1 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000; 2 Department of Clinical Physiology, Aarhus University Hospital, and Institute of Experimental Clinical Research, DK-8200 Aarhus N, Denmark; and 3 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892
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ABSTRACT |
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The role of AVP-V2 receptor (AVP-V2R)-dependent regulation of aquaporin-2 (AQP2) expression was evaluated in vasopressin-deficient Brattleboro (BB) rats. AQP2 levels were relatively high in BB rats (52 ± 8% of levels in Wistar rats), and treatment with the AVP-V2R antagonist SR-121463A (0.8 mg/day) for 48 h was associated with 1) increased urine output (170 ± 9%), 2), reduced AQP2 protein levels (42 ± 10% in whole kidney and 53 ± 8% in inner medulla), and 3) reduced AQP2 mRNA levels (36 ± 7%). In addition, the levels of AQP2 phosphorylated in the protein kinase A (PKA) consensus site (Ser256 of AQP2) was reduced to 3 ± 1% of control levels. Lithium (Li) treatment of BB rats for 1 mo, known to reduce adenylyl cyclase (AC) activity, downregulated AQP2 protein levels (15 ± 6%) and increased urine output (220%). Downregulation of AQP2 expression in response to SR-121463A or Li treatment indicates that AQP2 expression in BB rats depends in part on activation of AVP-V2Rs and that the signaling cascade(s) involves AC and hence cAMP. Complete water restriction of BB rats produced only a small increase in AQP2 mRNA (235 ± 33%) and AQP2 protein (156 ± 22%) levels. Immunoelectron microscopy confirmed the increase in AQP2 abundance but revealed no change in AQP2 apical plasma membrane labeling in response to thirsting. In conclusion, the expression and phosphorylation of AQP2 in BB rats are in part dependent on AVP-V2R signaling, and AVP-V2-mediated regulation of AQP2 trafficking and expression is effectively decoupled in BB rats, indicating differences in AVP-V2R-mediated regulation of AQP2 trafficking and expression.
arginine vasopressin; aquaporin; collecting duct; urinary concentrating mechanism; water transport
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INTRODUCTION |
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RENAL WATER EXCRETION IS REGULATED by vasopressin, which increases the water permeability in the collecting duct (15, 33). Recently, the water channel that predominantly mediates the water transport in the collecting duct in response to vasopressin, namely AQP2, has been identified (13). AQP2 is abundant in the apical plasma membrane and subapical vesicles in the collecting duct principal cells (35) and at a lower abundance in the connecting tubules (22). It is well established that AQP2 is the chief target for the action of vasopressin to regulate collecting duct water reabsorption and hence body water balance. Regulation of AQP2 is based in part on short-term mechanisms (within minutes) involving the trafficking of AQP2-bearing subapical vesicles to the apical plasma membrane [see recent review (36)]. In addition, AQP2 is regulated by long-term mechanisms (hours to days) that involve changes in the overall abundance of AQP2 in the collecting duct principal cells. This may reflect changes in transcription of the AQP2 gene, changes in the stability of AQP2 mRNA, or changes in the degradation of AQP2 protein [see recent review (31)]. Regulation of AQP2 transcription appears to involve a cAMP-responsive element in the 5' flanking element region of the AQP2 gene (20, 42, 46). Increased AQP2 expression was seen in normal rats subjected to water restriction or treated with a vasopressin V2-receptor agonist [1-desamino-8-D-arginine vasopressin (DDAVP)] for 24 h or more (18, 35, 41, 46). A marked increase in AQP2 expression was also observed in vasopressin-deficient Brattleboro (BB) rats treated with vasopressin for 5 days (7). Conversely, a decreased AQP2 expression was reported in normal rats treated with a vasopressin V2-receptor antagonist for 60 h or rats treated with lithium for 10 or 25 days (29, 30). Thus both short-term regulation (via trafficking) and long-term regulation (via abundance) of AQP2 seem to be tightly regulated by vasopressin and cAMP/protein kinase A (PKA) signal transduction pathway. However, some studies have revealed that AQP2 may also be regulated via vasopressin-independent mechanisms as well, which may be of significant physiological and pathophysiological importance. The first evidence came from the observation that water restriction of rats with lithium-induced nephrogenic diabetes insipidus (NDI) resulted in a much more pronounced increase in AQP2 expression than sustained DDAVP treatment, although both treatments corrected the polyuria (30). The second evidence is from water-loaded rats, which were at the same time treated with DDAVP via osmotic minipumps. Water loading markedly decreased AQP2 expression in these rats despite their relatively high plasma levels of circulating DDAVP (8). Additional evidence for a vasopressin-independent regulation of AQP2 also came from studies of experimental nephrotic syndrome, experimental acute renal failure, or experimental chronic renal failure (11, 24, 25). AQP2 protein levels were reduced in these conditions known to be associated with normal or increased plasma vasopressin levels.
