Vasopressin-dependent upregulation of aquaporin-2 gene expression in glucocorticoid-deficient rats

Takako Saito1, San-E Ishikawa1, Fumiko Ando1, Minori Higashiyama1, Shoichiro Nagasaka1, Sei Sasaki2, 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
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ABSTRACT
INTRODUCTION
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We determined alterations in renal aquaporin-2 (AQP2) gene expression in association with impaired water excretion in glucocorticoid-deficient rats. After adrenalectomy, Sprague-Dawley rats were administered aldosterone alone by osmotic pumps (glucocorticoid-deficient rats). As a control, both aldosterone and dexamethasone were administered. These animals were subjected to the studies on days 7-14. The expressions of AQP2 mRNA and protein in kidney of the glucocorticoid-deficient rats were increased by 1.6- and 1.4-fold compared with the control rats, respectively. An acute oral water load test verified the marked impairment in water excretion in the glucocorticoid-deficient rats. One hour after the water load, the expressions of AQP2 mRNA and protein were significantly reduced in the control rats, but they remained unchanged in the glucocorticoid-deficient rats. However, there was no alteration in [3H]arginine vasopressin (AVP) receptor binding and AVP V2 receptor mRNA expression in the glucocorticoid-deficient rats. A V2-receptor antagonist abolished the increased expressions of AQP2 mRNA and protein in the glucocorticoid-deficient rats. These results indicate that augmented expression of AQP2 participates in impaired water excretion, dependent on AVP, in glucocorticoid deficiency.

arginine vasopressin; water retention


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
REFERENCES

CLINICAL AND LABORATORY EXPERIMENTS have demonstrated that impairment in water excretion occurs in patients with hypopituitarism and adrenal insufficiency (1, 11, 15, 32) and in experimental models of glucocorticoid and mineralocorticoid deficiency (7, 13, 17, 22, 25, 31). The phenomenon has been investigated extensively, and there is evidence that arginine vasopressin (AVP) plays a role in impaired water excretion in such pathological states (1, 11, 13, 17). We showed the removal of water retention by the AVP V2-receptor antagonist in rats with glucocorticoid deficiency (13).

AVP produces the water permeability of renal collecting duct, and its action is mediated by cAMP (10, 16). Aquaporin-2 (AQP2) is a water channel in the apical collecting duct in the kidney (6, 30). The cAMP-responsive element is found in the 224 bp of the 5'-flanking region of the AQP2 gene, which contains the element necessary for the cAMP-induced regulatory mechanism (18, 34). AVP is known to be the important regulator of the transcription rates of the AQP2 gene (9, 18). When the cells are stimulated by AVP, AQP2 is translocated from cytoplasmic vesicles to apical plasma membranes, by shuttle trafficking, in collecting duct cells (19, 26, 36). AQP2 is again redistributed into cytoplasmic vesicles after removal of AVP stimulation (28). Recent studies have shown the upregulation of AQP2 mRNA expression in kidney in the pathological state of water retention in experimental models of the syndrome of inappropriate secretion of antidiuretic hormone (SIADH), liver cirrhosis with ascites, and congestive heart failure (2, 5, 20, 35).

The present study was undertaken to determine whether AQP2 is involved in pathogenesis of impaired water excretion in rats with experimental model of glucocorticoid deficiency. Also, we examined what alteration in AVP V2 receptors is found in glucocorticoid deficiency.


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Animals. Male Sprague-Dawley (SD) rats weighing 250-300 g were used in the present experiments. The animals were housed individually in metabolic cages at a temperature of 21-23°C with the lights on from 8 AM to 8 PM. Solid food and water were available ad libitum during the experimental period. Under light ether anesthesia, the animals were adrenalectomized through bilateral retroperitoneal incisions, and then osmotic pumps (Alza model 2ML4, Palo Alto, CA) were implanted subcutaneously along their backs. The adrenalectomized rats were divided into two groups. A group of glucocorticoid-deficient rats was administered aldosterone at a rate of 42 ng · 100 g-1 · h-1 by osmotic pumps throughout the experiments (4). The other group of rats (control) was administered both aldosterone (42 ng · 100 g-1 · h-1) and dexamethasone (58 ng · 100 g-1 · h-1) by osmotic pumps throughout the experiments. After the recovery, they were placed in cages with free access to water and rat chow. The two groups of rats were subjected to the following studies on days 7-14. One-milliliter blood collections were made to determine serum sodium and plasma osmolality (Posm). After the completion of sampling, the animals were killed by decapitation. Blood from the trunk was collected to measure plasma AVP levels. Both kidneys were removed to determine AQP2 mRNA and AQP2 protein expression, AVP V2 receptor mRNA expression, and AVP receptor binding. Posm was measured by freezing-point depression (model 3W2, Advanced Instruments, Needham Heights, MA). Plasma AVP levels were measured by radioimmunoassay, as described previously (12).

