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1 Department of Cell Biology, The acute effect
of treatment with the vasopressin
V2-receptor antagonist OPC-31260
(OPC) on aquaporin-2 (AQP2) distribution and expression in rat kidney
was examined. Immunofluorescence and semi-quantitative immunoelectron
microscopy revealed that 15 and 30 min of OPC treatment resulted in
significant reduction in apical plasma membrane labeling of AQP2, with
a concomitant increase in labeling of vesicles and multivesicular
bodies. In parallel, OPC treatment induced a large increase in urine
output [0.6 ± 0.2 vs. 8.3 ± 1.0 ml/h
(n = 4)]. Northern blotting
using a 32P-labeled AQP2 cDNA
probe and a digoxigenin-labeled AQP2 RNA probe revealed a band of
~1.6 kb corresponding to the predicted size of AQP2 mRNA. In control
experiments, thirsting increased, whereas water loading decreased AQP2
mRNA levels. Treatment of rats with OPC caused a significant reduction
in AQP2 mRNA within 30 min (52 ± 21%,
n = 8, P < 0.025) and 60 min (56 ± 7%,
n = 4, P < 0.001) of treatment compared
with intravenous saline-injected controls. Thus a very rapid reduction
in AQP2 mRNA was observed in response to vasopressin-receptor
antagonist treatment. The reduction in AQP2 mRNA persisted after 24 h
(40 ± 17%, n = 5, P < 0.05) of OPC treatment. There
was a parallel increase in diuresis and reduction in urine osmolality.
In conclusion, V2-receptor
blockade produced a rapid internalization of AQP2 parallel with a rapid
increase in urine output. Furthermore, OPC treatment caused a rapid and significant reduction in AQP2 mRNA expression, demonstrating that for
rapid regulation of AQP2 expression, modulation of AQP2 mRNA levels is
regulated via vasopressin-receptor signaling pathways.
aquaporin-2; collecting duct; nephrogenic diabetes insipidus; vasopressin antagonist
AQUAPORIN-2 (AQP2) (10) is localized in the
apical plasma membrane and in subapical vesicles in collecting duct
principal cells (32). AQP2 has been shown to be the predominant
vasopressin-regulated water channel of the kidney collecting duct and
is essential for urinary concentration (3, 4; for recent reviews see
Refs. 18, 29, 34). AQP2 is regulated both by short-term and long-term mechanisms. The short-term mechanism, i.e., the acute
vasopressin-induced increase in collecting duct water reabsorption, is
dependent on vasopressin-regulated trafficking of AQP2 between
intracellular vesicles and the apical plasma membrane (26, 31, 38, 45), whereas protein kinase A (PKA) phosphorylation of AQP2 only marginally increases water conductance (20, 22). Long-term regulation of AQP2
involves mechanisms that alter the expression of AQP2, thereby
modulating the acute response by changing the number of water channels
in the cell.
Increased AQP2 expression is seen in response to water restriction (12,
32) or direct vasopressin treatment (4), both of which cause parallel
increases in water permeability and AQP2 levels in collecting ducts in
Brattleboro rats (4). Lack of functional AQP2 in humans is associated
with very severe primary nephrogenic diabetes insipidus (NDI) (3).
Moreover, it has been shown that lithium treatment, hypokalemia, and
ureteral obstruction, all common causes of NDI, are associated with a
marked downregulation of AQP2 and reduction of AQP2 in the apical
plasma membrane (7, 8, 24, 25). A reduction in AQP2 levels therefore
appears to be a general mechanism in the development of multiple
acquired forms of NDI. Conversely, increased expression and apical
plasma membrane targeting of AQP2 are found in association with
conditions of extracellular fluid expansion, such as congestive heart
failure (36, 44) and liver cirrhosis (9). This is believed to be a
consequence of increased levels of circulating vasopressin. Thus AQP2
is critically involved both in short-term and long-term regulation of
normal body water balance and in multiple water balance disorders (for
recent reviews, see Refs. 18 and 34).
The signaling mechanisms involved in modulation of AQP2 expression have
proved to be very complex and are currently poorly understood. Although
it is well established that vasopressin can induce the expression of
AQP2 in vasopressin-deficient Brattleboro rats (4) as well as normal
rats (6), it has recently become clear that vasopressin-independent
mechanisms also are important. This was shown initially in a study (24)
where thirsting of rats with lithium-induced NDI produced a much
greater increase in AQP2 expression than did vasopressin treatment of
such NDI rats. Recently, the existence of vasopressin-independent
pathways was demonstrated in a extensive study (6). Rats with clamped high levels of vasopressin (vasopressin administered via osmotic minipumps) showed downregulation of AQP2 in response to water loading
despite the high levels of circulating vasopressin (a level of
vasopressin shown to induce complete antidiuresis and a
marked increase in AQP2 expression in the absence of water loading). Thus both vasopressin-mediated and vasopressin-independent mechanisms are involved in governing the levels of AQP2 expression.
