of AQP-2 mRNA in the kidneys in the presence or absence of exogenous
dDAVP. The animals were housed individually in metabolic cages at
temperature of 21-23°C with lights on from 8 AM to 8 PM. Food
and water were available ad libitum during the experimental period. One
day after, an osmotic minipump (Alza model 2001, Palo
Alto, CA) containing dDAVP (Kyowa-Hakko, Tokyo, Japan) or vehicle was
implanted subcutaneously along the back under ether anesthesia. dDAVP
was dissolved in 0.15 M NaCl at a concentration of 10 µM and infused
at a rate of 10 ng/h. The infusion of dDAVP or its vehicle was
continued for an additional 24 h. Four-hour urine collections were made to determine urine volume (UV) and urinary osmolality
(Uosmol). Blood collections (2 ml) were made before the start and at the end of experiments to measure
hematocrit, total protein, and serum sodium (Na). Also, body weight was
measured before the start and at the end of experiments. At the end of
experiments, the animals were killed by decapitation and both kidneys
were removed to measure AQP-2 mRNA expression and tissue contents of
AQP-2. Uosmol was measured by
freezing point depression (model 3W2, Advanced Instrument, Needham
Heights, MA).
In the second experiment, we determined whether AQP-2 mRNA is expressed
in the homozygous Brattleboro rats under 12-h dehydration. Male
homozygous Brattleboro rats weighing 250-300 g were housed individually in metabolic cages at temperature of 21-23°C
without water. Two-hour urine collections were made by gentle abdominal massage to determine UV, Uosmol,
and urinary excretion of AQP-2 and creatinine during the 12-h
observation period. Blood collections (1 ml) were made from tail blood
vessels at 4-h intervals to determine total protein, serum Na levels,
and plasma osmolality (Posmol). Body weight was also measured at 4-h intervals for 12 h. The animals were killed by decapitation at the end of the experiments. Plasma AVP
was measured by RIA using AVP RIA kits (13), and kidneys were removed
to measure AQP-2 mRNA expression and AQP-2 protein contents. Also,
plasma oxytocin was measured by RIA. As a control, we withheld water
from Long-Evans rats (300-340 g) for 64 h. This duration of water
deprivation was found to produce the same severity of dehydration,
which was assessed by the increases in serum Na levels,
Posmol, and total protein, as
observed in the Brattleboro rats that were water deprived for 12 h (see
Table 3). Blood and urine collections were made at 16- and 8-h
intervals for 64 h, respectively, to determine the same parameters as
described above. Also, body weight was measured every 16 h during the
observation period. The rats were then killed by decapitation at the
end of the experiments. Plasma AVP and oxytocin were measured, and
kidneys were removed to analyze AQP-2 mRNA and AQP-2 protein content in the medullary tissue. In addition, two additional studies were carried
out with shorter (6 h) and longer periods (24 h) of dehydration in the
homozygous Brattleboro rats to analyze expression of AQP-2 mRNA. The
12-h dehydration was barely tolerable for homozygous Brattleboro rats,
as Posmol increased to 317.0 ± 3.9 mosmol/kgH2O. Twenty-four-hour
dehydration was performed in the homozygous Brattleboro rats given some
water to permit long-time studies, and the dehydration was kept almost
equivalent to that in the 12-h dehydrated homozygous Brattleboro rats.
Also, 16-h water-deprived Long-Evans rats were prepared with a similar
extent of dehydration equivalent to the 6-h water-deprived homozygous
Brattleboro rats.
Northern blot analysis. The
experimental procedure was similar to that described previously (7).