A major problem in fully understanding the mechanisms involved in AQP2 regulation comes from observations using BB rats, which lack hypothalamic vasopressin secretion. Several studies indicate that AQP2 expression levels are quite high, corresponding to 30-40% of the levels seen in Long-Evans rats, the parent strain of BB rats (7, 23). This relatively high expression intuitively indicates the presence of a substantial vasopressin-independent induction of AQP2 expression but this has never been formally tested. Despite the relatively high levels of AQP2 protein expression the apical plasma membrane targeting is almost completely blocked in BB rats (7, 16, 39, 44). In the present study, we evaluated the potential role of AVP-V2-receptor-dependent and -independent regulation of AQP2 expression (mRNA or protein) using vasopressin-deficient BB rats. We first assessed the levels of AQP2 in BB relative to those seen in a normal strain of rats (we used Wistar rats for this purpose). We also examined in BB rats the effects of different treatments known to affect AQP2 expression in normal rats. 1) The levels of AQP2 expression were determined in BB rats and compared with those in normal rats. 2) BB rats were treated with a vasopressin V2R antagonist for 48 h to evaluate the potential role of vasopressin V2 receptors in regulation of AQP2 protein levels, AQP2 mRNA levels, and in the levels of AQP2 phosphorylated in the PKA consensus site (Ser256 of AQP2) in BB rats. 3) BB rats were treated with lithium for 1 mo, a treatment known to inhibit adenylyl cyclase and to dramatically reduce AQP2 expression in normal rats. 4) BB rats were completely water restricted for 48 h to evaluate the effect of this. 5) Finally, we evaluated whether there were major changes in the subcellular distribution of AQP2 at the electron microscopic level to relate this to the changes in expression in untreated and thirsted BB rats.
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MATERIALS AND METHODS |
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Experimental Animals
Adult female BB rats (~300 g; Harlan Sprague Dawley, Indianapolis, IN) with central diabetes insipidus were placed in individual metabolic cages for 4 days before the experiment for adaptation and during the entire experiment. Wistar rats were obtained from M & B (Ry, Denmark). All rats had free access to standard rat chow (Altromin, Lage, Germany) and tap water during the adaptation to the metabolic cages.Experimental Protocols
The following protocols were administered.Protocol 1. BB (n = 11) and Wistar rats (n = 12) had free access to standard rat chow and tap water.
Protocol 2. {5-Dimethylamino-1-[4-(2-methylbenzoylamino) benzoyl]-2,3,4,5-tetrahydro-1H-benzazepine} (OPC-31260) is a V2-selective nonpeptide vasopressin receptor antagonist (Otsuka, Tokyo, Japan). BB rats (n = 5) were given 5 g of food with 5 mg of OPC-31260 every 12 h for 24 or 48 h. Control BB rats (n = 5) were given 5 g of food without OPC-31260 every 12 h for 24 or 48 h. All rats had free access to water.
Protocol 3. {(1-[4-N-tert-butylcarbamoyl)-2-methoxybenzene sulfony]-5-ethoxy-3-spiro-[4-(2-morpholinoethoxy)cyclohexane]indol-2-one, fumarate: equatorial isomer} (SR-121463A) is another V2-selective nonpeptide vasopressin receptor antagonist (Sanofi Recherche, Paris, France). SR-121463A suspended in isotonic saline was subcutaneously administrated to BB rats (0.4 mg/rat) every 12 h for 24 or 48 h. In parallel, BB control rats received an isotonic saline injection every 12 h for 24 or 48 h. All rats had free access to food and water. From experimental (n = 5) and control (n = 5) rats, total kidney AQP2 protein levels and kidney inner medullary AQP2 mRNA levels were determined. From additional experimental (n = 7) and control (n = 7) rats, kidney inner medullary AQP2 protein and phosphorylated AQP2 protein levels were determined. Urine output was determined for all rats.