The complexity of the regulatory mechanisms, combined with the
importance of AQP2 in water balance physiology and pathophysiology (18), emphasizes the importance of detailed studies of each step in the
signaling pathways. The present study was undertaken to analyze the
initial events after acute in vivo treatment with the
vasopressin-receptor antagonist OPC-31260, by use of high-resolution immunocytochemistry, semi-quantitative Northern blotting, and functionally by monitoring the diuretic response. The purpose was to
test whether vasopressin-receptor antagonist treatment would cause
rapid reduction in AQP2 mRNA levels. This would demonstrate that acute
regulation of AQP2 expression is under the influence of vasopressin
receptors. Second, we examined the changes in the subcellular
localization of AQP2 immediately after vasopressin-receptor antagonist
treatment to investigate the offset response in vivo. The diuretic
effect of the antagonist treatment was continuously monitored.
Experimental Animals
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
OPC-31260 was given via the femoral vein (1 mg in 0.2 ml vehicle/animal) to animals briefly anesthetized with isofluran. During surgery, rats were placed on a heating table. Rats were then awaken for 15, 30, and 60 min. Anesthesia was then repeated and kidneys were either perfusion-fixed for immunocytochemistry or removed for RNA isolation and preparation of membrane fractions.
In other experiments, OPC was given orally (5 mg in 5 g food every 12 h) for 24 h. In separate experiments (protocols described below) rats were thirsted or water loaded for 48 h. For water loading, drinking water containing 300 mM sucrose was supplied. Prior to removal of the kidneys, rats (see protocol below) were anesthetized with intraperitoneal pentobarbital sodium (24).
Experimental Protocols
The following protocols were followed.Protocol 1. Rats were either thirsted for 48 h (n = 5 rats), water loaded for 48 h using 300 mM sucrose in drinking water (n = 5 rats), or had free access to water (n = 5).
Protocol 2. Rats were treated intravenously with OPC-31260 (1 mg) for 15 min (n = 9 rats). Control rats (shams) received intravenous saline (n = 8 rats). Kidneys were removed for RNA isolation. Additional rats were treated according to the same protocol, and kidneys were perfusion fixed for immunocytochemistry (OPC; n = 3 rats and controls, n = 4 rats).
Protocol 3. Rats were treated intravenously with OPC-31260 (1 mg) for 30 min (n = 8 rats). Control rats (shams) received intravenous saline (n = 4 rats). Kidneys were removed for RNA isolation. Additional rats were treated according to the same protocol, and kidneys were perfusion fixed for immunocytochemistry (OPC; n = 4 rats and controls; n = 4 rats).
Protocol 4. Rats were treated intravenously with OPC-31260 (1 mg) for 60 min (n = 4 rats). Control rats (shams) received intravenous saline (n = 4 rats).
Protocol 5. Rats were maintained for 5 days on standard rat diet (5 g twice a day), followed by 24 h on food containing OPC-31260 (5 mg OPC-31260/5 g food) given twice daily (n = 5 rats). Control rats were maintained on standard rat diet (n = 5 rats).
Immunocytochemistry
Immunocytochemistry was performed essentially as previously described (30, 31). Kidneys were perfusion fixed with 0.1% or 0.2% glutaraldehyde plus 2% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, via the abdominal aorta. Tissue blocks were prepared from kidney inner medulla. The blocks were infiltrated with 2.3 M sucrose/2% paraformaldehyde for 30 min, mounted on holders, and rapidly frozen in liquid nitrogen. Frozen tissue blocks were either directly used for cryosectioning or subjected to a cryosubstitution and Lowicryl HM20 embedding prior to ultramicrotomy. Cryosubstitution was performed as described previously (28, 35). The frozen samples were freeze-substituted 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
to 70°C and then rinsed in pure methanol for 24 h at
70 to 45°C and infiltrated at 45°C with Lowicryl HM20 and methanol 1:1, 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.