Acute oral water load. Acute oral water loads were performed in the glucocorticoid-deficient rats and the control rats to determine the effect of glucocorticoid deficiency on renal water excretion. The water load was studied 2 h after removal of food and water. The rats were given 30 ml/kg of deionized water by gastric tubes under light ether anesthesia. The animals were then placed in individual metabolic cages (Natsume, Tokyo, Japan) and allowed to awaken. Spontaneously voided urine samples were collected at 20-min intervals for 3 h. The final urine samples were obtained by gentle abdominal massage at the end of experiments. Urine volume and urinary osmolality (Uosm) were measured, and percent excretion water-loaded and basal and minimal Uosm were recorded. Uosm was measured by freezing-point depression.

A few days later, the acute oral water loads (30 ml/kg) were again carried out. On that occasion, the rats were killed by decapitation 1 h after the water loads. This was done because our previous studies showed that the maximal urine flow and minimal Uosm occurred 40-60 min after the oral water load (27, 33). Trunkal blood and both kidneys were obtained to determine plasma AVP levels and expressions of AQP2 mRNA and protein. The same experiments were performed in the glucocorticoid-deficient rats and the control rats receiving 30 mg/kg of the nonpeptide AVP V2-receptor antagonist 5-dimethylamino-1-[4-(2-methylbenzoylamino) benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine hydrochloride (OPC-31260; Otsuka Pharmaceutical, Osaka, Japan) (37). An acute oral water load was started 2 h after the oral administration of OPC-31260.

Northern blot analysis. The experimental procedure was performed by the modified method from that described previously (5). Total cellular RNA from whole kidneys was extracted by the acid guanidium thiocyanate-phenol-chloroform method (3). Total RNA (20 µg) was denatured with formamide and formaldehyde at 65°C for 15 min, then electrophoresed on a 1% agarose-2.2 M formaldehyde gel, and then blotted onto nylon membrane filters (Hybond-N+, Amersham, Buckinghamshire, UK). The cDNA probes for rat AQP2 and beta -actin were labeled with digoxigenin (DIG)-11-UTP by the random primed labeling method (DIG DNA labeling kit, Roche, Tokyo, 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× saline sodium citrate (SSC) 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 incubated by chemiluminescent substrate (Tropix, Bedford, MA) and exposed to Kodak X-Omat film (Eastman Kodak, Rochester, NY) for 4 h at 37°C. The films were analyzed by densitometry (CS-9000, Shimadzu, Kyoto, Japan) to show the quantitative comparison. All the data were calculated to show the ratio of density of AQP2 mRNA to that of beta -actin mRNA and further expressed as a percent increase in the density compared with the density of control.

Western blot analysis. The expression of AQP2 protein in the kidney medulla was determined by Western blot analysis as described previously (28, 30). 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, 0.1 mM phenylmethylsulfonyl 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). The samples were solubilized in a sample loading buffer; 3% SDS, 65 mM Tris · HCl, 10% glycerol, and 5% 2-mercaptoethanol, and heated at 70°C for 5 min. They were separated by SDS-PAGE by using 10% polyacrylamide gels and were transferred to polyvinyl membranes (Immobilon; Millipore, Bedford, MA). The blots were incubated with an antiserum (1:100 dilution) against 15 COOH-terminal synthetic peptides of rat AQP2 (Tyr0-AQP2 [V257-A271]). After rinsing, 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 (ECL) substrate and exposed to Hyperfilm ECL to visualize the immunoreactive bands.