Protocol 4. Lithium chloride (Bie & Berntsen, Aarhus, Denmark) was added to the chow to give a concentration of 40 or 60 mM lithium/kg dried food (30). BB rats received food containing 40 mM lithium/kg for the first 10 days and then food containing 60 mM lithium/kg for 20 days (n = 5). This protocol has previously been shown to induce NDI in normal Wistar rats (5, 30). Control BB rats were maintained on standard rat diet (n = 6). All rats had access to a salt block to prevent sodium depletion.
Protocol 5. BB rats were thirsted for 48 h (n = 11). Control BB rats had free access to water for 48 h (n = 12).
Tissue Preparation
Kidneys were rapidly removed during halothane anesthesia. The right kidney was processed for membrane fractionation. Inner medulla from the left kidney was dissected and processed for total RNA extraction.Membrane Fractionation For Immunoblotting
Kidneys were homogenized (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, pH 7.2, containing 8.5 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride) by using an ultra-turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany) at maximum speed for 10 s, and the homogenate was centrifuged in an Eppendorf centrifuge at 4,000 g for 15 min at 4°C to remove whole cells, nuclei, and mitochondria. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet containing membrane fractions enriched for both plasma membranes and intracellular vesicles (30, 32). Gel samples (Laemmli sample buffer containing 2% SDS) were made of this pellet.Primary Antibodies
The following antibodies were used: 1) an affinity-purified antibody raised against a peptide corresponding to amino acids 250-271 of rat AQP2 and diluted to 1:2,000 (32, 34, 35); and 2) an affinity-purified antibody raised against peptides corresponding to amino acids 253-262 of rat AQP2 and in which Ser256 in the PKA phosphorylation consensus site was phosphorylated (4, 38). Unlike the previous antibody, which both recognizes phosphorylated and nonphosphorylated AQP2, the latter is specific to phosphorylated AQP2 and was only used diluted to 1:200 in protocol 3.Electrophoresis and Immunoblotting
Samples of membrane fractions from total kidney were run on 12% polyacrylamide minigels (Bio-Rad Mini Protean II). For each gel an identical gel was run in parallel and subjected to Coomassie staining to assure identical loading (41). The other gel was subjected to immunoblotting. After transfer by electroelution to nitrocellulose membranes, blots were blocked with 5% milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20, pH 7.5) for 1 h and incubated with the primary antibody. The labeling was visualized with horseradish peroxidase-conjugated secondary antibody (diluted 1:3,000, P448, DAKO, Glostrup Denmark) by using the enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, UK).Preparation of RNA Samples and Northern Blotting
Total RNA was extracted from kidney inner medulla by using a RNeasy Mini kit (Qiagen, Studio City, CA). Quantification of the message for AQP2 was performed by using a digoxigenin-labeled AQP2 RNA probe. The full-length human cDNA encoding for human AQP2 was kindly provided by Dr. P. Deen (Nijmegen University, The Netherlands). The synthesis and digoxigenin-labeling of AQP2 RNA probe were performed by in vitro transcription by using a Maxiscript in vitro transcription kit (Ambion, Austin, TX).RNA samples (7 µg) were denatured and separated by electrophoresis on a gel agarose (1.2%) containing 0.6 M formaldehyde. Equal RNA loading was verified by visual inspection after coloration with ethidium bromide. The RNA were transferred overnight from gel to nylon membranes (Hybond-N, Amersham Life Science), which were then baked in a vacuum oven (2 h at 80°C). Blots were placed in a glass hybridization tube containing 5× saline sodium citrate (SSC), 50% formamide, 0.1% sarcosyl, 0.02% SDS, and 2% blocking solution (blocking reagent in maleic acid, Boehringer, Mannheim, Germany). Prehybridization was performed at 55°C for 30 min in a hybridization oven. The digoxigenin-labeled AQP2 RNA probe was then added to prehybridization medium, and membranes were incubated overnight at 55°C. The blots were washed twice at 25°C in 2× SSC, 0.1% SDS for 5 min, and twice at 68°C in 0.1× SSC, 0.1% SDS for 15 min. Blots were then equilibrated for 1 min in maleic acid solution containing 0.3% Tween 20 and blocked for 30 min. After incubation for 30 min with anti-digoxigenin-alkalinephosphatase conjugate (Boehringer), blots were washed twice for 15 min in maleic acid solution containing 0.3% Tween 20 and equilibrated for 5 min in 0.1 M Tris · HCl, 0.1 M NaCl. The bands were visualized by using a chemiluminescent substrate (CSPD, Boehringer).