Semithin (0.85 µm) cryosections and ultrathin (80 nm) Lowicryl sections, cut on a Reichert Ultracut FCS, were preincubated with PBS containing 0.1% skimmed milk and 0.05 M glycine (for light microscopy) or with TBST (0.05 M Tris, pH 7.4, 0.1% Triton X-100) containing 0.1% sodium borohydride and 0.05 M glycine followed by incubation with TBST containing 2% BSA or 0.2% skimmed milk (for electron microscopy). The preincubation was followed by incubation with affinity-purified antibodies against AQP2 (100-800 ng IgG/ml). For fluorescence microscopy, the labeling was visualized using fluorescein-conjugated secondary antibody (Z205, diluted 1:40; DAKO, Copenhagen, Denmark). For immunoelectron microscopy, goat-anti-rabbit-gold (GAR10 or GAFR, 10-nm colloidal gold particles; Biocell Research Laboratories, Cardiff, UK) was used, and grids were stained in uranyl acetate for 10 min and in lead citrate for 5 s. Immunolabeling controls using nonimmune IgG as primary antibody substituting for anti-AQP2 revealed a complete absence of labeling. Fluorescence microscopy was performed using a Leica Laborlux S microscope, and electron microscopy was performed using a Philips CM100 electron microscope.
Semiquantitation of AQP2 Immunogold Labeling
Electron micrographs were taken of the apical part of inner medullary collecting duct (IMCD) principal cells from animals treated with OPC-31260 (intravenously) for 30 min (n = 4) and sham-operated animals receiving saline treatment (intravenously) for 30 min (n = 4). Electron micrographs were printed at a final magnification of ×63,000. The number of gold particles associated with apical plasma membranes, intracellular vesicles, and multivesicular bodies was determined. Gold particles in structures that could not be identified distinctly as vesicles or multivesicular bodies or apical plasma membrane were counted separately. This is likely to represent labeling in tangentially sectioned vesicles and labeling in tangential sections of part of rough endoplasmic reticulum. There was only a weak background labeling, since only occasionally gold particles were observed in nuclei and in the lumen. Immunolabeling controls using preadsorbed anti-AQP2 or nonimmune IgG also revealed low background labeling.Purification of Total RNA
Total RNA from inner medulla was extracted by the acid guanidinium thiocyanate-phenol-chloroform method with modifications (2). One frozen inner medulla was homogenized (Ultra-Turrax T8, IKA Labortechnik) for 30 s with 1 ml solution D [741 mg/ml guanidinium thiocyanate, 39 mM sodium citrate (pH 7.0), 0.78% sarcosyl, 7 µl/ml
-mercaptoethanol], 100 µl of 2 M sodium
acetate (pH 4.0), and 1 ml water-saturated phenol. Subsequently, 0.15 volume CHCl3/isoamylalcohol (49:1)
was added to the homogenate. The final suspension was cooled on ice for
30 min, and samples were then centrifuged at 4,000 g for 30 min at 4°C. The aqueous phase containing the RNA was mixed with 1 vol isopropanol and left at
20°C for 15 min followed by centrifugation at 4,000 g for 30 min at 4°C. The resulting
RNA pellet was washed in 70% ethanol and finally dissolved in 50 µl
TE buffer (10 mM Tris, 0.1 mM EDTA, pH 7.4) and frozen.
Northern Blot Analysis
Northern blot analyses were performed using an [
-32P]dCTP-labeled
human AQP2 cDNA probe and a digoxigenin-labeled RNA probe. Total RNA
was separated on a 1% agarose and 2% or 6% formaldehyde gel
followed by blotting on a nylon membrane filter (Hybond-N; Amersham Life Science, Buckinghamshire, UK). To reveal the size of AQP2
mRNA, a digoxigenin-labeled RNA molecular weight marker (Boehringer,
Mannheim, Germany) was used. Prehybridization was performed at 55°C
for 30 min in 5× SSC, 50% formamide, 0.1% sarcosyl, 0.02% SDS,
and 2% blocking solution (blocking reagent in maleic acid,
Boehringer). 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; Ambion, Austin, TX). After
hybridization, blots were washed in 2× SSC + 0.1% SDS at room
temperature for two 5-min periods followed by washing 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-digoxiginin-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: disodium
3-(4-methoxyspiro{1,2-dioxetane-3,2'-(5'-chloro)tricyclo[3.3.1.13,7]decan}-4-yl)
phenyl phosphate (CSPD, Boehringer).
Northern blot analysis using the radioactive probe was performed by
prehybridization at 42°C for 2 h in 5× SSPE, 5×
Denhardt's solution, 0.5% SDS, 50% formamide, and 67 µg/ml
denatured salmon sperm DNA. After prehybridization, the blots were
hybridized with an
[-32P]dCTP-labeled
human AQP2 cDNA probe (specific activity, 3,000 Ci/mmol, Amersham) at
42°C for 24 h. cDNA probe labeling was performed using
random-primed method (Prime-It II Random Primer Labeling Kit,
Stratagene). After hybridization, the blots were washed in 2× SSC + 0.1% SDS at room temperature for 15 min followed by washing in
1× SSC + 0.1% SDS at 68°C for 30 min. Finally, the blots
were exposed to high-performance autoradiography film at
70°C for 24 h.
