Low aquaporin-2 levels in polyuric DI +/+ severe mice with constitutively high cAMP-phosphodiesterase activity

Jørgen Frøkiaer1,2, David Marples2,3, Heinz Valtin4, John F. Morris5, Mark A. Knepper6, and Søren Nielsen2

1 Department of Clinical Physiology, Aarhus University Hospital and Institute of Experimental Clinical Research, 2 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark; 3 Department of Physiology, University of Leeds, LS2 9NQ Leeds, United Kingdom; 4 Department of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755-3836; 5 Department of Anatomy, University of Oxford, Oxford OX1 3QX, United Kingdom; and 6 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892


    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

In the renal collecting duct, vasopressin acutely activates cAMP production, resulting in trafficking of aquaporin-2 water channels (AQP2) to the apical plasma membrane, thereby increasing water permeability. This acute response is modulated by long-term changes in AQP2 expression. Recently, a cAMP-responsive element has been identified in the AQP2 gene, raising the possibility that changes in cAMP levels may control AQP2 expression. To investigate this possibility, we determined AQP2 protein levels in a strain of mice, DI +/+ severe (DI), which have genetically high levels of cAMP-phosphodiesterase activity, and hence low cellular cAMP levels, and severe polyuria. Semiquantitative immunoblotting of membrane fractions prepared from whole kidneys revealed that AQP2 levels in DI mice were only 26 ± 7% (±SE) of those in control mice (n = 10, P < 0.01). In addition, semiquantitative Northern blotting revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice compared with control mice (43 ± 6% vs. 100 ± 10%; n = 6 in each group, P < 0.05). AQP3 levels were also reduced. The mice were polyuric and urine osmolalities were accordingly substantially lower in the DI mice than in controls (496 ± 53 vs. 1,696 ± 105 mosmol/kgH2O, respectively). Moreover, there was a linear correlation between urine osmolalities and AQP2 levels (P < 0.05). Immunoelectron microscopy confirmed the markedly lower expression of AQP2 in collecting duct principal cells in kidneys of DI mice and, furthermore, demonstrated that AQP2 was almost completely absent from the apical plasma membrane. Thus expression of AQP2 and AQP2 trafficking were severely impaired in DI mice. These results are consistent with the view that in vivo regulation of AQP2 expression by vasopressin is mediated by cAMP.

collecting duct; nephrogenic diabetes insipidus; water balance; vasopressin


    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

COLLECTING DUCT WATER permeability has been shown to be regulated both by short-term and long-term mechanisms (16, 19). Acutely, vasopressin acts via vasopressin V2 receptors linked to adenylyl cyclase, causing a rise in cytosolic cAMP. Ultimately, this cascade results in the transfer of aquaporin-2 (AQP2) (10), the vasopressin-regulated water channel of the collecting duct (3, 4, 15, 24, 27), from intracellular vesicles to the apical plasma membrane, thereby increasing the water permeability of the collecting duct. Phosphorylation of serine-256 of the AQP2 protein by cAMP-dependent protein kinase (PKA) appears to be involved in AQP2 trafficking in AQP2-transfected LLC-PK1 cells (9, 14), implying a direct role of AQP2 phosphorylation.

This acute response is modulated by long-term changes in AQP2 expression in response to the state of hydration, thought to be at least partly mediated by vasopressin. Indeed, chronic vasopressin infusion in rats markedly increases AQP2 expression levels (4, 5, 31). This finding raises the possibility that cAMP may increase the transcription of the AQP2 gene as well as the vesicular trafficking of AQP2. In support of this hypothesis, recent studies have identified a putative cAMP responsive element (CRE) in the 5'-untranslated region of the AQP2 gene (12). On the other hand, it has recently been shown that water loading causes severe downregulation of AQP2 expression despite high circulating DDAVP levels maintained by continuous subcutaneous infusion via osmotic minipumps ("vasopressin escape" phenomenon) (5). This finding supports earlier evidence for the existence of vasopressin-independent mechanisms for the regulation of AQP2 expression (20).

Downregulation of AQP2 has been shown to be associated with the development of severe polyuria in multiple forms of acquired nephrogenic diabetes insipidus (8, 20, 21). To investigate the role of cAMP in the control of AQP2 transcription we used a strain of mice, called "DI +/+ severe" (DI), which have genetically high levels of rolipram-sensitive cAMP-phosphodiesterase (cAMP-PDE; type IV) activity, low levels of cytosolic cAMP levels, and which are unable to raise the cytosolic cAMP in response to vasopressin (32). These mice have extensive polyuria and low urine osmolality compared with the control mice (1, 2, 11, 32).