AVP receptor binding. The experimental procedure was modified from the methods of Jard et al. (14). Renal medullary tissues were homogenized in a Potter-Elvehjen apparatus in 10 vol of homogenized solution (0.32 M sucrose, 5 mM Tris · HCl, 2 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride). After the homogenization, the samples were centrifuged twice at 1,000 g for 10 min at 4°C. The supernatants were then centrifuged at 250,000 g for 30 min at 4°C, and the pellets were suspended in the ice-cold binding buffer [50 mM Tris · HCl (pH 7.4), 5 mM MgCl2, 1 mg/ml bovine serum albumin]. Two hundred microliters of the binding buffer containing 2 × 10-9 M [3H]AVP (specific activity, 67.6 Ci/mmol; New England Nuclear, Boston, MA) in the absence or presence of cold AVP (1 × 10-9~1 × 10-6 M) were added to the tubes, and then the reaction was initiated by the addition of the membrane fraction (100-200 µg) at 4°C for 60 min. After the incubation, 3 ml of the ice-cold binding buffer were added. The mixture was then filtered through glass microfiber filters (Whatmen 2.4-cm GF/C; Whatman, Maidstone, UK). The filters were rinsed three times with 3 ml of ice-cold binding buffer. They were put into counting vials, and 10 ml of scintillation solution were added. The radioactivity was counted with a liquid scintillation counter (model LSC-671, Aloka, Tokyo, Japan). Protein was measured by a Bio-Rad protein assay kit.

Semiquantitative multiprimer RT-PCR. Total RNA was isolated from whole kidney in the same manner as in Northern blotting. Reverse transcription was performed by using a cDNA synthesis kit (SuperScript Preamplification System for first-strand cDNA synthesis, GIBCO-BRL, Life Technology, Rockville, MD). It was carried out in a 20-µl reaction volume containing 5 µg total RNA, 6 µl of buffer, 1 µl of RNase inhibitor, 1 µl of deoxynucleotide mixture, 5 µl of random primer, and 1 µl (200 U) of SuperScript. Reaction tubes were incubated at 42°C for 50 min, and the reaction was stopped by heating at 70°C for 15 min. Then the reaction tubes were placed on ice until the addition of PCR reagents. PCR was carried out by using 1 µl cDNA stock solution in the presence of 0.01U/µl r-Taq enzyme (Takara, Otsu, Japan), 0.2 µM gene-specific primer, 200 µM dNTPs, 10 mM Tris · HCl, 1.5 mM MgCl2, 50 mM KCl, and 0.1% Triton X-100, and cycled 24 cycles (57°C for 30 s, 72°C for 5 min, 95°C for 60 s), starting at 95°C for 1 min and finishing at 72°C for 5 min. The gene-specific primers of AQP2 and beta -actin were made according to the study of Yasui et al. (38). The primers of AQP2 were 5'-AGTGCTGGCTGAGTTCTTGG-3' (antisense) and 5'-GCTGTGGCGTTGTTGTGGAG-3' (sense), those of AVP V2 receptors were 5'-CCTCCTACATGATCCTGGCCATGAG-3' (antisense) and 5'-TGCACCAGGAAGAAGGGTGCCCAGCA-3' (sense), and those of beta -actin were 5'-TGAACCCTAAGGCCAACCGT-3' (antisense) and 5'-GCTAGGAGCCAGGGCAGTA-3' (sense). The product sizes of AQP2, V2 receptor, and beta -actin were 344, 523, and 635 bp, respectively. Twenty microliters of the PCR solution were separated on a 2% agarose gel and stained with ethidium bromide. Each RT-PCR product was subjected to restriction-enzyme analysis to verify the specificity (data not shown). When the RTase was omitted from the RT reaction solution, no products appeared, confirming that the products were produced from mRNA and not contaminating genomic DNA. All semiquantitative multiprimer (SQM) RT-PCRs were made at least three times with three different RNA preparations. All reaction solutions (except RNA and enzymes) in the SQM RT-PCR were premixed to eliminate errors during pipetting. The Hi-Lo DNA Marker (Abetec, Tokyo, Japan) was used on all agarose gels as a molecular weight marker.