Membrane Fractionation for Immunoblotting
The inner medulla was dissected from each kidney, minced finely, and homogenized in 10 ml of dissecting buffer [0.3 M sucrose, 25 mM imidazole, and 1 mM EDTA, pH 7.2, and containing the protease inhibitors leupeptin (8.5 µM) and phenylmethylsulfonyl fluoride (1 mM)] with 5 strokes of a motor-driven Potter-Elvehjem homogenizer, at 1,250 rpm (24, 26). This homogenate was centrifuged in a Beckman L8M centrifuge at 4,000 g for 15 min at 4°C. To increase membrane yield, the pellet was rehomogenized with 3 strokes, and the centrifugation was repeated. The supernatants were pooled, then centrifuged at 200,000 g for 1 h. The resultant pellet was resuspended in dissecting buffer.Electrophoresis and Immunoblotting
Samples of membrane fractions from inner medulla (1 µg/lane) were subjected to SDS-PAGE (21) using 12% polyacrylamide minigels (Bio-Rad Mini Protean II), and transferred to nitrocellulose paper by electroelution. Blots were blocked with 5% milk in PBS with Tween 20 (PBS-T: 80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated overnight at 4°C with antibody raised against the COOH-terminal 22 amino acids of AQP2 (either serum, diluted 1:1,000, or affinity purified with the immunizing peptide, diluted to 40 ng IgG/µl in PBS-T plus 1% bovine serum albumin). After washing in PBS-T, the blots were incubated for 1 h with horseradish peroxidase-conjugated secondary antibody (P448, diluted 1:3,000; DAKO). After final washing in PBS-T, AQP2 was visualized using enhanced chemiluminescence (ECL; Amersham International, Buckinghamshire, UK).Statistical Analysis
Densitometry of Northern and immunoblots. For Northern and immunoblots, samples from OPC-31260-treated, thirsted, and water-loaded animals were run on gels with corresponding samples from sham/control animals. Films were scanned using a UMAX VISTA-S8 scanner and Adobe Photoshop Software. The scanning was performed using ECL exposures that gave bands in lower gray scale where there is a linear correlation between signal and protein levels (24). The labeling density was quantitated using specially written software (26). AQP2 labeling in the samples from the experimental animals was calculated as a fraction of the mean sham/control value for that film.Densitometry of AQP2 protein levels. Both the 29-kDa and the 35- to 50-kDa bands (corresponding to nonglycosylated and the glycosylated AQP2) were scanned (24, 31, 32). Values were corrected by densitometry of Coomassie-stained preliminary gels.
Densitometry of AQP2 mRNA levels. The band of ~1.6 kb corresponding to AQP2 mRNA on the autoradiography film was scanned. Values were corrected by densitometry of 18S and 28S rRNA bands visualized by ethidium bromide on the same blot. Values are presented in the text as means ± SE. Comparisons between groups were made by unpaired t-test. P < 0.05 was considered significant.
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Short-Term Effects of V2-Receptor Antagonist Treatment on AQP2 Distribution
As shown in Fig. 1, intravenous treatment of rats with the V2-receptor antagonist OPC-31260 (OPC) induced a rapid increase in urine output (n = 4), consistent with what was previously described (46).
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The localization of AQP2 water channel protein in kidney inner medulla of control rats and rats treated with OPC for 15 min and 30 min was investigated by immunofluorescence microscopy using 0.85-µm cryosections (Fig. 2). These studies showed that in control rats, AQP2 labeling was mainly associated with apical plasma membrane domains and subapical vesicles (Fig. 2A) in collecting duct principal cells. After 15 min of OPC treatment, apical labeling was reduced with a concomitant increase in labeling of vesicles (Fig. 2B). After 30 min of OPC treatment, only sparse labeling of the apical plasma membrane domains was observed, with vesicular labeling observed deeper in the cells (Fig. 2C). A striking feature was the concentration of labeling in larger structures representing clusters of vesicles and multivesicular bodies (see below). Immunolabeling controls were negative (Fig. 2D).