Total cellular RNA from whole kidneys was extracted by the acid
guanidium thiocyanate-phenol-chloroform method (3). Total RNA (15 µg)
was denatured with formamide and formaldehyde at 65°C for 15 min
and then electrophoresed on a 1% agarose-2.2 M formaldehyde gel and
blotted onto nylon membrane filters (Hybond-N+, Amersham,
Buckinghamshire, UK). Prehybridization was conducted at 42°C for 2 h in 5× SSPE (1× SSPE is 0.15 M NaCl, 0.01 M
Na2HPO4,
and 0.001 M EDTA, pH 7.4), 50% formamide, 4× Denhardt's, and 40 µg/ml denatured salmon sperm DNA. The cDNA probes for rat AQP-2 and
-actin were labeled with
[
-32P]dCTP
(specific activity, 3,000 Ci/mmol, Amersham) by random primed labeling
method (Random primer DNA labeling kit, Takara, Otsu, Japan). The
filters were hybridized at 42°C for at least 12 h with the probes.
After hybridization, the filters were washed twice in 2× SSC
(1× SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.0) and
0.1% SDS for 20 min at room temperature and then washed three times
with 0.2× SSC and 0.1% SDS at 42°C. The filters were exposed
to Kodak X-OMAT film (Eastman Kodak, Rochester, NY) for 48 h at
70°C. The films were analyzed by densitometry (CS-9000, Shimadzu, Kyoto, Japan) to obtain a quantitative comparison. All the
data were calculated to show the ratio of density of AQP-2 mRNA to that
of -actin mRNA and further expressed as a percent increase in the
density compared with the density of control.
Western blot analysis. The expression
of AQP-2 protein in the kidney medulla was determined by Western blot
analysis, as described previously (22, 24). Membranes were prepared
from rat kidney medulla by homogenization in a Potter Elvehjem
apparatus. After homogenization in 10 vol of 0.32 M sucrose, 5 mM
Tris · HCl, 2 mM EDTA, and 0.1 mM
phenylmethylsulphonyl fluoride, two centrifugations (1,000 g, 10 min) were applied. The
supernatants were then centrifuged at 250,000 g for 30 min, and the pellets were
suspended in the same buffer (membrane fraction). This fraction is
considered to contain not only plasma membranes but also subapical
vesicles. The samples were solubilized in a sample loading buffer (3%
SDS, 65 mM Tris · HCl, 10% glycerol, 5%
2-mercaptoethanol) and heated at 70°C for 5 min. They were
separated by SDS-PAGE using 10% polyacrylamide gels and were
transferred to polyvinyl membranes (Immobilon; Millipore, Bedford, MA).
The blots were incubated with an antibody (1:100 dilution) against 15 carboxy-terminal synthetic peptides of rat AQP-2
[Tyr0-aquaporin-2
(V257-A271)]. After they were rinsed, the blots were immersed
with a 1:10,000 dilution of goat anti-rabbit horseradish peroxidase-conjugated antibodies. The blots were incubated with the
enhanced chemiluminescence substrate and exposed to Hyperfilm ECL to
visualize the immunoreactive bands.
RIA of AQP-2. Tissue and urinary AQP-2
immunoreactivities were measured by a specific RIA using the antibody
against the synthetic peptide
[Tyr0-aquaporin-2
(V257-A271)] corresponding to the 15-amino acid sequence of the
COOH terminal of AQP-2, as described earlier (14, 21, 23). The peptide
was radioiodinated with 125I by
chloramine-T. Homogenized solutions were prepared from rat kidney
medulla with 10 vol of sample buffer (0.05 M sodium phosphate, pH 7.4, 2 mM EDTA, 0.1 mM phenylmethylsulphonyl fluoride, 1 µg/ml leupeptin,
1 µg/ml pepstatin) by homogenization in a Potter Elvehjem apparatus.
They were then diluted with the buffer in a final dilution of
1:40-1:160 and subjected to the measurement of AQP-2. Also, urine
samples were diluted by the buffer in a final dilution of 1:2-1:32
and then employed to the assay. For the assay, 0.1 ml of the sample,
0.1 ml of the assay buffer (0.05 M sodium phosphate, pH 7.4, 0.08 M
sodium chloride, 0.01 M EDTA, 0.1% bovine serum albumin, 0.5%
NP-40, and 0.01% sodium azide), and 0.1 ml of the antibody (final dilution, 1: 12,000) were incubated at 4°C for 48 h
followed by the addition of 0.1 ml of radiolabeled synthetic peptide
(~10,000 counts/min) and further incubation at 4°C
for 48 h. Bound and free quantities of radiolabeled ligands were
separated by the double-antibody method. The minimal detectable
quantity of AQP-2 was 0.86 pmol/tube. The intra- and interassay
coefficients of variation were <10%. The serial dilution curves of
the tissue and urine samples were parallel to that of the standard
(data not shown).