Statistical analysis. Values of percent excretion water loaded, Uosm, serum sodium, Posm, plasma AVP levels, ratio of the density of AQP2 mRNA expression to that of beta -actin mRNA, and density of AQP2 protein in immunoreactive bands were analyzed by Student's t-test. A P value of <0.05 was considered significant.


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Serum sodium levels were 139.3 ± 0.3 meq/l in the glucocorticoid-deficient rats, a value less than that of 146.5 ± 0.8 meq/l in the control rats (n = 6, P < 0.05). Figure 1 shows the expression of AQP2 mRNA and protein in the kidney of the glucocorticoid-deficient and the control rats. A transcript at 1.5 kb was seen in both rats, which expresses AQP2 mRNA (Fig. 1A). Also, a 4.4-kb transcript was detected, that is, alternative splicing or polyadenylation variants, as described previously (6, 30). The expression of AQP2 mRNA increased by 164.2 ± 3.7% in the glucocorticoid-deficient rats, compared with the control rats (n = 6, P < 0.01 ). Similar results were obtained with AQP2 protein in Western blot analysis (Fig. 1B). Immunoblots showed the bands at 29 and 36-45 kDa. High-molecular-weight-mass band of 36-45 kDa was the glycosylated form of AQP2 protein (30). Also, the densitometric analysis showed that the glucocorticoid-deficient rats had increased AQP2 protein in renal medulla by 137.2 ± 5.8% compared with that of the control rats (n = 6, P < 0.05).


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Fig. 1.   Expression of aquaporin-2 (AQP2) mRNA and protein in the kidney of the glucocorticoid-deficient (GD) rats and the control rats under ad libitum water drinking (n = 6). A: Northern blot analysis. There are 2 transcripts at 1.5 and 4.4 kb. B: Western blot analysis using an antibody against rat AQP2. There are 2 bands of 29 and 36-45 kDa, corresponding to predicted molecular weight mass of AQP2 protein and glycosylated form of AQP2 protein, respectively.

Table 1 shows the results of an acute oral water load test. Percent excretion water loaded was 38.7 ± 11.7% in the glucocorticoid-deficient rats, which was less than that of 139.3 ± 10.1% in the control rats (n = 6, P < 0.01). The glucocorticoid-deficient rats had impaired water excretion. The minimal Uosm in the glucocorticoid-deficient rats was as high as 379.4 ± 50.8 mosmol/kgH2O, a value significantly greater than that of 138.0 ± 25.5 mosmol/kgH2O in the control rats (P < 0.01). The administration of 30 mg/kg OPC-31260 totally restored the impairment in water excretion in the glucocorticoid-deficient rats, as the percent excretion water loaded was 137.5 ± 10.0% and the minimal Uosm 83.6 ± 7.0 mosmol/kgH2O. The plasma AVP level was 2.6 ± 0.8 pg/ml in the glucocorticoid-deficient rats allowed free access to water and was reduced to only 1.4 ± 0.1 pg/ml 60 min after the oral water load. In the control rats this maneuver decreased plasma AVP levels from 2.9 ± 0.5 to 1.2 ± 0.1 pg/ml.

                              
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Table 1.   Changes in percent excretion water-loaded, Uosm, Posm, and plasma AVP levels in acute oral water load test (30 ml/kg) in the glucocorticoid-deficient rats

The expression of AQP2 mRNA and protein 1 h after an acute oral water load in the glucocorticoid-deficient rats is shown in Fig. 2. The expression of AQP2 mRNA was still increased in the glucocorticoid-deficient rats, compared with that in the control (Fig. 2A). The desitometric analysis revealed a 141.5 ± 17.3% increase in the AQP2 mRNA expression in the glucocorticoid-deficient rats (n = 6, P < 0.05). When the study was carried out in the rats receiving 30 mg/kg OPC-31260, an increase in the expression of AQP2 mRNA totally disappeared in the glucocorticoid-deficient rats (89.3 ± 8.8%, n = 6). This finding was in concert with the marked water diuresis as shown in Table 1. Similar results were obtained by the Western blotting, as shown in Fig. 2B. A 29-kDa protein increased in the glucocorticoid-deficient rats, compared with the control. Densitometric analysis resulted in a 120.3 ± 6.8% increment in AQP2 protein (n = 6, P < 0.05). The administration of OPC-31260 completely abolished the increase in AQP2 protein in the glucocorticoid-deficient rats.