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Immunoelectron microscopy using ultrathin HM20 Lowicryl sections (Figs. 3-5; Table 1) confirmed the subcellular distribution of AQP2, as seen by immunofluorescence microscopy. Extensive AQP2 labeling was associated with the apical plasma membrane in control rats (Fig. 3, A and B). After 15 or 30 min of OPC treatment, less labeling was associated with the apical plasma membrane, with some cells showing almost complete absence of labeling in the apical plasma membrane (Figs. 4 and 5A), whereas other cells showed weak labeling, although the labeling in the apical plasma membrane was much less than in control animals. Most labeling was associated with vesicles and multivesicular bodies (Fig. 5, B and C). To further evaluate the changes in the subcellular distribution of AQP2, semiquantitation of AQP2 immunogold labeling was performed in the apical part of IMCD principal cells from control rats and rats treated with OPC-31260 for 30 min (Table 1). In control rats, the fraction in the apical plasma membrane of the total labeling was 0.21 ± 0.01 (n = 4), whereas in OPC-treated animals the fraction was markedly reduced to 0.037 ± 0.001 (n = 4). In contrast, the fraction of labeling in intracellular vesicles and multivesicular bodies in OPC-treated animals was higher than in control animals. Thus V2-receptor blockade produced a significant internalization of AQP2 with reduction of apical plasma membrane labeling, as shown by immunoelectron microscopy. This is consistent with the shuttle mechanism as described previously (26, 31, 38, 45).
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Expression of AQP2 mRNA and Protein in Thirsted and Water-Loaded Rats
To evaluate the Northern blotting and hybridization procedures including the probes, control experiments were performed with thirsted or water-loaded rats, conditions known to be associated with changes both in AQP2 protein and mRNA levels (12, 23, 32). Total RNA purified from kidney inner medulla was analyzed by Northern blotting (Fig. 6, A and B). As shown in Fig. 6A, a band of ~1.6 kb was detected, consistent with the predicted size of AQP2 mRNA (10). Occasionally a weak higher molecular weight band with a size between 2.8 kb and 5.3 kb was also observed, which may correspond to a splicing variant or a polyadenylation variant of AQP2 mRNA, as previously described (10). Compared with control animals, thirsting induced a 2.8-fold increase in AQP2 mRNA expression (~1.6-kb band), whereas water loading decreased the expression of AQP2 mRNA to 54 ± 15% of AQP2 levels in control and to 20 ± 5% of that observed in thirsted animals (Table 2). Corresponding changes were observed for AQP2 protein levels (Fig. 7A). Immunoblots of membrane fractions from kidney inner medulla of animals subjected to thirsting or water loading for 48 h revealed a major band at 29 kDa, corresponding to nonglycosylated AQP2, and a broad band at 35-50 kDa, corresponding to glycosylated forms of AQP2 (24, 31, 32). Consistent with the increase in AQP2 mRNA expression, AQP2 protein expression increased significantly in thirsted relative to water-loaded animals (P < 0.005), consistent with previous observations (12, 32, 39, 41).
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Expression of AQP2 mRNA in Rats Treated with V2-Receptor Antagonist, OPC-31260
To investigate whether the expression of AQP2 mRNA is regulated under the influence of the vasopressin V2 receptor, rats were treated with the vasopressin V2-receptor antagonist, OPC-31260, for 15 min, 30 min, 60 min (1 mg iv), and 24 h (5 mg/12 h orally). Northern blotting of total RNA purified from kidney inner medulla of OPC-treated animals and sham animals showed a band of ~1.6 kb corresponding to AQP2 mRNA (Figs. 8 and 9). No reduction in AQP2 mRNA expression was observed in response to 15 min of OPC treatment compared with shams (Table 2). However, a significant reduction in AQP2 mRNA expression levels was observed after 30 min of treatment (Fig. 8), and the reduction persisted after 60 min (Fig. 9). The reduction corresponded to 48 ± 6% (n = 8, P < 0.025) of control levels after 30 min and to 44 ± 3% (n = 4, P < 0.001) after 60 min. The effect on urine production of OPC treatment was also determined in the same rats. Rats were kept in metabolic cages for 60 min after intravenous OPC-31260 administration or saline administration, and the urine was collected. As shown in Table 3, OPC treatment induced a marked increase in urine output [0.6 ± 0.2 vs. 8.3 ± 1.0 ml/h (n = 4)]. In parallel, a decrease in urine osmolality was observed (659 ± 145 vs. 177 ± 18 mosmol/kgH2O). AQP2 protein expression in rats treated with OPC for 60 min (1 mg iv) was analyzed by immunoblotting. In contrast to the significant reduction in AQP2 mRNA levels observed after 60 min, no significant change in AQP2 protein expression was noted (Fig. 7B). The rapid downregulation of AQP2 mRNA expression within 30 min (Figs. 8 and 9), strongly suggests a major role of V2-receptor signaling pathways in modulating AQP2 mRNA levels.