It can be hypothesized that the polyuria of DI mice might be a consequence of low AQP2 expression levels, or reduced trafficking of AQP2 to the apical plasma membrane, or a combination of both. The purpose of the present study was to examine the levels of AQP2 expression and its subcellular distribution in collecting duct principal cells to evaluate these possibilities. This information will allow us to evaluate whether cAMP plays a significant role in the intracellular signaling pathways controlling AQP2 expression. Furthermore, in the context of the evidence for both vasopressin-dependent and vasopressin-independent signals for regulation of AQP2 expression, it remains a possibility that cAMP may play a role in both pathways or even act as a final common pathway.

The results show that polyuric DI mice have low total AQP2 expression and, moreover, low levels of AQP2 in the apical plasma membrane of collecting duct principal cells. Since mice of this strain have constitutively high levels of cAMP-PDE activity, and hence low cellular cAMP levels, these results strongly support the view that cAMP is a critical component in the long-term regulation of AQP2 and AQP3 gene transcription as well as mediates the acute shuttling of AQP2 to and from its active site, namely the apical plasma membrane. The very low levels of AQP2 expression in DI mice, in the order of 10-15% in severely polyuric male DI mice, raise the possibility that cAMP acts as a common final pathway in controlling AQP2 expression.


    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Experimental animals. Mice were kept in the Animal Care Facility of the Anatomy Department, Oxford University. Mice had free access to standard mouse diet and water. The abbreviation "DI" was used to describe the DI +/+ severe mice. For controls, normal mice were used and are described as "controls."

Antibodies. The affinity-purified antibody to AQP2 has previously been described (25). Briefly, peptides corresponding to the 22 COOH-terminal amino acids of rat AQP2 were synthesized with the addition of a cysteine residue at the carboxy terminus. Peptides were conjugated to keyhole limpet hemocyanin and used to immunize rabbits. Crude antisera were affinity purified using a column on which 2 mg of the synthetic peptide was immobilized via a sulfhydryl linkage to the NH2-terminal cysteine (Immunobilization kit no. 2; Pierce, Rockford, IL). Antibodies to AQP3 and AQP1 were prepared using the same procedures (6, 31), and both antibodies were affinity purified as previously described. All antibodies have been characterized for immunoblotting, immunocytochemistry, and immunoelectron microscopy previously (4, 6, 25, 26, 31).

Membrane fractionation for immunoblotting. The kidneys from male and female DI mice and from male and female control mice were removed and frozen in liquid nitrogen. After thawing on ice, the kidneys were minced finely, and homogenized in dissecting buffer (0.3 M sucrose, 25 mM imidazole, 1 mM EDTA, 8.5 µM leupeptin, and 1 mM phenylmethylsulfonyl fluoride, pH 7.2) with five strokes of a motor-driven Potter-Elvehjem homogenizer, at 1,250 rpm. To increase yields, the pellet was rehomogenized with three strokes, and the centrifugation was repeated. The supernatants were pooled and centrifuged at 200,000 g for 1 h. The resultant pellet was resuspended in dissecting buffer, and assayed for protein concentration using the method of Lowry. Gel samples (in Laemmli sample buffer containing 2% SDS) were made from this membrane preparation.

Electrophoresis and immunoblotting. Samples were loaded 10 µg/lane onto 12% polyacrylamide SDS-PAGE gels and run on minigel systems. After transfer by electroelution to nitrocellulose membranes, blots were blocked for 1 h with 5% skimmed milk in PBS-T (80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20, pH 7.5), washed three times in PBS-T over 25 min, and subsequently incubated with affinity-purified anti-AQP2, anti-AQP3, or anti-AQP1 overnight. After a wash as above, the blots were incubated at room temperature for 1 h with horseradish peroxidase-conjugated secondary antibody (P448, diluted 1:3,000; Dako, Glostrup, Denmark). After a final wash as above, antibody binding was visualized using an enhanced chemiluminescence system (ECL; Amersham International). Controls were made with exchange of primary antibody to antibody preabsorbed with immunizing peptide (100 ng per 40 ng IgG), or with preimmune serum (diluted 1:1,000). All controls were without labeling (not shown). Identical gels were run in parallel and were stained with Coomassie blue.

