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Original Studies |
Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School, Tochigi 329-0498, Japan
Address all correspondence and requests for reprints to: San-e Ishikawa, M.D., Division of Endocrinology and Metabolism, Department of Medicine, Jichi Medical School, 3311–1 Yakushiji Minamikawachi-machi, Tochigi 329-0498, Japan. E-mail: saneiskw{at}jichi.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In response to AVP, concentrated urine is produced by water reabsorption across the renal collecting duct (15). Sasaki et al. (16, 17) recently cloned a complementary DNA of the apical collecting duct water channel, aquaporin-2 (AQP-2), from rat and human kidneys. AQP-2 is an AVP-regulated water channel; it is translocated from the cytoplasmic vesicles to the apical plasma membranes, by shuttle trafficking, in collecting duct cells when the cells are stimulated by AVP (18, 19, 20), and is again redistributed into the cytoplasmic vesicles after removal of AVP stimulation (21). Also, AQP-2 is in part excreted into the urine (16, 17), which is measurable by RIA or Western blot using a specific antibody against AQP-2 (22, 23). We demonstrated that urinary excretion of AQP-2 is significantly increased by exogenous and endogenous AVP, and urinary AQP-2 excretion has a positive correlation with plasma AVP levels (22, 24, 25). The measurement of urinary excretion of AQP-2 is an useful tool for diagnosing central diabetes insipidus in the hypertonic saline infusion test (24).
The present study was therefore undertaken to determine whether urinary excretion of AQP-2 is of value to diagnose the pathological state of water retention and hyponatremia. We measured urinary excretion of AQP-2 by RIA in normal subjects and patients with impaired water excretion.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Ten patients with water retention and 7 control subjects were
studied. The control group had 7 normal volunteers, aged 22–25 yr;
they were 6 males and 1 female. Profiles of the group of 10 patients
are shown in Table 1
; they were 6 males
and 4 females whose ages ranged from 55–75 yr (mean ± SE,
67.1 ± 3.8 yr). The mean serum sodium (Na) level was 128.6
± 3.8 mEq/L when the patients were admitted to Jichi Medical School
Hospital. Urinary osmolality (Uosm) was as high as 468.2 ± 115.1
mosmol/kg H2O despite the decrease in plasma osmolality
(Posm). The plasma AVP level was 1.3 ± 0.4 pg/mL, although Posm
was reduced to 267.3 ± 7.5 mosmol/kg H2O, and it was
not significantly different from the value of 1.4 ± 0.2 pg/mL in
the control subjects (n = 20) (26). There was no abnormality in
renal function. Two patients were diagnosed with SIADH whose adrenal
function was normal. Two patients were diagnosed with the central
salt-wasting syndrome. Patients 5–10 had decreased levels of plasma
ACTH (8.7 ± 2.3 pg/mL) and serum cortisol (2.6 ± 0.8
µg/dL) with reduced urinary excretion of 17-OHCS. These 6 patients
were diagnosed with hypopituitarism. Particularly, isolated ACTH
deficiency was found in the patients 6 and 7. The present study was
approved by the ethical committee of Jichi Medical School Hospital for
human study. We obtained informed consent from all subjects before they
joined the present protocol.
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An acute water load test was carried out in two groups of subjects. After an overnight fast, the study was started at 0800 h. The subjects were allowed to drink water freely before the start of the present protocol. After urination, blood was taken. Thereafter, water (20 mL/kg) was given orally for 30 min. Thirty-minute urine collections were made during the 240-min observation period. Blood samples were collected at 60-min intervals for 240 min. Urine samples were subjected to measurements of urine volume, Uosm, and urinary excretions of creatinine and AQP-2. Posm and plasma AVP levels were determined in blood samples.
In addition, an acute oral water load test was performed in the six patients with hypopituitarism in the absence and presence of 20 mg hydrocortisone. Hydrocortisone was administered at 0500 h, and the oral water load test was started at 0800 h. The protocol was the same as that described above.