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Fig. 2.   Expression of AQP2 mRNA and protein in the kidney of the GD rats and the control rats 1 h after an acute oral water load (30 ml/kg body wt; n = 6). A: Northern blot analysis of AQP2 mRNA. B: Western blot analysis of AQP2 protein. Left: control rats. Middle: GD rats receiving the vehicle for 5-dimethylamino-1-[4-(2-methylbenzoylamino) benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine hydrochloride (OPC-31260). Right: GD rats receiving 30 mg/kg OPC-31260.

Membrane fraction was prepared from the kidney medulla of the glucocorticoid-deficient and the control rats to determine whether glucocorticoid deficiency affects [3H]AVP receptor binding. Figure 3 shows a Scatchard analysis of AVP receptor binding. The equilibrium dissociation constant (Kd) was 1.51 × 10-10 and 1.75 × 10-10 M in the control and the glucocorticoid-deficient rats, respectively. The maximal binding capacity (Bmax) was 8.45 and 7.29 pmol/mg protein in the control and the glucocorticoid-deficient rats, respectively. There was no difference in Kd and Bmax between the two groups of rats.


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Fig. 3.   Scatchard analysis of 3H-labeled arginine vasopressin (AVP) binding to membrane fractions from renal medullary tissues of the control (open circle ) and GD rats ().

The expression of AVP V2 receptor mRNA and AQP2 mRNA analyzed by SQM RT-PCR in the glucocorticoid-deficient rats is shown in Fig. 4. The expression of AQP2 mRNA in the glucocorticoid-deficient rats was higher than that of control rats. The densitometric analysis revealed a 140.3 ± 9.5% increase in the glucocorticoid-deficient rats (n = 6, P < 0.05), which was in concert with the study in Northern blot described earlier. In contrast, the expression of AVP V2 receptor mRNA seemed likely to be somewhat high in the glucocorticoid-deficient rats compared with the control rats, but there was no significance between the control and the glucocorticoid-deficient rats.


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Fig. 4.   Expression of AVP V2 receptor mRNA and AQP2 mRNA in the kidney of the GD and control rats, analyzed by semiquantitative multiprimer RT-PCR (n = 6). Left: control rats. Right: GD rats. MW, molecular wt. markers.


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Impaired water excretion occurs in patients with hypopituitarism and isolated ACTH deficiency and experimental models of glucocorticoid deficiency (1, 7, 11, 13, 15, 17, 22, 25, 31, 32). In this pathological setting there is hyponatremia to various extents. Nonsuppressible release of AVP is found despite hypoosmolality, which should suppress AVP release to undetectable levels in humans and rats (1, 8, 11, 13, 17, 21). In addition, a study performed in adrenalectomized homozygous Brattleboro rats showed that AVP-independent factors are involved in the impaired water excretion associated with glucocorticoid deficiency (7). Almost two decades ago we demonstrated that the AVP V2-receptor antagonist promptly normalized renal water excretion in the experimental rats with glucocorticoid deficiency (13). Also, it is evident that the defect in renal water excretion could be corrected by replacement of glucocorticoid hormone in these animals and patients (7, 11, 13, 15). Therefore, nonsuppressible secretion of AVP plays a crucial role in impaired water excretion in glucocorticoid deficiency.