Statistical analysis. Values of body
weight, urine volume, Uosmol,
Posmol, serum Na, hematocrit,
total protein, plasma AVP levels, urinary excretion of AQP-2, ratio of
the density of AQP-2 mRNA expression to that of -actin mRNA, and
tissue contents of AQP-2 protein were analyzed by Student's
t-test. A
P value <0.05 was considered significant.
| |
RESULTS |
Table 1 shows effect of exogenous dDAVP on
body weight, hematocrit, total protein, and serum Na levels in the
homozygous Brattleboro rats in which dDAVP was infused subcutaneously
for 24 h. During the 24-h observation period, body weight, hematocrit, total protein, and serum Na remained unchanged in the presence or
absence of dDAVP.
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Table 1.
Changes in body weight, hematocrit, total protein, and serum Na in
homozygous Brattleboro rats receiving dDAVP or vehicle
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Urine volume and Uosmol in the
homozygous Brattleboro rats receiving dDAVP are shown in Table
2. A diuretic state was found in the rats
receiving only the vehicle for dDAVP. Subcutaneous infusion of dDAVP
markedly decreased urine volume and increased Uosmol after the start of
infusion. These levels persisted throughout the rest of the experiment
in both groups of rats.
Figure 1 shows the expression of AQP-2 mRNA
in the kidney of homozygous Brattleboro rats. A transcript at 1.5 kb
was seen in the homozygous Brattleboro rats, as expected for AQP-2
mRNA. After the administration of dDAVP, there was a major increase in
transcript of 1.5 kb. Also, a 4.4-kb transcript was detected, which may
be attributed to alternative splicing or polyadenylation variants of
AQP-2 mRNA, as described previously (24). Thus upregulation of AQP-2
mRNA was evident. The expression of AQP-2 mRNA significantly increased
by 2.3-fold in the homozygous Brattleboro rats receiving dDAVP compared
with the rats receiving the vehicle for dDAVP (125.0 ± 13.9 vs.
54.4 ± 4.3%, n = 4 in each group,
P < 0.05).
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Fig. 1.
Northern blot analysis of expression of rat aquaporin-2 (AQP-2) mRNA in
kidney of homozygous Brattleboro rats.
Lanes
1 and
2, ad libitum water drinking;
lanes
3 and
4, 24 h after subcutaneous infusion of
1-deamino-8-D-arginine
vasopressin (dDAVP; 10 ng/h).
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|
Western blotting showed that the dDAVP infusion remarkably increased
AQP-2 protein in renal medulla (Fig. 2;
n = 4 in each group). Immunoblots
showed bands at 29 and 36-45 kDa. The high molecular mass band of
36-45 kDa was the glycosylated form of AQP-2 protein (2, 24).
Tissue contents of AQP-2 protein were determined by RIA. AQP-2 protein
was 2.2 ± 0.2 µg/g tissue wt (n = 4) in the homozygous Brattleboro rats, a value significantly less
than that for Sprague-Dawley rats described previously (22). Subcutaneous infusion of dDAVP significantly increased AQP-2 protein to
6.0 ± 1.0 µg/g tissue wt
(n = 4, P < 0.05).
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Fig. 2.
Western blot analysis of AQP-2 protein in kidney of homozygous
Brattleboro rats. Lanes
1 and
2, ad libitum water drinking;
lanes
3 and
4, 24 h after subcutaneous infusion of
dDAVP (10 ng/h).