Semiquantitative Northern blot analysis. Total RNA was prepared from inner medulla using a RNeasy Mini Kit (Qiagen). Northern blot analysis was performed using digoxigenin-labeled RNA probes [rat AQP2 and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH)]. Total RNA was separated on a 1% agarose, 2% formaldehyde gel followed by blotting on a nylon membrane filter (Hybond-N; Amersham Life Science, Buckinghamshire, UK). Prehybridization was performed at 55°C for 30 min in 5× SSC (with 50% formamide, 0.1% sarcosyl, and 0.02% SDS) and 2% blocking solution (blocking reagent in maleic acid; Boehringer, Mannheim, Germany). 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, Ambien). After hybridization, blots were washed in 2× SSC + 0.1% SDS at room temperature for two 5-min periods followed by wash 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-digoxigenin-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, CSPD (Boehringer).

Quantitation of AQP2, AQP1, and AQP3 expression. ECL films were scanned using a Umax Vista-S8 scanner and Corel Photopaint Software, and both the 29-kDa and the 35- to 50-kDa band (corresponding to nonglycosylated and glycosylated AQP2; Ref. 30) were scanned. For AQP3, the affinity-purified anti-AQP3 recognized both nonglycosylated and glycosylated AQP3, corresponding to the 27-kDa and the 33- to 40-kDa bands, respectively. Both bands were scanned as described previously (6). Similarly, for AQP1 both the 29-kDa and the 35- to 50-kDa bands were scanned. The labeling density was quantitated using specially written software (available upon request). Bands from gels made with serial dilutions of protein from inner medulla, processed as above, were found to be linear over a wide range (20). For quantitation of AQP2 expression, ECL exposures were chosen that gave bands in the control samples that were close to the top of the linear range. Equal loading among lanes was confirmed by scanning and densitometry of identical SDS-PAGE gels run in parallel to the gels for immunoblots and stained with Coomassie blue.

Densitometry of AQP2 mRNA levels. The band of ~1.6 kb corresponding to AQP2 mRNA was scanned. Values were corrected by densitometry of GAPDH visualized using a digoxigenin-labeled probe for human GAPDH on separate identical blots.

Preparation of tissue for immunocytochemistry. Immunocytochemistry was performed as previously described (20). Kidneys were perfusion fixed with 4% paraformaldehyde in 0.1 M sodium cacodylate buffer, pH 7.4. The kidneys were postfixed for 2 days in 2% paraformaldehyde. Tissue blocks were cut from the kidney inner medulla (close to the junction between inner and outer medulla) and infiltrated for 30 min with 2.3 M sucrose + 2% paraformaldehyde. Blocks were subsequently mounted on holders and rapidly frozen in liquid nitrogen. The blocks were used either directly for cryosectioning or for cryosubstitution and Lowicryl HM20 plastic resin embedding.

Semithin (0.85 µm) cryosections cut on a Reichert Ultracut FCS were preincubated with PBS containing 1% skimmed milk and 0.05 M glycine, then incubated with affinity-purified antibodies against AQP2 (100-600 ng IgG/ml) in PBS containing 1% skimmed milk and 0.3% Triton X-100. The labeling was visualized as previously described (20, 21) using horseradish peroxidase-conjugated secondary antibody (P448, 1:100, Dako), and sections were counterstained with Meier counter- stain.

The frozen samples were freeze substituted (28) 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°C to -70°C, and then rinsed in pure methanol for 24 h at -70°C to -45°C and infiltrated at -45°C with Lowicryl HM20 and methanol at 1:1, then 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.

Ultrathin sections (Reichert Ultracut FCS) placed on nickel grids (with or without Formvar coating) were quickly dipped in absolute ethanol containing saturated NaOH, washed extensively in water, and then incubated in TBST (0.05 M Tris, 0.1% Triton X-100, 0.1% sodium borohydride, and 50 mM glycine) for 10 min and in TBST solution containing 2% BSA for 10 min. The grids were then incubated with affinity-purified anti-AQP2, and the labeling was visualized using goat-anti-rabbit-gold (GAR 10, 10-nm colloidal gold particles; BioCell Research Laboratories, Cardiff, UK), and grids were stained with uranyl acetate. Immunolabeling controls were performed using nonimmune IgG and revealed absence of labeling. Semiquantitation of the immunogold labeling was performed on kidneys from two controls and two DI mice. Ultrathin sections were prepared from cryosubstituted and Lowicryl HM20-embedded tissue. Sections were obtained from proximal parts of the kidney inner medulla. The sections were labeled with affinity-purified anti-AQP2 in parallel, and very low antibody concentrations were used to assure extremely low background labeling (this is essential due to the very low labeling density in the DI mice). Electron micrographs were obtained at a primary magnification of ×15,500 or ×21,000, and the gold particle distribution was determined. Light microscopy was performed using a Leica Laborlux S microscope, and electron microscopy was performed using Philips CM100 and Philips EM208 electron microscopes.