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To examine whether AQP2 mRNA levels would further decrease with prolonged OPC treatment, we extended OPC treatment for 24 h. The reduction in AQP2 mRNA levels was maintained at 60 ± 15% of control levels after 24 h (n = 5, P < 0.05; Table 2). Urine output was determined in the same animals (Table 3). After 24 h of OPC treatment, urine production was increased fivefold from 0.32 ± 0.05 to 1.59 ± 0.64 ml/h (Table 3) with a decrease in urine osmolality. The ability of 24-h OPC treatment to maintain reduced AQP2 mRNA levels further substantiates a role of V2-receptor signaling pathways in regulating AQP2 expression. However, the absence of a further reduction in AQP2 mRNA levels after 24 h of OPC treatment is also consistent with the existence of additional vasopressin-independent mechanisms involved in long-term regulation of AQP2 expression.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Immunofluorescence and immunoelectron microscopy demonstrated that vasopressin V2-receptor antagonist treatment in vivo caused a rapid retrieval of AQP2 from the apical plasma membrane into vesicles and multivesicular bodies. This is consistent with a critical role of water channel retrieval in the reduction of collecting duct water permeability. In parallel, treatment with the V2-receptor antagonist was associated with development of extensive polyuria and reduced urine osmolality. Semiquantitative Northern blotting showed a rapid reduction in AQP2 mRNA levels in response to V2-receptor antagonist treatment. This demonstrates that for rapid regulation of AQP2 expression, mRNA levels are tightly regulated via vasopressin receptors. The reduction in AQP2 mRNA levels were maintained after continuous OPC treatment for 24 h, which supports the view that V2-receptor signaling pathways also play an important role in the long-term regulation of AQP2 expression. However, the absence of further reduction in AQP2 mRNA levels after 24 h treatment is also consistent with the known existence of additional vasopressin-independent mechanisms involved in long-term regulation of AQP2 expression.
Retrieval of AQP2 in Response to Vasopressin Receptor Antagonist Treatment
Body water balance is tightly regulated by vasopressin, which increases the water permeability of the collecting duct. As expected (46), vasopressin V2-receptor blockade induced severe polyuria, as shown in Fig. 1 and Table 3. Immunocytochemistry revealed that short-term treatment with the V2-receptor antagonist produced a rapid internalization of AQP2 from the apical plasma membrane to intracellular vesicles and multivesicular bodies. This demonstrates that the offset response to vasopressin action in vivo in normal rats involves AQP2 internalization. These data are in agreement with previous observations of an increased apical plasma membrane targeting of AQP2 in response to vasopressin treatment in vivo (26, 38, 45) and confirm the internalization of AQP2 in response to vasopressin removal in an in vitro model of isolated perfused IMCD (31). A reduction in AQP2 plasma membrane labeling in response to removal of vasopressin or forskolin has also been clearly demonstrated in cell systems transfected with AQP2 (16, 17). Reduction in AQP2 plasma membrane labeling has also been observed after long-term treatment with OPC-31260 for 3 days (12), although this study did not allow evaluation of the acute steps in AQP2 trafficking.The extensive accumulation of AQP2 in vesicles in response to treatment with vasopressin-receptor antagonist is consistent with the hypothesis that at least part of the vesicular AQP2 is available for recycling in vasopressin-triggered externalization. However, the observation that multivesicular bodies were also significantly labeled after antagonist treatment suggests that not all intracellular AQP2 is available for recycling, since multivesicular bodies are thought to represent a site for removal/degradation of cell contents. Consistent with the present data, AQP2 has also been found in multivesicular bodies (25, 32), e.g., in conditions of AQP2 downregulation such as hypokalemia, where AQP2-labeled multivesicular bodies are prominent in collecting duct principal cells.
AQP2 contains a consensus site for PKA phosphorylation in the cytoplasmic COOH terminus. It has recently been shown that this Ser256 PKA phosphorylation site may be involved in the vasopressin-induced trafficking of AQP2 from intracellular vesicles to the plasma membrane in AQP2-transfected LLC-PK1 cells (17). Thus it appears likely that PKA phosphorylation of AQP2 in vesicles would trigger externalization of AQP2 and insertion into the apical plasma membrane. This is supported by recent studies indicating a very rapid vasopressin-stimulated phosphorylation of AQP2 in situ (as early as 1 min after vasopressin stimulation) using slices of kidney papilla (G. Nishimoto, M. Yasui, D. Li, M. Zelenina, S. Nielsen, A. Aperia, and A. C. Nairn, unpublished observations), fully within the time course of vasopressin-stimulated water permeability (19, 43).