Uosm and Posm were measured by freezing point depression (model 3W2, Advanced Instrument, Needham Heights, MA). The concentrations of creatinine in urine samples were measured by automatic clinical analyzer (model 736, Hitachi, Tokyo, Japan). Plasma AVP levels were measured by RIA using AVP RIA kits (Mitsubishi Chemistry, Tokyo, Japan) (9). Urinary AQP-2 was determined as described below.
RIA of urinary AQP-2
The RIA of urinary AQP-2 was performed by the method described
in our previous reports (22, 24). Urinary AQP-2-like immunoreactivity
was measured by a specific RIA that used the polyclonal antibody
against a synthetic portion of the C-terminal of human AQP-2 raised in
rabbits. A synthetic peptide
(Tyr0-aquaporin-2[V257-A271]) corresponding to the
15-amino acid sequence of the C-terminal of AQP-2 was radioiodinated
with iodine-125 (New England Nuclear, Boston, MA) by the chloramine-T
method. For the assay, 0.1 mL of the urine sample (diluted 1–8 times)
or a standard, 0.1 mL assay buffer [0.05 mol/L sodium phosphate (pH
7.4), 0.08 mol/L sodium chloride, 0.01 mol/L ethylenediamine
tetraacetate, 0.5% BSA, 0.5% Nonidet P-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 the
radiolabeled synthetic peptide (
10,000 cpm) and further incubation
at 4 C for 48 h. Bound and free quantities of radiolabeled ligand
were separated by the double antibody method. The serial dilution curve
of the urine samples was parallel to that of the standard (data not
shown). Each sample was analyzed in duplicate. We changed the antibody
against a 15-amino acid sequence of the C-terminal of human AQP-2 for
the RIA, and the data shown in the present study were less than those
that we previously reported (24). For the antibody used in the present
study, the intra- and interassay coefficients of variation were less
than 10%. The minimal detectable quantity of AQP-2 was 0.86 pmol/tube,
and an amount equivalent to 6.9 pmol/tube caused 50% inhibition of
binding of the radiolabeled ligand.
Statistical analysis
Urine volume, Uosm, Posm, plasma AVP, and excretion of urinary AQP-2 values were expressed as the mean ± SE. All values were compared with two-way ANOVA and Fisher’s t test. P < 0.05 was considered significant.
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Results |
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shows the urinary excretion
of AQP-2 in the normal volunteers and the patients with water retention
under ad libitum water drinking. Urinary excretion of AQP-2
was 153.3 ± 28.1 fmol/mg creatinine in the normal volunteers. Its
excretion was 2.8-fold greater in the patients with water retention
than that in the normal volunteers.
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and Figs. 2 and 3. An
acute oral water load (20 mL/kg) produced water diuresis in the control
subjects, since the percent excretion of the water load was
70.2 ± 7.8% during the 4-h observation period. As shown in Table 2, the minimal Uosm was as low as 94.4 ± 9.8 mosmol/kg
H2O, which was responsible for the decrease in plasma AVP
levels after the reduction in Posm. Water diuresis was evident, as the
free water clearance (CH2O) significantly increased after
the acute oral water load in the control subjects (Fig. 2). In
contrast, the percent excretion of the water load was only
29.9 ± 4.6% in the patients with water retention, a value
significantly less than that in the control subjects (P
< 0.01). Basal levels of Uosm were 501.9 ± 75.3 mosmol/kg
H2O. The minimal Uosm was not as sufficiently reduced, as
its value of 387.8 ± 61.1 mosmol/kg H2O was
significantly greater than that in the control subjects
(P < 0.01). The free water clearance remained below
0.6 mL/min after the oral water load, as shown in Fig. 2. This impaired
water excretion was in concert with the nonsuppressible levels of
plasma AVP despite the hypoosmolality in the patients with water
retention.
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. Basal levels of urinary AQP-2 were 516.9 ± 137.5 fmol/mg
creatinine in the patients with water retention, which was
significantly greater than that of 153.3 ± 28.1 fmol/mg
creatinine in the control subjects (P < 0.05). The
maneuver of an acute oral water load gradually decreased urinary
excretion of AQP-2, followed by the gradual increase toward the level
before the start of the water load in both groups of subjects. The
nadir was obtained at 60–90 min and was significantly greater in the
patients with water retention than that in the control subjects. As the
nadir appeared at different times in individuals, the minimal
urinary excretion of AQP-2 was determined (Fig. 3b). The
difference in the minimal urinary AQP-2 between the two groups of
subjects was more evident. In the patients with water retention, the
minimal urinary AQP-2 decreased to only 253.9 ± 54.3 fmol/mg
creatinine, a value significantly greater than that in the control
subjects (P < 0.01).