Statistics. For densitometry of immunoblots, samples from kidneys of DI mice of each gender were run together with samples from the corresponding controls. AQP2, AQP1, and AQP3 labeling in the samples from the DI mice was calculated as a fraction of the mean value of the samples from the controls for that gel. Parallel Coomassie-stained gels were subjected to densitometry and used for correction of potential minor differences in loading. Values are presented in the text as means ± SE. Comparisons between groups were made by unpaired t-tests. P < 0.05 was considered significant.


    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Reduced expression of AQP2 and AQP2 mRNA levels in male DI mouse kidneys. To test whether DI mice manifest a decrease in AQP2 expression, semiquantitative immunoblotting of kidney membranes was performed using anti-AQP2 antibodies. Immunoblotting using membrane fractions prepared from total mouse kidney revealed that the affinity-purified anti-AQP2 antibody exclusively recognized 29-kDa and 35- to 50-kDa bands (Fig. 1), corresponding to nonglycosylated and glycosylated forms of AQP2, respectively (30). The AQP2 bands were ablated by use of antibody previously absorbed with immunizing peptide (not shown), consistent with previous evidence in rat tissue (8, 20, 21).


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Fig. 1.   A: representative immunoblot comparing aquaporin-2 (AQP2) expression in kidneys from 5 male DI +/+ severe mice (DI-Male) and from 5 male control mice (control). Membranes were prepared from total kidneys and loaded at 10 µg/lane. Antibody labels 29-kDa and 35- to 50-kDa bands corresponding, respectively, to nonglycosylated and glycosylated forms of AQP2 (see METHODS). Also shown are densitometry of AQP2 expression (B) and urine osmolality (C) from male DI mice (n = 5) and male control mice (control, n = 9). Values were obtained by densitometry of immunoblots, and expression is presented as fraction of expression in controls (control).

Immunoblotting of membrane fractions prepared from kidneys of male DI mice and control mice revealed markedly lower AQP2 expression levels in DI male mice (Fig. 1A). Both AQP2 bands (29 and 35-50 kDa) were decreased. Densitometry (Fig. 1B) revealed that in male DI mice (n = 5) AQP2 expression levels were reduced to 13 ± 4% of that in male controls (100 ± 17%, n = 5). Consistent with this, male DI mice were severely polyuric, and consequently, the urine osmolalities were low, averaging 347 ± 42 compared with 1,740 ± 80 mosmol/kgH2O in urine from control mice (Fig. 1C). Thus there is a marked downregulation of AQP2 expression in kidneys of polyuric male DI mice.

Total RNA purified from kidney inner medulla of male DI mice and male control mice was analyzed by semiquantitative Northern blotting. As shown in Fig. 2, a band of ~1.6 kb was detected, consistent with the predicted size of AQP2 mRNA (10). Compared with control animals, semiquantitative Northern blotting revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice, i.e., 43 ± 6% (n = 6) vs. 100 ± 10% (n = 6; P < 0.05).


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Fig. 2.   A: Northern blot showing AQP2 mRNA in whole kidney from control mice and male DI mice. Digoxigenin-labeled human AQP2 mRNA probe labels a band of 1.6 kb consistent with the predicted size of AQP2 mRNA (10). B: a parallel blot identical to the blot shown in A was hybridized with a cDNA probe of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a control for loading of kidney mRNA levels. C: semiquantitative densitometric analysis revealed a significantly lower AQP2 mRNA expression in kidneys from DI male mice compared with control mice of 43 ± 6% vs. 100 ± 10%.

Differential AQP2 protein expression levels in male vs. female DI mice. Although both male and female DI mice are known to be polyuric (2, 11), previous studies have indicated a significant difference in the concentrating ability of males and females, with males displaying a more severe polyuria (32). To examine whether this difference correlates with changes in AQP2 expression, a differential analysis of AQP2 levels in male and female DI mice was undertaken. As shown in Fig. 3A, the decrease in AQP2 levels in females relative to female control mice was not as extensive as seen in males. The AQP2 expression in female DI mice was 39 ± 12% (n = 5, Fig. 3A) of that of female controls (100 ± 17%, n = 4). In males (n = 5), AQP2 expression was reduced to 13 ± 4% of levels in male controls (Fig. 1, A and B). Combining data from females and males confirmed a significant decrease both in AQP2 expression and urine osmolality in DI mice (Fig. 4). A direct comparison of the expression levels between male and female DI mice confirmed a significantly lower AQP2 expression in male DI mice (Fig. 5A). Densitometry (Fig. 5B) of the immunoblot revealed that AQP2 levels in kidneys of male DI mice constituted only 36 ± 11% of the levels in female DI mice (100 ± 13%). Consistent with the lower AQP2 expression in male compared with female DI mice, urine osmolalities were significantly lower in male compared with female DI mice (347 ± 42 vs. 615 ± 31 mosmol/kgH2O in males and females, respectively) (Figs. 1C and 3B).