In the present study we confirmed impaired renal water excretion in the glucocorticoid-deficient rats. The percent excretion water loaded was reduced to 38.7% (mean), and the minimal Uosm remained as high as 379.4 mosmol/kgH2O (mean). These values were markedly different from those in the control rats. The radioimmunoassayable plasma levels of AVP were high compared with the Posm in the glucocorticoid-deficient rats. Plasma AVP levels were not sufficiently suppressed by an acute oral water load in the glucocorticoid-deficient rats, which suppressed plasma AVP levels to 1.2 ± 0.1 pg/ml in the control. As indicated the impairment in water excretion, the expression of AQP2 mRNA and protein in kidney was significantly increased in the glucocorticoid-deficient rats compared with the control. The enhanced expression of AQP2 mRNA and protein totally disappeared in the glucocorticoid-deficient rats after receiving the AVP V2-receptor antagonist, in association with the normalization of water excretion. This finding strongly indicates that the augmentation in AQP2 mRNA expression is dependent on plasma AVP. In addition, urinary excretion of AQP2 is exaggerated in the patients with hypopituitarism, particularly in hypofunction of the pituitary-adenocortical axis (27). Because ~3% of AQP2 in renal collecting duct cells is excreted into the urine (23), and urinary excretion of AQP2 positively correlates with plasma AVP levels (29), urinary AQP2 excretion allows for an understanding of AVP dependency. Upregulation of AQP2 mRNA expression in kidney was evident in the glucocorticoid-deficient rats, which could be tightly linked with exaggerated urinary excretion of AQP2 in the pituitary-adenocortical dysfunction. Such an upregulation is also found in other pathological states of impaired water excretion, including the syndrome of inappropriate secretion of antidiuretic hormone, liver cirrhosis with ascites, and congestive heart failure (2, 5, 20, 35).

What mechanisms underlie in the upregulation of AQP2 mRNA expression in the glucocorticoid-deficient rats? Augmented antidiuresis has been understood as dependent on nonsuppressible levels of plasma AVP compared with hypoosmolality, though plasma AVP levels are not so high, in most settings of water retention (12, 13, 27, 33). An information of the augmentation could be simply accepted by the kidney. If so, how does the kidney distinguish such nonsuppressible levels of plasma AVP from those under normal regulation? As a result, the exaggerated action of AVP is found. We examined [3H]AVP receptor binding in the glucocorticoid-deficient rats. Bmax was ~14% less than that in the control, although it was not significantly different. Kd values were almost equal in these two groups. The expression of AVP V2 receptor mRNA was somewhat stimulated in the glucocorticoid-deficient rats, which might be due to the decrease in receptor numbers. Therefore, at the receptor site the initiation of intracellular signaling is kept in the normal range under the chronically relative AVP excess condition. In the literature, chronic treatment with small amounts of dDAVP for a few days in rats, which did not induce short-term desensitization, increased AVP-induced cAMP production by 30% (24). In the present study, it is clear that the expression of AQP2 mRNA was upregulated in the glucocorticoid-deficient rats. Chronically nonsuppressible levels of plasma AVP do not always downregulate the V2 receptor function and could rather maintain its long-term stimulation on V2 receptor-mediated signaling. In patients with impaired water excretion, the exaggerated urinary excretion of AQP2 was more marked and seemed likely to be dissociated from the positive correlation between urinary excretion of AQP2 and plasma AVP levels in the normal subjects (27). Renal action of AVP is more manifest than that expected from the plasma AVP levels despite hypoosmolality in the patients with impaired water excretion. However, there is no evidence that other factors except for AVP are involved in regulation of AQP2 in renal collecting duct cells. These findings cannot rule out the unknown mechanism for enhancing postreceptor signaling of AVP. Further study will be necessary to elucidate the existence of an enhancer system in the upregulation of AQP2 in pathological state of impaired water excretion, including glucocorticoid deficiency.

In conclusion, we demonstrated impaired water excretion in glucocorticoid-deficient rats. Nonsuppressible release of AVP was found despite hypoosmolality. The expression of AQP2 mRNA and protein was upregulated, and the AVP V2-receptor antagonist totally abolished the increases in their expression. AVP V2 receptor binding capacity and its mRNA expression were maintained. The AQP2 mRNA expression was more manifest in the kidney than that expected from the plasma AVP levels. These results indicate that the upregulation of AQP2 plays a crucial role in impaired water excretion, dependent on AVP, in glucocorticoid deficiency.


    ACKNOWLEDGEMENTS

The present study was supported by grants from the Ministry of Education, Science, and Culture of Japan (no. 10770557). A portion of this study was presented at the 81st Annual Meeting of The Endocrine Society in San Diego, CA, June 12-15, 1999.


    FOOTNOTES

Address for reprint requests and other correspondence: S. Ishikawa, Div. 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).

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.

Received 14 July 1999; accepted in final form 7 April 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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