Quantitation of AQP2 Protein or mRNA Levels
Enhanced chemiluminescence films with bands within the linear range were scanned (30) by using a AGFA Arcus II scanner (AGFA-Gevaert, Leverkusen, Germany) and Corel Photopaint Software (Corel, Toronto, ONT) to control the scanner. The total kidney expression of AQP2 in the experimental animals was calculated as a fraction of control levels, which were normalized to 100%.Immunoblotting. Both the 29- and the 35- to 50-kDa bands corresponding to nonglycosylated and the glycosylated of AQP2 were scanned, as described (30). The labeling density was corrected by densitometry of identical Coomassie-stained gels run in parallel.
Northern blotting. The band of ~1.6 kb corresponding to AQP2 mRNA was scanned. Values were corrected for potential differences in loading of total RNA in the specific lines by densitometry of 18S and 28S bands visualized by ethidium bromide on the same gel.
Results are expressed as means ± SE. Differences were analyzed by unpaired t-test. P values < 0.05 were considered significant.Preparation of Tissue For Immunocytochemistry
The kidneys from untreated BB rats (n = 4) and BB rats thirsted for 48 h (n = 4) were fixed by retrograde perfusion via the abdominal aorta with periodate-lysine-paraformaldehyde (PLP; 0.01M NaIO4, 0.075M lysine, 2% paraformaldehyde, in 0.0375 M Na2HPO4 buffer, pH 6.2). Kidneys were postfixed for 1 h, and tissue blocks were infiltrated for 30 min with 2.3 M sucrose containing 2% paraformaldehyde, mounted on holders, and rapidly frozen in liquid nitrogen.Immunoelectron Microscopy
The frozen samples were freeze substituted in a Reichert Auto Freeze-substitution Unit (Reichert, Vienna, Austria) (37, 45). Briefly, the samples were sequentially equilibrated over 3 days in methanol containing 0.5% uranyl acetate at temperatures gradually increasing fromSemiquantitation of AQP2 Immunogold Labeling
Electron micrographs were taken of the apical part of inner medullary collecting duct principal cells from BB rats that were either untreated (n = 4) or thirsted for 48 h (n = 4). Electron micrographs were printed at a final magnification of ×63,000. The total area of the cytoplasm was determined by using a lattice square test system, with the size of the squares of 30 × 30 mm. To ensure that the immunogold quantitation was performed on comparable tissue sampling, the length of the apical plasma membrane and the area of the cell (excluding nuclei) were determined. The total labeling density was calculated as the total number of gold particles per cytoplasm area. The length of the apical plasma membrane as a fraction of the area of cytoplasm in untreated and thirsted BB rats was not different: 0.12 ± 0.04 and 0.10 ± 0.04, respectively. This indicates equal sampling, i.e., same apical surface-to-cytoplasmic area ratio for both sets of animals. The length of the apical plasma membrane was determined by a manual tracing device, and the number of gold particles per length apical plasma membrane was calculated. The number of gold particles associated with apical plasma membranes, intracellular vesicles, and multivesicular bodies was determined. ![]() |
RESULTS |
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Total Kidney AQP2 Protein Expression in BB Rats is Relatively High Compared with Wistar Rats
As shown in Fig. 1A, BB rats have a ninefold higher urine output than Wistar rats (191 ± 24 vs. 21 ± 1 ml/24 h, P < 0.001). To assess AQP2 protein levels in BB rats relative to normal Wistar rats, semiquantitative immunoblotting was performed. As shown in Fig. 1, B and C, AQP2 protein levels were relatively high in BB rats, corresponding to 52 ± 8% of those seen in Wistar rats (100 ± 13%, P < 0.01).
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Total Kidney AQP2 Protein and mRNA Expression are Decreased in BB Rats Treated With the V2-Receptor Antagonist SR-121463A
To determine whether the quite high expression levels of AQP2 protein in BB rats (compared with normal Wistar rats) is associated with AVP-V2-receptor dependent or -independent signaling, we examined the effects of vasopressin V2-receptor antagonist treatment on urine output and AQP2 expression. We used two different compounds, OPC-31260 or SR- 121463A, both of which previously have been shown to be efficient (29, 40). As shown in Fig. 2, urine output was markedly increased in response to 24 h of treatment with OPC-31260 or SR-121463A. After 48 h of treatment, urine output remained markedly elevated in rats treated with SR-121463A, whereas the OPC-treated rats returned to control levels.