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Fig. 3.   Densitometry of AQP2 expression (A) and urine osmolality (B) from female DI mice (n = 5) and female control mice (control, n = 4). Values are obtained by densitometry of immunoblots, and expression is presented as fraction of expression in controls (control).


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Fig. 4.   Densitometry of AQP2 expression (A) and urine osmolality (B) from DI mice (n = 10) and control mice (control, n = 9). Both genders are included. Values are obtained by densitometry of immunoblots, and expression is presented as fraction of expression in controls.


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Fig. 5.   A: immunoblot comparing AQP2 expression in kidneys from 5 male DI mice (DI-Male) and 5 female DI mice (DI-Female). Membranes were prepared from total kidneys and loaded at 10 µg/lane. Antibody labels 29-kDa and 35- to 50-kDa bands corresponding, respectively, to nonglycosylated and glycosylated forms of AQP2. B: densitometry of AQP2 expression. Values are obtained by densitometry of immunoblot, and expression is presented as fraction of expression in controls. In males, AQP2 expression is 36 ± 11% of expression in female (100 ± 13%) DI mice.

Urine osmolality correlates with AQP2 protein expression in DI mice. As demonstrated in Figs. 1C and 3B, the urine osmolality is markedly lower in both male and female DI mice compared with control mice. In Fig. 6, the relationship between urine osmolality and relative AQP2 expression in both male and female DI as well as male and female control mice is depicted. The analysis revealed a significant correlation (R2 = 0.6). Thus the results are consistent with the view that low AQP2 levels and hence low levels in the apical plasma membrane (see below) are a significant factor determining the high urine output and low urine osmolality observed in DI mice.


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Fig. 6.   Urine osmolality in DI (n = 10) and control (n = 9) mice plotted as function of AQP2 expression. Both genders are included. Linear regression analysis reveals a significant correlation (R2 = 0.6). This increase in AQP2 expression and increase in urine osmolality is similar to the parallel increase in AQP2 and urine osmolality previously shown in Brattleboro rats in response to long-term (5 days) vasopressin treatment. Long-term treatment with vasopressin results in a 3- to 4-fold increase in AQP2 expression and a 4- to 5-fold increase in urine osmolality (to ~1,100 mosmol/kgH2O) (4).

Immunocytochemistry and immunoelectron microscopical analysis of AQP2 distribution. cAMP plays a pivotal role in the acute hydrosmotic response to vasopressin by inducing the transfer of AQP2 from intracellular vesicles to its active site in the apical plasma membrane (22, 25, 29, 35). It would be expected that this process might be disturbed in the kidneys of the DI mice having low cytosolic cAMP levels. To investigate whether there are major differences in the subcellular distribution of AQP2 in the kidney collecting duct of DI mice, immunocytochemistry and immunoelectron microscopy were performed. Immunocytochemistry (Fig. 7) confirmed the markedly lower AQP2 expression in the inner medullary collecting ducts of kidneys from DI mice (Fig. 7B), compared with that in control mice (Fig. 7A). The sparse labeling in DI mice was primarily intracellular. Immunolabeling controls revealed absence of labeling (Fig. 7C). Immunoelectron microscopy was used to analyze the subcellular distribution of AQP2 in kidneys from control mice (Fig. 8) and DI mice (Fig. 9). Qualitative analysis of multiple inner medullary collecting duct cells in kidneys of several DI mice uniformly revealed a significant reduction in overall labeling (confirming the light microscopical observations). In addition, a very marked reduction in immunogold labeling of the apical plasma membrane of collecting duct principal cells was observed (Fig. 9). This was consistently seen in all collecting duct principal cells in the different DI mice. The remaining labeling (albeit markedly reduced in abundance) was present in vesicles (Fig. 9). In contrast, significant AQP2 labeling was consistently found in the apical plasma membrane of collecting duct principal cells in kidneys from control mice (Fig. 8). Semiquantitation of the immunogold labeling (703 gold particles) revealed that in controls ~43% of the labeling was associated with the apical plasma membrane compared with 4% in the DI mice. Importantly, the AQP2 labeling density was extremely low in DI mice compared with the controls. Thus there was a marked reduction in labeling of the apical plasma membrane (being the "active" site of AQP2 to allow water to be reabsorbed from the collecting duct lumen) in DI mice. The immunocytochemistry and immunoelectron microscopy confirmed the low AQP2 protein levels and demonstrated that there was indeed impaired delivery of AQP2 to the apical plasma membrane in DI mice. This supports the view that both the short- and long-term regulation of AQP2 are severely disturbed in DI mice with low cytosolic levels of cAMP in kidney collecting duct cells.