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Changes in body weight, urine volume,
Uosmol, total protein, serum Na,
and Posmol after dehydration in
the homozygous Brattleboro and Long-Evans rats are shown in Table
3. Basal levels of serum Na,
Posmol, and total protein were
somewhat high in the homozygous Brattleboro rats compared with the
Long-Evans rats. The homozygous Brattleboro rats were dehydrated for 12 h. Uosmol increased from 200.3 ± 12.5 to 649.3 ± 36.9 mosmol/kgH2O. Serum Na and
Posmol levels were remarkably
elevated, and body weight decreased during the 12-h water deprivation
period. These results indicated a severe dehydration state in the
homozygous Brattleboro rats. Similar dehydration was obtained in the
Long-Evans rats, which were water deprived for 64 h. This was confirmed
by the increases in total protein, serum Na, and
Posmol, which were similar to
those in the 12-h dehydrated homozygous Brattleboro rats. This maneuver increased Uosmol from 1,274.1 ± 221.1 to 3,352.4 ± 95.6 mosmol/kgH2O in the Long-Evans
rats. In response to an increase in
Posmol, plasma AVP levels markedly
elevated to 2.7 ± 0.9 from 0.2 ± 0.1 pg/ml
(n = 6, P < 0.01) in the Long-Evans rats. In
contrast, plasma AVP was not detectable before and after the
dehydration in the homozygous Brattleboro rats
(n = 4). Plasma oxytocin levels were 6.5 ± 2.4 and 8.7 ± 1.2 µU/ml in the 12-h dehydrated
Brattleboro rats (n = 3) and the 64-h
dehydrated Long-Evans rats (n = 3), respectively, and no significant difference was obtained.
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Table 3.
Changes in body weight, urine volume, Uosmol, total
protein, serum Na, and Posmol after water deprivation
in homozygous Brattleboro and Long-Evans rats
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Figure 3 depicts the expression of AQP-2
mRNA in the dehydrated Brattleboro and Long-Evans rats. A transcript at
1.5 kb was seen in the homozygous Brattleboro rats after the 12-h
dehydration, and it was not quantitatively different from that in the
Brattleboro rats under ad libitum water drinking conditions. The
expression of AQP-2 mRNA in the 12-h dehydrated Brattleboro rats was
112.5 ± 7.4% of that in the Brattleboro rats drinking water freely
(n = 4 in each group, not
significant). The 1.5-kb transcript in the Long-Evans rats under ad
libitum water drinking conditions was 172.3 ± 13.2% of that in the
Brattleboro rats (n = 4 in each group,
P < 0.05). In the 64-h dehydrated
Long-Evans rats, the expression of AQP-2 mRNA was evident, inasmuch as
the AQP-2 mRNA expression increased by 254.3 ± 8.5% after the 64-h
water deprivation (n = 6, P < 0.05). Similar results were
obtained in the 6-h water deprived Brattleboro rats and the 16-h
dehydrated Long-Evans rats (P < 0.05). In addition, there was no alteration in AQP-2 mRNA expression in
the 24-h water deprived homozygous Brattleboro rats, inasmuch as its
expression was 125.6 ± 3.7% of that in the Brattleboro rats under
ad libitum water drinking (n = 4, in
each group, not significant).
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Fig. 3.
Northern blot analysis of expression of rat AQP-2 mRNA in kidney of
homozygous Brattleboro and Long-Evans rats.
A: homozygous Brattleboro rats.
Lanes
1 and
2, control;
lanes
3 and
4, 12-h dehydration.
B: Long-Evans rats.
Lanes
5 and
6, control;
lanes
7 and
8, 64-h dehydration.
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We determined tissue AQP-2 protein by Western blot (Fig.