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Because SR-121463A exerted a more pronounced diuretic effect during the
conditions used, we determined the effect of treatment for 48 h
with this compound on the expression levels of AQP2. Semiquantitative
immunoblotting revealed a marked decrease in AQP2 protein levels in
kidney inner medulla from BB rats treated with SR-121463A compared with
control BB rats: 53 ± 8 (n = 7) vs.
100 ± 8% (n = 7), P < 0.01 (Fig. 3, A and B).
A second set of animals was used to determine total kidney AQP2 protein
levels. Consistent with the reduction in kidney inner medullary AQP2
levels total kidney AQP2 levels also were decreased in response to
treatment with SR-121463A: 42 ± 10% (n = 5) of
control levels (100 ± 20%, n = 5, P < 0.05; Fig. 3C). Thus the relative high AQP2
expression levels in BB rats is to a large extent dependent
on AVP-V2-receptor activation. To further establish and
investigate the observed decrease in AQP2 expression in response to
SR-121463A treatment of BB rats, we examined the effect of this
treatment on AQP2 mRNA abundance. For this purpose, AQP2 mRNA levels
were determined in kidney inner medulla. As shown in Fig.
4, A and B, AQP2
mRNA levels were significantly decreased in rats treated with
SR-121463A for 48 h (n = 4) to 36 ± 7%
(P < 0.05) of levels in control rats (100 ± 21%, n = 5). The decreased AQP2 mRNA and protein
expression in response to 48 h of AVP-V2-receptor
blockade demonstrate that activation of AVP-V2 receptors
(i.e., via endogenous oxytocin or other vasopressin-like substances) is
involved in regulation of AQP2 expression in BB rats.
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In normal rats, the AVP-V2 receptors regulate the
trafficking of AQP2 between the apical plasma membrane and the
intracellular vesicles, which involves phosphorylation and
dephosphorylation of Ser256 in AQP2 (4,
38). Moreover the levels of phosphorylated AQP2 are known
to be regulated by AVP-V2 receptors (4,
38). To determine the changes in phosphorylated AQP2
levels in response to AVP-V2-receptor blockade, membrane
fractions of kidney inner medulla from control BB rats and BB rats
treated with SR-121463A for 48 h were prepared. Immunoblots were
probed with an antibody that exclusively recognizes phosphorylated AQP2
(4). As demonstrated in Fig.
5, A and B,
phosphorylated AQP2 levels were dramatically reduced in BB rats treated
with SR-121463A to 3 ± 1% of levels in control BB rats (100 ± 17%, P < 0.001). This severe decrease suggests
that even in BB rats the phosphorylation of AQP2 is in large part
dependent on the AVP-V2-receptors.
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Total Kidney AQP2 Protein Expression is Decreased in BB Rats Treated With Lithium
Because lithium treatment has been shown to inhibit adenylyl cyclase activity (5) and lithium-induced NDI in Wistar rats is associated with severe downregulation of AQP2 (30), we tested the effect of prolonged lithium treatment of BB rats on AQP2 expression and urine output. Urine output increased markedly in BB rats treated with lithium for 1 mo (103 ± 4 vs. 35 ± 6 ml/8 h in the BB controls rats, P < 0.001, Fig. 6A). Immunoblotting and densitometric analysis of total kidney AQP2 protein levels revealed a severe decrease in AQP2 protein levels in BB rats treated with lithium for 1 mo to 15 ± 7% (P < 0.001) of levels in BB controls (100 ± 10%, Fig. 6, B and C). Consistent with this, a significant decrease in the expression of AQP2 mRNA was found in kidney inner medulla from lithium-treated BB rats (45 ± 7 vs. 100 ± 10%, P < 0.01; Fig. 7, A and B). This decrease in expression of AQP2 protein and mRNA in lithium-treated BB rats suggests that the AVP-V2-receptor-dependent regulation of AQP2 may involve adenylyl cyclase and cAMP/PKA signaling transduction pathways.