The stoichiometry of AQP2 phosphorylation may also be very important. It is speculated that phosphorylation of more than one monomer within the homotetramer may be required to trigger externalization to the plasma membrane. The offset response remains to be characterized, and internalization of AQP2 by endocytosis in response to vasopressin removal or vasopressin-receptor antagonist treatment may potentially require dephosphorylation. Evaluation of the role of AQP2 phosphorylation in trafficking will require knowledge of the subcellular compartment in which AQP2 undergoes phosphorylation. Furthermore, it is also unknown whether PKA phosphorylation of other components is involved in the vasopressin response. It has been shown that cAMP stimulates protein kinase activity in homogenates of bovine kidney. Moreover, the ability of purified PKA to phosphorylate various membrane proteins was demonstrated (5). In saponin-permeabilized outer medullary collecting duct segments, vasopressin treatment was shown to induce phosphorylation of at least two proteins of 45 and 66 kDa (11). Thus it can be speculated that in addition to AQP2, other proteins may also be phosphorylated and play distinct roles in mobilizing the hydrosmotic response to vasopressin. Although studies have indicated that both the onset and offset vasopressin responses are regulated (33), the regulation of the offset response remains unknown. Our data support the view that vasopressin withdrawal, here performed by vasopressin-receptor antagonist treatment, is critical to the internalization of AQP2.
Vasopressin Regulation of AQP2 Expression
We here provide direct evidence for a rapid downregulation of AQP2 mRNA in response to vasopressin-receptor antagonist treatment in vivo, pointing to an important role for vasopressin V2-receptor signaling pathways in rapid downregulation of AQP2 expression. Consistent with these results, it has previously been shown that treatment with vasopressin for 60 min increases the expression of AQP2 mRNA (13) and long-term treatment of DDAVP results in a several-fold increase in AQP2 protein expression in Brattleboro rats (4) and in mRNA and protein expression in normal rats (6).The AQP2 gene contains a cAMP-responsive element (CRE) (42), and several studies have evaluated the importance of vasopressin receptor signaling pathways and cAMP on AQP2 promoter-reporter constructs in cultured kidney cells (15, 27, 47). Hozawa et al. (15) found that treatment with cAMP-enhancing agents (vasopressin or forskolin and 3-isobutyl-1-methylxanthine) significantly induced AQP2 promoter activity in LLC-PK1 cells (which normally express V2-receptors) and IMCD cells transfected with an AQP2 promoter-luciferase construct. Moreover, in IMCD cells, deletion of the CRE of the transfected AQP2 promoter construct resulted in partial loss of cAMP responsiveness, and removal of the remaining binding sites for transcriptional activators (including AP1) completely abolished cAMP responses. By using chloramphenicol acetyltransferase fused with a fragment of the human AQP2 promoter (AQP2-CAT) as a reporter gene, Yasui et al. (47) showed that treatment with vasopressin, DDAVP, and cAMP all resulted in markedly increased CAT activity in LLC-PK1 cells. Furthermore, activation of the adenylate cyclase-coupled V2 receptor induced phosphorylation of CRE binding protein (CREB), presumably PKA catalytic subunits, and leads to expression of another transcriptional factor c-Fos. Binding of these factors to CRE and AP1 lead to increased AQP2-CAT promoter activity. Finally, mutations in CRE and AP1 in AQP2-CAT markedly reduced promoter activity, confirming that vasopressin activation of the AQP2-CAT promoter requires intact CRE and AP1. Since LLC-PK1 cells do not express functional AQP2, the relative importance of the signal mechanism to other potential regulators of AQP2 expression cannot be evaluated. Matsumura et al. (27) recently used an AQP2 mRNA-expressing collecting duct cell line established from a transgenic mouse harboring the temperature-sensitive simian virus SV40. By using a luciferase assay performed with various 5'-flanking regions of the human AQP2 gene, they found that CRE appeared to be the predominant element responsible for cAMP-induced transcriptional regulation (27). Thus all of these studies point directly to an important role for V2-receptor signaling pathways, including cAMP, in the regulation AQP2 expression (15, 27, 47). In addition, transcriptional regulation changes in AQP2 mRNA levels may be speculated also to depend on factors that influence mRNA stability. The very rapid downregulation of AQP2 mRNA in response to vasopressin-receptor antagonist treatment would be consistent with this view. However, further studies are necessary to characterize this in detail.
Other renal genes containing CRE have also shown rapid changes in mRNA expression (14, 37). In transgenic mice carrying a bovine growth hormone (bGH) gene fused with the promoter of the phosphoenolpyruvate carboxykinase (PEPCK) gene, a twofold increase in the level of endogenous renal PCK mRNA was observed within 7 h of onset of acidosis (14). Similarly, in transgenic mice carrying the bGH gene fused with a segment of the promoter of PEPCK, treatment with cAMP for 90 min caused a twofold increase in PEPCK mRNA in the liver (37).