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Fig. 7.   Immunocytochemical localization of AQP2 in kidney inner medulla. A: kidney from a control mouse (male). Collecting duct principal cells are heavily labeled (arrows), whereas intercalated cells (arrowhead), thin limbs, and vascular structures are unlabeled. Within collecting duct principal cells, AQP2 labeling is localized to the apical plasma membrane domains and subapical intracellular vesicles. B: in male DI mice, AQP2 labeling is very sparse. Labeling is localized in the subapical region. C: immunolabeling controls using nonimmune IgG reveal no labeling. Magnification, ×1,000.


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Fig. 8.   Immunoelectron microscopical localization of AQP2 in kidney inner medullary collecting duct principal cells from male control mouse. Abundant AQP2 labeling of the apical plasma membrane (arrows) and intracellular vesicles (arrowheads) is seen. Inset: immunolabeling controls using nonimmune IgG reveal no labeling. Magnification, ×71,000.


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Fig. 9.   Immunoelectron microscopical localization of AQP2 in kidney inner medullary collecting duct principal cells from male DI mouse. Only sparse AQP2 labeling of intracellular vesicles (arrowheads) is seen, with virtually no labeling of the apical plasma membrane. Inset: immunolabeling controls using nonimmune IgG reveal no labeling. Magnification, ×71,000.

Dysregulation of other water channels. Several transporters have been shown to be regulated by vasopressin via V2 receptors and, hence, cAMP. It has been shown that AQP3 expression can be induced by continuous infusion of vasopressin via osmotic minipumps, whereas the expression of the proximal tubule/descending thin limb water channel AQP1 remains unchanged (31). The expression pattern of AQP1 and AQP3 was therefore examined. As demonstrated in Fig. 10, there was no change in AQP1 expression in immunoblots of membrane fractions from kidneys of male DI mice compared with male controls (Fig. 10A). In contrast, there was a lower AQP3 expression in kidneys of male DI mice compared with controls (Fig. 10B). Immunolabeling controls using peptide absorption of the affinity-purified anti-AQP3 antibody revealed a complete absence of labeling (not shown). Densitometry revealed that AQP3 expression was 26 ± 7% of levels in kidneys from control mice (100 ± 16%; Fig. 10C). Thus, in addition to the marked downregulation of the apical collecting duct water channel AQP2, there is also a significant reduction in the basolateral collecting duct water channel AQP3. These results strongly suggest that the expression of both channels is regulated by cAMP and that dysregulation of both channels may play a critical role in the severe polyuria and concentrating defect associated with the increased cAMP-PDE activity in this strain.


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Fig. 10.   A: immunoblot comparing AQP1 expression in kidneys from 5 male DI mice (DI-Male) and from 5 male control mice. Membranes were prepared from total kidneys and loaded at ~10 µg/lane. B: immunoblot comparing AQP3 expression in kidneys from 5 male DI mice (DI-Male) and from 5 male control mice. Membranes were prepared from total kidneys and loaded at ~10 µg/lane. A prominent band was observed at ~26-28 kDa, and higher molecular weight bands were also seen, which are likely to represent glycosylated and/or multimeric AQP3. C: densitometry of AQP3 expression in male DI mice (n = 5) and control mice (n = 5). Values are obtained by densitometry of immunoblots, and expression is presented as fraction of expression in controls.