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Total Kidney AQP2 Expression is Increased in Thirsted BB Rats
To determine whether water restriction affects AQP2 expression in BB rats as it does in normal rats, semiquantitative immunoblotting was performed in BB rats subjected to 48 h of thirsting (complete water restriction). BB rats thirsted for 48 h showed a marked decrease in urine output during the second day (6 ± 1 ml/24h, P < 0.001) compared with BB control rats (167 ± 15 ml / 24h). As shown in Fig. 8, A and B, this was associated with a significant increase in AQP2 protein levels to 156 ± 22% (P < 0.05) of the levels in BB controls rats (100 ± 14%). To investigate whether the increase in AQP2 protein levels during 48 h of thirsting partially resulted from an increase in AQP2 mRNA abundance, the levels of AQP2 mRNA in kidney inner medulla were also determined. As shown in Fig. 8, C-D, AQP2 mRNA levels were significantly increased in BB thirsted rats to 235 ± 33% (P < 0.05) of levels in BB control rats (100 ± 17%).
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Immunoelectron Microscopic Localization of AQP2 in Thirsted and Untreated BB Rats
Immunoelectron microscopy was also performed on inner medulla from control BB rats and BB rats subjected to complete water restriction for 48 h. In control BB rats immunogold labeling of AQP2 in collecting duct principal cells was mainly associated with intracellular vesicles whereas the apical plasma membrane exhibited very sparse labeling (Fig. 9). Semiquantitation revealed that 1.0 ± 0.1% of the total labeling was associated with the apical plasma membrane (Table 1). Consistent with the immunoblotting, there was a significant increase in the total labeling density in collecting duct principal cells from thirsted BB rats compared with control BB rats (Fig. 10, Table 1). The fraction of AQP2 in the apical plasma membrane was 3.5 ± 1.8%, demonstrating that no major or significant increase in the fractional plasma membrane labeling was observed in response to thirsting. Similarly, there was no significant change in the linear density of AQP2 in the apical plasma membrane in the control BB rats and in the thirsted BB rats (Table 1).
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The very low level of AQP2 in the apical plasma membrane both in control BB rats and in thirsted BB rats demonstrates that there is no or very low AVP-V2-receptor-mediated targeting of AQP2 despite significant AVP-V2-receptor-mediated AQP2 expression (i.e., sensitive to AVP-V2-receptor blockade). Thus this indicates that there is an almost complete decoupling between the AVP-V2-receptor-mediated regulation of AQP2 trafficking and AQP2 expression in BB rats.
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DISCUSSION |
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This study demonstrated that AQP2 expression is remarkably high in vasopressin-deficient BB rats, corresponding to ~50% of the levels in normal Wistar rats. Moreover, treatment with an AVP-V2-receptor antagonist induced a marked reduction in AQP2 mRNA and AQP2 protein levels. This marked downregulation of AQP2 in response to AVP-V2-receptor antagonist treatment strongly indicates that the relatively high expression of AQP2 in vasopressin-deficient BB rats is mediated in part via activation of AVP-V2-receptors. Moreover, the treatment with AVP-V2-receptor antagonist also induced a dramatic reduction in the levels of AQP2 which is phosphorylated in the PKA phosphorylation consensus site (Ser256). Thus the expression of AQP2 and the phosphorylation of AQP2 in BB rats is in part dependent on AVP-V2-receptor signaling. Prolonged lithium-treatment, known to reduce adenylyl cyclase activity, was associated with severe downregulation of AQP2 protein and mRNA levels in BB rats indicating that the signaling cascade involves adenylyl cyclase and hence cAMP. This is also consistent with the view that AQP2 expression in BB rats is dependent in part on AVP-V2-receptor signaling. Immunoelectron microscopy demonstrated that ~1% of collecting duct AQP2 is present in the apical plasma membrane of collecting duct principal cells in untreated BB rats and that this does not increase significantly in thirsted BB rats. Thus there was no significant AVP-V2-receptor-mediated trafficking of AQP2 to the apical plasma membrane. Furthermore, the normal AVP-V2-mediated regulation of both AQP2 trafficking and of AQP2 expression is effectively decoupled in BB rats. This supports the view that there are differences in AVP-V2-receptor-mediated regulation of AQP2 trafficking and in AVP-V2-receptor regulation of AQP2 expression.