In addition to the long-term regulation of AQP2 expression directly by vasopressin, altered expression of AQP2 mRNA and protein is also seen in response to water restriction and water loading (12, 32, 39). Consistent with this, here we show that AQP2 mRNA and protein expression increases in response to thirsting and decreases in response to water loading (Figs. 6 and 7; Table 2). It remains, however, unclear whether the effects of thirsting or water loading are entirely dependent on vasopressin V2-receptor activation and deactivation, respectively. Certainly, the observation that Brattleboro rats, which lack vasopressin secretion, do not mount an increase in AQP2 expression in response to thirsting (41) would be consistent with an important role of vasopressin receptor signaling pathways. However, there are also several lines of evidence in favor of involvement of vasopressin-independent factors in the thirsting response. First, it has been shown that thirsting results in a much higher increase in AQP2 expression in lithium-induced NDI rats than does DDAVP treatment (although DDAVP was even more effective than thirsting in reducing the water intake and urine output) (24). Second, recent studies using a rat model for vasopressin escape showed that in the continued presence of high levels of vasopressin, water loading resulted in a severe downregulation of AQP2 expression (6). Thus these studies indicate that changes in AQP2 expression in response to water loading or thirsting may be at least in part dependent on signaling mechanisms other than the V2-receptor signaling pathways or at least of the proximal parts of this signaling cascade including the vasopressin V2-receptor. It cannot be excluded that thirsting/water loading may influence, for example, cAMP levels by altering the activity of phosphodiesterases or prostaglandins. It has recently been shown that dehydration of rats results in an increased binding of one or more transcriptional factors, possibly including CREB, to the CRE of the AQP2 promoter in nuclear extracts from rat kidney papilla (27). Thus thirsting may activate the same transcriptional factors but possibly also ones other than those mediating the V2-receptor activation.
Our results demonstrated a maintained reduction in AQP2 mRNA levels after 24 h of vasopressin-receptor antagonist treatment, consistent with the view that modulation of AQP2 expression is dependent on V2-receptors. However, the absence of a further reduction in AQP2 mRNA levels would be consistent with the view that long-term modulation of AQP2 expression may not be mediated exclusively by V2-receptors. In conclusion, our results clearly demonstrate that the V2-receptor signaling pathway is critically involved in the acute regulation of AQP2 mRNA expression and also plays a role in long-term regulation of AQP2 mRNA expression.
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ACKNOWLEDGEMENTS |
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We thank Trine Møller, Annette Blak Rasmussen, Mette Vistisen, and Hanne Weiling for expert technical assistance.
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FOOTNOTES |
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Support for this study was provided by the Karen Elise Jensen Foundation; The Medical Faculty, University of Aarhus; Novo Nordisk Foundation; and University of Aarhus Research Foundation. The OPC-31260 compound was kindly provided by Otsuka Pharmaceutical, Tokyo, Japan.
Address for reprint requests: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus, Denmark.
Received 24 September 1997; accepted in final form 7 May 1998.
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REFERENCES |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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T. E. N. Jonassen, D. Promeneur, S. Christensen, J. S. Petersen, and S. Nielsen Decreased vasopressin-mediated renal water reabsorption in rats with chronic aldosterone-receptor blockade Am J Physiol Renal Physiol, February 1, 2000; 278(2): F246 - F256. [Abstract] [Full Text] [PDF]
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B. M. Christensen, M. Zelenina, A. Aperia, and S. Nielsen Localization and regulation of PKA-phosphorylated AQP2 in response to V2-receptor agonist/antagonist treatment Am J Physiol Renal Physiol, January 1, 2000; 278(1): F29 - F42. [Abstract] [Full Text] [PDF]
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 S. NIELSEN, T.-H. KWON, B. M. CHRISTENSEN, D. PROMENEUR, J. FRØKI&Aelig;R, and D. MARPLES Physiology and Pathophysiology of Renal Aquaporins J. Am. Soc. Nephrol., March 1, 1999; 10(3): 647 - 663. [Abstract] [Full Text]
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D. Marples, J. Frokiaer, and S. Nielsen Long-term regulation of aquaporins in the kidney Am J Physiol Renal Physiol, March 1, 1999; 276(3): F331 - F339. [Abstract] [Full Text] [PDF]
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D. Marples, B. M. Christensen, J. Frokiaer, M. A. Knepper, and S. Nielsen Dehydration reverses vasopressin antagonist-induced diuresis and aquaporin-2 downregulation in rats Am J Physiol Renal Physiol, September 1, 1998; 275(3): F400 - F409. [Abstract] [Full Text] [PDF]
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