Next, we compared the acute oral water load test in the patients with
hypopituitarism in the absence and presence of hydrocortisone
replacement. The patients included four panhypopituitarism and two
isolated ACTH deficiency. The results of an acute oral water load test
are shown in Table 3 and Fig. 4. Before treatment with hydrocortisone,
the percent excretion of the water load was only 31.5 ±
5.9% during the 4-h observation period. In contrast, hydrocortisone
replacement enormously improved water excretion, as the percent
excretion of the water load increased to 102.5 ± 17.4% during
the 4-h observation period. As shown in Table 3, the minimal Uosm
decreased to 108.5 ± 8.0 mosmol/kg H2O in the
patients treated with hydrocortisone, a value significantly lower than
that in the untreated patients with hypopituitarism. There was no
difference in the minimal levels of Posm and plasma AVP between the
presence and absence of hydrocortisone.
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shows urinary excretion of AQP-2 after the acute oral water
load in the patients with hypopituitarism. The exaggerated urinary
excretion of AQP-2 throughout the 4-h observation period was evident in
the absence of hydrocortisone replacement (Fig. 4a). Both the basal and
the nadir levels of urinary AQP-2 were high in the untreated patients
with hypopituitarism. The minimal urinary excretion of AQP-2 was
determined (Fig. 4b), as the nadir appeared at different times.
Hydrocortisone replacement significantly reduced the minimal urinary
excretion of AQP-2 after the acute oral water load, as the minimal
urinary AQP-2 excretions were 224.7 ± 69.7 and 49.4 ± 20.5
fmol/mg creatinine in the patients before and after hydrocortisone
replacement (P < 0.05). |
Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Clinical and laboratory experiments have demonstrated that impaired ability to excrete a water load occurs in patients with SIADH, liver cirrhosis with ascites, congestive heart failure, and adrenal insufficiency. (1, 2, 3, 4, 5, 6, 7, 8, 27) Persistent elevation of plasma AVP levels has been shown despite hypoosmolality, which should suppress the osmotic release of AVP to undetectable levels (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 27). These pathological states are associated with hyponatremia to a various extent. RIA of AVP enables reliable measurement of plasma AVP levels, but the extent that plasma AVP varies in pathological states of impaired water excretion is not as great. In most clinical settings, the nonsuppressible levels of plasma AVP are estimated to be relatively high compared to the reduced Posm (14, 26). We are often faced with the dilemma of elucidating the exact role of AVP in the impaired renal excretion of water.
AQP-2 is the AVP-dependent water channel of collecting duct cells and
is recycling between the cytoplasmic vesicles and the apical plasma
membranes in the cells (18, 19, 20, 21, 28, 29). Recently, AQP-2 is partly
excreted into the urine, which is approximately 3% of AQP-2 in the
collecting duct cells (22, 23, 24, 25). In normal subjects, urinary excretion
of AQP-2 is changeable in a wide range in physiological conditions, and
its variation positively correlates with that of plasma AVP levels
(24). Rai et al. (25) showed that there is no difference in
urinary excretion of AQP-2 among the varying ages, ranging 24–76 yr.
The difference in basal levels of urinary excretion of AQP-2 was
evident in three groups of subjects. Urinary excretion of AQP-2 was
2.8-fold greater in the patients with water retention than that in the
normal subjects, and it was one eighth less in the patients with
central diabetes insipidus than in the normal subjects (24). The
maneuver of an acute oral water load of 20 mL/kg BW totally suppressed
plasma AVP levels and produced water diuresis in the normal subjects.