AVP-V2-Receptor-Mediated Regulation of AQP2 Expression in BB Rats
The previous observations that AQP2 expression levels were relatively high in vasopressin-deficient BB rats compared with their parent strain (7, 23) were puzzling because vasopressin induces the expression of AQP2 directly and via thirsting (7, 35). Thus in vasopressin-deficient animals one would expect AQP2 expression levels to be quite low, or, alternatively, that vasopressin-independent regulation may play a significant role in maintaining AQP2 expression levels. To address these issues, here we treated BB rats with a potent AVP-V2-receptor antagonist (SR-121463A) for 2 days and looked at changes in urine output and AQP2 mRNA and protein expression. This AVP-V2-receptor antagonist has been shown to inhibit specifically the AVP-V2 receptor and not the AVP-V1 receptor (40). Treatment with this compound for 2 days produced a marked increase in urine production consistent with previous evidence (40). This supports the view that there is a level of activation of the AVP-V2 receptor in BB rats. Importantly AVP-V2-receptor antagonist treatment produced a marked reduction in total AQP2 protein expression in BB rats (Fig. 3), demonstrating that AQP2 expression in BB rats is in part mediated via AVP-V2 receptors. This was further supported by the observed reduction in AQP2 mRNAs expression (Fig. 4), which suggests that the reduction in AQP2 protein levels is in part due to reduced transcription. A potentially increased degradation of AQP2 may also contribute to the overall reduction.AQP2 contains a consensus phosphorylation site for PKA (Arg-Arg-Gln-Ser) at Ser256 in its amino sequence. Recent studies have shown that phosphorylation of AQP2 at Ser256 is involved in regulation of AQP2 trafficking (4, 38). Indeed immunoelectron microscopy revealed that in kidneys from BB rats phosphorylated AQP2 was almost exclusively present in intracellular vesicles and that 2 h of a AVP-V2-receptor agonist (DDAVP) treatment induced a dramatic trafficking of phosphorylated AQP2 from intracellular vesicles to the apical plasma membrane. The treatment of BB rats with an AVP-V2-receptor antagonist (in the present study) allowed us to identify what stimulates the activation of PKA in BB rats and hence the phosphorylation of AQP2. The dramatic reduction in phosphorylated AQP2 seen in response to blockade of AVP-V2-receptors demonstrated that AVP-V2-receptors are involved. This reduction in the levels of phosphorylated AQP2 in response to AVP-V2-receptor antagonist treatment may result from either an increase in phosphatase activity or a reduced PKA activity.
The ligand responsible for the activation of AVP-V2-receptors in BB rats is presently not defined, but a series of studies have demonstrated that oxytocin acts as an antidiuretic hormone (1, 3, 6, 10, 27) and is present in BB rats in concentrations of ~5 pM (10) (see below). At this concentration there is no effect on collecting duct water permeability in tubules dissected from untreated BB rats (3), which is obviously consistent with the fact that BB rats are severely polyuric. However, all of the studies determining the action of oxytocin on AVP-V2-receptors have focused on antidiuretic effects and hence direct changes in the osmotic water permeability. The present study strongly suggests that the stimulation of AVP-V2-receptors, which are sufficient to induce AQP2 expression, are not sufficient to induce targeting (and hence changes in water reabsorption). Thus it may be hypothesized that oxytocin is present at sufficient levels in BB rats to stimulate AQP2 expression although this needs to be further established in future studies.
Lithium treatment for 30 days dramatically reduced AQP2 protein levels to ~15% of control levels, which is a much more substantial downregulation than the 60% reduction seen in response to 2 days of AVP-V2-receptor antagonist treatment. Consistent with this, treatment of Wistar rats with the AVP-V2-receptor antagonist (OPC-31260) also reduced AQP2 protein levels ~50% (29) whereas lithium treatment for 25 or 35 days produced a dramatic decrease to ~5-10% of control levels (26, 30). Although the difference in downregulation between lithium and of AVP-V2-receptor antagonist treatment (both in BB and Wistar rats) could be taken as clear evidence for a vasopressin-independent regulation of AQP2 further studies are necessary to further evaluate this.
It remains possible that both the AVP-V2-receptor-dependent and -independent pathways are mediated via cAMP. Several investigators have demonstrated that a cAMP-responsive element is present in the 5'-flanking region of the AQP2 gene and that this is involved in the regulation of AQP2 gene expression (20, 42, 46). Recently, we demonstrated that in a strain of mice (DI +/+ Severe mice), which have high levels of cAMP phosphodiesterase activity, and hence low levels of cytosolic cAMP (19), had severely reduced expression of AQP2 (12). In male DI +/+ Severe mice AQP2 expression levels were very low (<15% of control), which would be consistent with the view that AVP-V2-receptor-dependent and possible also -independent regulation could be mediated via cAMP. This is consistent with the observations described above in lithium-treated rats. The nature of the AVP-V2-receptor-independent regulation remains to be identified.