    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

It has previously been demonstrated that the renal collecting ducts of DI mice have markedly decreased intracellular levels of cAMP and a failure to increase intracellular cAMP in response to vasopressin (1, 2, 11). The present study demonstrated that these mice manifest a marked suppression of AQP2 protein abundance and AQP2 mRNA levels. The low AQP2 protein levels correlated closely with low urinary osmolality in these mice. Furthermore, AQP2 levels were lower in male than in female DI mice in concordance with the lower urine osmolalities in male compared with female DI animals. These results support the view that cAMP plays an important role in the regulation of AQP2 expression. The very low AQP2 protein and AQP2 mRNA levels, especially in male DI mice, strongly support the view that cAMP may represent a final common pathway for modulating AQP2 expression, presumably through regulation of gene transcription. Furthermore, immunoelectron microscopy revealed low labeling of the apical plasma membrane of collecting duct principal cells in kidneys of DI mice, providing direct evidence that delivery of AQP2 to the apical plasma membrane is also severely impaired in the DI mice. In addition, the expression level of AQP3, the basolateral water channel of the collecting duct prin-cipal cell, was decreased in the DI mice. Thus the present study provides evidence that intracellular cAMP levels regulate 1) AQP2 trafficking, 2) AQP2 protein and AQP2 mRNA expression, and 3) AQP3 expression.

Reduced expression of AQP2 protein and AQP2 mRNA in DI mice. As determined both by semiquantitative immunoblotting and by immunocytochemistry, the expression of AQP2 protein was significantly reduced in kidneys from DI mice compared with control mice. In addition, semiquantitative Northern blotting revealed a significantly lower AQP2 mRNA expression in kidneys from DI mice compared with control mice. These findings are consistent with the fact that the DI mice have high nucleotide PDE activity and consequently low levels of cytosolic cAMP (2, 11). Thus the finding of low AQP2 levels in the kidneys of DI mice suggests that cAMP is important for regulation of AQP2 expression, most likely via cAMP-dependent regulation of AQP2 gene transcription. This possibility is discussed in more detail below. The mechanisms responsible for the difference in AQP2 expression and urine production between male and female DI mice remain unknown. It may be speculated that the defects in these mice may render them more sensitive to a potential effect of sex hormones. Although a potential effect of such hormones on the regulation of AQP2 and urine production has not been directly examined, a recent study indicated that there was a difference in urine excretion of AQP2 between male and female individuals (7), raising this as a possibility.

Involvement of PDEs in vasopressin regulation of water transport. Vasopressin binds to V2 receptors present in the basolateral membrane of collecting duct principal cells. Acting through the GTP-binding protein Gs, the interaction of vasopressin with the V2 receptor activates adenylyl cyclase, which accelerates the production of cAMP from ATP (16). Subsequently, cAMP binds to the regulatory subunit of PKA, resulting in dissociation of the regulatory subunit from the catalytic subunit. This activates the catalytic subunit, which phosphorylates various proteins including AQP2. AQP2 is then translocated from intracellular vesicles to the apical plasma membrane, thereby increasing the water permeability of the apical plasma membrane. cAMP-dependent PDE inactivates cAMP by hydrolyzing it to 5'-AMP, to be recycled into a cellular pool of adenylyl nucleotides. In a series of studies, Dousa and Valtin (2, 11, 13) and their associates analyzed the role of PDEs in DI mice, which are completely unresponsive to high levels of arginine vasopressin (AVP) to increase cytosolic levels of cAMP. When measured in microdissected segments of outer medullary (13) and inner medullary (11) collecting duct, the cAMP-PDE activity was significantly higher in DI mice than in normal mice. Rolipram, a selective inhibitor of the isozyme PDE IV, fully restored the AVP-stimulated cAMP accumulation (11). In addition, the authors demonstrated a parallel increase in the density of intramembranous particle clusters (thought to be an index of water channels present in the plasma membrane) in freeze-fracture replicas of the apical plasma membrane and demonstrated that the antidiuretic response to AVP was fully restored (2). The activity of PDE IV was found to be fourfold higher in the DI mice (2, 33). The use of cilostamide, a selective inhibitor of the isozyme PDE III, suggested a role also for PDE III in the defect (11). The critical role of PDEs was further substantiated in subsequent studies using renal cells in vitro by Dousa and colleagues (17, 33, 34).

In the DI mouse, both the activation of adenylyl cyclase and the content of cAMP substrate ATP are normal, but there is a fourfold increase in PDE types III and IV, and hence low baseline levels of cAMP, and a lack of any rise in cAMP levels in response to vasopressin even at high concentrations (11). Our results demonstrating low AQP2 expression in DI mice strongly suggest that cAMP plays a pivotal role in AQP2 gene transcription. Recently, it was demonstrated that a CRE is present in the 5'-untranslated flanking region of the AQP2 gene, suggesting that changes in intracellular levels of cAMP may represent an important pathway regulating AQP2 expression (12, 23, 36). Thus the lower expression of AQP2 in DI mice is likely to reflect an impaired synthesis of AQP2, although other mechanisms cannot be ruled out. For example, it appears possible that the absence of, or reduction in, phosphorylation of AQP2 may also induce trafficking of AQP2 into a degradative pathway.