4). In the Long-Evans rats, dehydration
markedly increased AQP-2 protein in renal medulla compared with the
rats under ad libitum water drinking
(n = 6 in each group,
P < 0.05). In contrast, tissue AQP-2
protein was detectable to lesser extent in the homozygous Brattleboro
rats. There was no difference in AQP-2 protein in the homozygous
Brattleboro rats between the presence and absence of water
(n = 4 in each group). Similar results
were obtained with AQP-2 radioimmunoreactivity. Tissue contents of
AQP-2 were 2.2 ± 0.3 and 3.4 ± 0.8 µg/g tissue wt in the
homozygous Brattleboro rats under ad libitum water drinking conditions
and 12 h after water deprivation, respectively, and these values were
much less than those of Long-Evans rats. Tissue content of AQP-2 was
greater in the dehydrated Long-Evans rats than those in the control
condition (13.5 ± 0.7 vs. 5.6 ± 0.2 µg/g tissue wt;
n = 4, P < 0.001).
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Fig. 4.
Western blot analysis of AQP-2 protein in kidney of homozygous
Brattleboro and Long-Evans rats. Lanes
1 and
2, homozygous Brattleboro rats,
control; lanes
3 and
4, homozygous Brattleboro rats, 12-h
dehydration; lanes
5 and
6, Long-Evans rats, control;
lanes
7 and
8, Long-Evans rats, 64-h
dehydration.
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Finally, urinary excretion of AQP-2 is shown in Fig.
5. Urinary excretion of AQP-2 was markedly
greater in the Long-Evans rats than the homozygous Brattleboro rats
during the water deprivation period. Water deprivation caused a gradual
increase in urinary AQP-2 in the Long-Evans rats. In contrast, urinary
excretion of AQP-2 in the homozygous Brattleboro rats remained low
during the dehydration period. We analyzed a regression line between
urinary excretion of AQP-2 and serum Na, which is closely related to
AVP release. There was a positive correlation
(r = 0.51, P < 0.01) in the Long-Evans rats but
not in the homozygous Brattleboro rats (r = 0.098, not significant; data are
shown prior to the analysis).
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Fig. 5.
Urinary excretion of AQP-2 (UAQP-2) in homozygous Brattleboro rats and
Long-Evans rats after water deprivation.
A: 2-h urine collections during 12-h
observation period (n = 4) for
dehydrated, homozygous Brattleboro rats.
B: 8-h urine collections during 64-h
observation period (n = 6) for
dehydrated Long-Evans rats. * P < 0.05 vs. ad libitum water drinking. Values are means ± SE.
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| |
DISCUSSION |
Exogenous and endogenous AVP stimulate the expression of AQP-2 mRNA in
the kidney (1, 7, 8). A cAMP-responsive element was found in the
5'-flanking region of the AQP-2 gene, and the 224-bp
5'-flanking region contains the elements necessary for cAMP-induced regulatory mechanisms (17, 27). Because AVP is well known
to increase cAMP levels in collecting duct cells (12, 15), it is highly
likely that AVP regulates the transcription rates of the AQP-2 gene
(10, 17). This is firmly supported by use of the nonpeptide AVP
receptor antagonist OPC-31260, which blocked the AVP-induced expression
of AQP-2 mRNA (7, 9, 30). Immunocytochemistry and immunoelectron
microscopy demonstrated that at least a 15-min infusion of AVP led to
translocation of AQP-2 to the apical plasma membranes from the
cytosolic vesicles of collecting duct cells (31). In the present study,
we detected the expression of AQP-2 mRNA and AQP-2 protein in the
homozygous Brattleboro rats under ad libitum water drinking. A
subcutaneous infusion of dDAVP produced a marked antidiuresis and
stimulated the expression of AQP-2 mRNA and increased AQP-2 protein in
the kidney of homozygous Brattleboro rats. Similar studies were
performed by DiGiovanni et al. (4), and Sabolic et al. (20) showed that
there was a sharp increase in the number of gold particles on the
apical plasma membrane of principal cells from homozygous Brattleboro
rats after the 30-min AVP treatment. However, exogenous AVP did not
noticeably increase AQP-2 protein in homozygous Brattleboro rats
analyzed by Western blotting. The difference could be derived from the
duration of AVP treatment in our study.