Also, this maneuver reduced urinary AQP-2 excretion to 41.8 ±
14.8 fmol/mg creatinine. In contrast, an acute water load test did not
significantly produce water diuresis in the patients with water
retention, as the percent excretion of the water load was
markedly less and the nadir of Uosm was significantly higher than those
in the control subjects. Similar results were obtained with urinary
excretion of AQP-2. The minimal levels of urinary excretion of AQP-2
were significantly greater than those in the control subjects.
Nonsuppressible levels of urinary excretion of AQP-2 were similar in
the patients with SIADH and those with central salt-wasting syndrome.
As shown in Table 2, plasma AVP levels were not as high in the patients
with impaired water excretion whose Posm was hypotonic. Although an
acute oral water load further decreased Posm, plasma AVP levels were
not suppressed to undetectable levels in these patients. Therefore,
there was no correlation between plasma AVP levels and urinary
excretion of AQP-2 in the patients with water retention. The finding of
the exaggerated urinary excretion of AQP-2 may be tightly linked with
the up-regulation of AQP-2 messenger ribonucleic acid expression in the
kidneys in the experimental models of SIADH, liver cirrhosis, and
congestive heart failure (30, 31, 32, 33). As mentioned above, the antidiuretic
action of AVP is dominant, and the impaired water excretion may not
solely depend upon the plasma level of AVP. The antidiuretic action of
AVP is a key to augment antidiuresis, because the exaggerated urinary
excretion of AQP-2 reflected the action of AVP in collecting duct
cells. These findings therefore indicate that urinary excretion of
AQP-2 accounts for the cellular action of AVP in renal collecting duct
cells and is a potent marker for the diagnosis of water metabolism
dependent upon AVP.
In the present study urinary excretion of AQP-2 was exaggerated in the patients with hypopituitarism, which was closely linked with the impaired water excretion. After hydrocortisone replacement, renal water excretion was normalized, and urinary excretion of AQP-2 was reduced to levels comparable to those in control subjects. In the last 2 decades clinical and laboratory studies have demonstrated the involvement of AVP in impaired water excretion and hyponatremia in primary and secondary adrenal insufficiency (8, 34, 35, 36, 37). Plasma levels of AVP were relatively elevated despite hypoosmolality in the patients with primary and secondary adrenal insufficiency and the adrenalectomized animals (8, 34, 35, 36, 37). Hypothalamic AVP messenger ribonucleic acid expression was increased in the animals after adrenalectomy, and corticosterone replacement reduced its expression to that in the sham-operated animals (38, 39). Also, the administration of an AVP antidiuretic antagonist remarkably improved renal water excretion in the adrenalectomized rats receiving deoxycorticosterone (40). The measurement of urinary excretion of AQP-2 strongly supported the pathological role of AVP in the impaired water excretion in these adrenal insufficiency. Hydrocortisone treatment decreased the exaggerated urinary AQP-2 excretion to the normal value and normalized water excretion in the patients with hypopituitarism. These results indicate that urinary excretion of AQP-2 participates in the understanding of AVP dependency and that AVP plays a major role in the impaired water excretion in hypopituitarism, particularly in pituitary-adrenal hypofunction.
In conclusion, we demonstrated that exaggerated urinary excretion of AQP-2 was found in the patients with water retention and remained high after an acute oral water load. Similar results were obtained in the group of patients with hypopituitarism. However, hydrocortisone replacement decreased urinary excretion of AQP-2 to the normal value, in association with the normalization of renal water excretion. These results indicate that urinary excretion of AQP-2 is a potent marker for the diagnosis of impaired water excretion, dependent upon AVP, in the patients with water retention and hypopituitarism.
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Footnotes |
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Received May 4, 1998.
Revised June 23, 1998.
Accepted July 14, 1998.
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References |
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, Control subjects (n = 7); •,
patients with water retention (n = 10). *, P
< 0.01; **, P < 0.05 (vs. control
subjects). Values are the mean ± SEM.
, Absence of hydrocortisone; 
, presence of
hydrocortisone. *, P < 0.01; **,
P < 0.05 (vs. in the presence of
hydrocortisone). a, Time course during the 4-h observation period. b,
Basal, minimal and last values of UAQP-2. Values are the mean ±
SEM.