Role of cAMP in AQP2 trafficking. Immunocytochemistry revealed a marked reduction in AQP2 labeling in collecting ducts from kidneys of DI mice compared with kidneys from control animals. This is consistent with the marked downregulation of AQP2 determined by semiquantitative immunoblotting and is consistent with the parallel decrease in urine osmolality (Figs. 1C and 3B). Moreover, immunocytochemistry and immunoelectron microscopy demonstrated markedly reduced AQP2 labeling of the apical plasma membrane (as well as intracellular vesicles) consistent with impaired targeting and delivery of AQP2 to the plasma membrane in response to vasopressin and with the observed increase in urine production and reduced urine osmolality. This strongly suggests that both AQP2 expression levels and AQP2 targeting (already well established from previous investigations) to the apical plasma membrane are critically dependent on intracellular cAMP levels. This is consistent with recent studies concerning the role of cAMP in PKA-mediated phosphorylation of AQP2 itself for trafficking. It was shown that the serine residue at position 256 (S256) in the cytoplasmic COOH terminus of AQP2 is the consensus site for PKA phosphorylation (9, 18). By introducing a serine-to-alanine mutation into AQP2 by PCR-directed mutagenesis in LLC-PK1 cells, it was recently shown that PKA failed to phosphorylate AQP2 and prevented vasopressin- or forskolin-stimulated translocation of AQP2 from the intracellular site to the cell surface (9, 14). This indicates that vasopressin-induced exocytosis of AQP2 is dependent on the presence of the S256 PKA phosphorylation site (9). The present studies extend this by demonstrating in vivo that kidneys with low cAMP levels in the collecting ducts (kidneys of DI mice) have a marked reduction in AQP2 in the apical plasma membrane in vivo.

Reduced AQP3 expression in kidneys of DI mice. Semiquantitative immunoblotting also revealed a decrease in AQP3 protein expression in kidneys of male DI mice to one-third of control levels (Fig. 10). Thus there was a decrease in the two collecting duct principal cell water channels AQP2 and AQP3. The results indicate that intracellular cAMP is likely to be an important factor in the regulation of AQP3 expression and raise the possibility that AQP3 gene transcription may also be, at least in part, dependent on cytosolic levels of cAMP. However, a CRE element has not been demonstrated for AQP3. Thus it is at present unclear whether a potential role of cAMP in AQP3 expression is a primary or a secondary effect. The parallel decrease of both AQP2 and AQP3 protein levels is consistent with previous studies showing that long-term treatment of rats with exogenous vasopressin in osmotic minipumps is associated with increased AQP3 as well as AQP2 expression, indicating that the expression of both channels can be regulated by vasopressin (31). Furthermore, prolonged dehydration is associated with a parallel increase in both AQP2 and AQP3 (6). In contrast AQP1 expression levels are not changed by vasopressin treatment or dehydration (31). The lack of altered AQP1 expression in DI mice compared with controls is consistent with this.

Ecelbarger et al. (5) recently used a model where rats were implanted with osmotic minipumps administrating DDAVP, and the rats were subsequently water loaded for several days. In this model, there was a parallel increase in both AQP2 and AQP3 protein expression in response to DDAVP treatment alone prior to water loading (this is consistent with the results in the present study showing a lower expression of both AQP2 and AQP3 in DI mice). But after initiating water loading in the continued presence of DDAVP treatment, a marked downregulation of AQP2 but not of AQP3 was observed (5). This indicates that during vasopressin escape, which presumably takes place via a vasopressin-independent mechanism, the regulation of AQP2 and AQP3 differs. Although further studies are warranted to clarify this, it is possible that cAMP is important for regulation of both AQP2 and AQP3 expression, whereas the induced downregulation of AQP2 alone during vasopressin escape may be independent of cAMP.


    ACKNOWLEDGEMENTS

We thank Mette Vistisen, Gitte Christensen, Annette Blak Rasmussen, and Zhila Nikrozi for expert technical assistance.


    FOOTNOTES

Support for this study was provided by the Karen Elise Jensen Foundation, Novo Nordisk Foundation, Danish Medical Research Council, University of Aarhus Research Foundation, the University of Aarhus, the University of Leeds, and the intramural budget of the National Heart, Lung, and Blood Institute, National Institutes of Health.

J. Frøkiaer, D. Marples, and S. Nielsen contributed equally to this study.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus, Denmark.

Received 5 March 1998; accepted in final form 24 September 1998.


    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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