The purpose of the present study was to clarify whether the
AVP-independent factor also controls the expression of AQP-2 mRNA in
kidney using homozygous Brattleboro rats. We demonstrated in experimental rats with syndrome of inappropriate secretion of antidiuretic hormone receiving dDAVP and liquid diet that
the expression of AQP-2 mRNA is continuously increased during the 14-day observation period (7). Its expression was upregulated in the
kidney as a result of long-term administration of AVP. Because in such
a state AVP receptor binding could be downregulated (29), the
possibility of the involvement of AVP-independent factor could not be
ruled out (5). Renal medullary tissue tonicity is an important factor
for urinary concentrating (11). Although we did not actually measure
tissue tonicity of inner medulla, Xu et al. (30) determined tissue
tonicity of renal medulla of the experimental rats with myocardial
infarction in which AQP-2 mRNA expression was also upregulated. They
did not suggest the involvement of tissue tonicity in regulation of
AQP-2 mRNA expression.
Therefore, we added an additional experimental protocol. The severity
of dehydration as determined by the increases in serum Na,
Posmol, and total protein was
almost equal between the two groups of homozygous Brattleboro and
Long-Evans rats. Under this condition, there was a large difference in
increases in Uosmol between the
two groups of rats (3,352 vs. 649 mosmol/kgH2O), which had to be
based on the presence or absence of endogenous AVP. We recognized that
the Uosmol of 649 mosmol/kgH2O was the maximum obtained in the homozygous Brattleboro rats. The expression of AQP-2
mRNA was manifest in the 64-h dehydrated Long-Evans rats. However, no
increases in AQP-2 mRNA expression and AQP-2 protein were found in the
homozygous Brattleboro rats after 12 h of water deprivation. Also, the
modified 24-h water deprivation did not alter AQP-2 mRNA expression.
This was consistent with the study by Sabolic et al. (20), who reported
no change in AQP-2 protein after an overnight dehydration in
Brattleboro rats. Approximately 3% of AQP-2 of renal collecting duct
cells is excreted into urine, and urinary AQP-2 excretion has a
positive correlation with plasma AVP levels (14, 19, 21, 23). Also,
urinary excretion of AQP-2 seems likely to depend on the contents of
AQP-2 protein in renal collecting duct cells. Urinary excretion of
AQP-2 did not alter in response to water deprivation in the homozygous
Brattleboro rats, but increased gradually depending on the duration of
water deprivation, reflecting central release of AVP in the Long-Evans rats. Although the severity of dehydration was similar in two groups of
rats, some other metabolic difference could not be ruled out because of
the different duration of water deprivation. We also demonstrated the
similar results under mild water deprivation in the homozygous
Brattleboro rats (6-h deprivation) and in the Long-Evans rats (16-h
dehydration). Also, we measured plasma oxytocin levels in both groups
of dehydrated rats, because oxytocin may play an AVP-like role in renal
collecting duct (6). There was no increase in plasma oxytocin levels in
the 12-h dehydrated homozygous Brattleboro rats and the 64-h dehydrated
Long-Evans rats, respectively, suggesting that oxytocin is not involved
in AQP-2 gene expression under water-deprived conditions. These
findings indicate the lack of an AVP-independent mechanism for
upregulating AQP-2 mRNA expression in renal collecting duct cells.
| |
ACKNOWLEDGEMENTS |
The present study was supported by grants from the Ministry of
Education, Science and Culture of Japan (No. 10770557).
| |
FOOTNOTES |
The study was presented as a poster at the 31st Annual Meeting of the
American Society of Nephrology in Philadelphia, PA, October 25-28,
1998, and an abstract was published (J. Am. Soc. Nephrol. 9, Suppl.:
20A, 1998)
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: S. Ishikawa,
Division of Endocrinology and Metabolism, Dept. of Medicine, Jichi
Medical School, 3311-1 Yakushiji Minamikawachi-machi, Tochigi,
329-0498, Japan (E-mail: saneiskw{at}jichi.ac.jp).
Received 23 July 1998; accepted in final form 13 April 1999.
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