Effect of Thirsting
BB rats have severe diabetes insipidus due to an autosomal recessive trait caused by mutation in AVP-neurophysin gene and resulting in the inability to release vasopressin. In the present study we demonstrated that prolonged water deprivation increases both AQP2 mRNA and protein abundance in BB rats. Because it has been shown that water deprivation does not induce any changes in hypothalamic content of vasopressin mRNA in BB rats (21, 28) or any increased release of vasopressin, the rise in AQP2 expression in response to thirsting suggests that factors other than vasopressin are able to modulate the AQP2 expression levels via AVP-V2-receptor-dependent pathways. As described above oxytocin has been shown to act as an antidiuretic hormone, and treatment with oxytocin has been shown to increase the water permeability of amphibian bladders (9, 43); in BB rats oxytocin has been shown to increase urinary osmolality (1, 6, 10, 27) and to increase the water permeability of isolated perfused inner medullary collecting ducts (3, 17). The plasma concentrations of oxytocin in BB rats have been shown to increase from ~5 to 10-40 pM in response to 24 h of water restriction (10). Thus it is conceivable that oxytocin levels may increase in response to thirsting and induce an increase in AQP2 expression without inducing a substantial increase in AQP2 trafficking. Interestingly, Chou et al. (3) demonstrated in an elegant study that isolated perfused inner medullary collecting ducts from BB rat kidneys responded to 20 pM of oxytocin by increasing the osmotic water permeability 2.8-fold. However, unlike in tubules dissected from either untreated or thirsted Sprague-Dawley rats (which all respond), this was only seen in tubules dissected from thirsted BB rats and not in tubules dissected from nonthirsted BB rats (3). This is not easily explained, but it is possible that thirsting may upregulate some of the components in the signaling cascades including those involved in upregulation and targeting of AQP2. The increased expression of AQP2 found in this study in response to thirsting is consistent with this hypothesis. However, the relatively small increase in AQP2 abundance in response to thirsting indicates that this probably plays an insignificant role for the selective increase in osmotic water permeability in tubules from thirsted BB rats. The demonstration of an increase in AQP2 levels in the subapical domain of collecting duct principal cells (but not in the apical plasma membrane) supports previous light microscopic observations of an increased labeling of AQP2 in the subapical domains of collecting duct principal cells from kidneys of thirsted BB rats (3). It should be emphasized that the severe reduction in urine output of thirsted BB rats is likely to be due to a severe reduction in glomerular filtration rate (14).The observation that treatment of BB rats with the AVP-V2-receptor antagonist SR- 121463A [chosen due to its high potency (Fig. 2)] reduced AQP2 expression is consistent with the possibility that oxytocin may stimulate AQP2 expression via AVP-V2 receptors in vasopressin-deficient BB rats at concentrations lower than those necessary to stimulate trafficking. Indeed, it was recently demonstrated that SR-121463A could inhibit oxytocin action in BB rats (2). Bankir and colleagues (2) showed that the reduction in urine output seen in response to 5 days of oxytocin treatment was completely abolished by SR-121463A (2).
Decoupling of AVP V2-Receptor-Mediated Regulation of Trafficking and Expression of AQP2
In the present study we showed that AVP-V2-receptor antagonist treatment markedly reduces AQP2 protein and mRNA expression, which demonstrates that AQP2 expression in BB rat kidneys is dependent (in part) on AVP-V2-receptor activation. In contrast there was no AVP-V2-receptor-induced targeting of AQP2 to the apical plasma membrane (Fig. 10) in untreated BB rats, consistent with previous observations (7, 16, 39, 44). Combined, these results strongly indicate that there is an almost complete dissociation between the AVP-V2-receptor-mediated induction of AQP2 expression and AQP2 targeting in BB rats. It appears likely that oxytocin may play a significant role in this, but further studies are necessary to define whether oxytocin or possible other antidiuretic substances are responsible for the activation of AVP-V2 receptors, and hence AQP2 expression, in BB rats. ![]() |
ACKNOWLEDGEMENTS |
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The authors thank Mette Vistisen, Zhila Nikrozi, Inger Merete Paulsen, Helle Høyer, and Gitte Christensen 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, Novo Nordic Foundation, Danish Medical Research Council, University of Aarhus Research Foundation, the University of Aarhus, the Commission of the European Union (EU-TMR Program and EU-Biotech Program), and the intramural budget of the National Heart, Lung, and Blood Institute.
Address for reprint requests and other correspondence: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).
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 13 August 1999; accepted in final form 29 March 2000.
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