Abstract
ANG II plays a major role in renal water and sodium regulation. In the immortalized mouse renal collecting duct principal cells (mpkCCDcl4) cell line, we treated cells with ANG II and examined aquaporin-2 (AQP2) protein expression, trafficking, and mRNA levels, by immunoblotting, immunofluorescence, and RT-PCR. After 24-h incubation, ANG II-induced AQP2 protein expression was observed at the concentration of 10−10 M and increased in a dose-dependent manner. ANG II (10−7 M) increased AQP2 protein expression and mRNA levels at 0.5, 1, 2, 6, and 24 h. Immunofluorescence studies showed that ANG II increased the apical membrane targeting of AQP2 from 30 min to 6 h. Next, the signaling pathways underlying the ANG II-induced AQP2 expression were investigated. The PKC inhibitor Ro 31–8220 (5 × 10−6 M) and the PKA inhibitor H89 (10−5 M) blocked ANG II-induced AQP2 expression, respectively. Calmodulin inhibitor W-7 markedly reduced ANG II- and/or dDAVP-stimulated AQP2 expression. ANG II (10−9 M) and/or dDAVP (10−10 M) stimulated AQP2 protein levels and cAMP accumulation, which was completely blocked by pretreatment with the vasopressin V2 receptor (V2R) antagonist SR121463B (10−8 M). Pretreatment with the angiotensin AT1 receptor (AT1R) antagonist losartan (3 × 10−6 M) blocked ANG II (10−9 M)-stimulated AQP2 protein expression and cAMP accumulation, and partially blocked dDAVP (10−10 M)- and dDAVP+ANG II-induced AQP2 protein expression and cAMP accumulation. In conclusion, ANG II regulates AQP2 protein, trafficking, and gene expression in renal collecting duct principal cells. ANG II-induced AQP2 expression involves cAMP, PKC, PKA, and calmodulin signaling pathways via V2 and AT1 receptors.
Keywords: signaling pathway, water channel, losartan
angiotensin causes vasoconstriction, increased blood pressure, and release of aldosterone from the adrenal cortex. ANG II has renal receptors (AT1) on the proximal tubule, thick ascending limb (TAL) of the loop of Henle, and the collecting duct (4, 5, 20). In addition, there are AT1 receptors on the afferent and efferent glomerular arterioles and the glomerular mesangium which regulate glomerular filtration rate, renal blood flow, and glomerular membrane permeability. In the collecting duct, ANG II increases expression and activity of epithelial sodium channels (ENaC) in the cortical collecting ducts (15) and facilitates AVP-stimulated urea permeability by activating PKC in the rat terminal inner medullary collecting duct (IMCD) (8). In the TAL and collecting duct, ANG II regulates several pathways including PKC, intracellular calcium, and metabolites of arachidonic acid (2). It is well known that AVP modulates solute-free water excretion by both short-term and long-term regulation of aquaporin-2 water channels (AQP2) in the principal cells of the collecting duct (16). There is evidence showing a relationship between ANG II and AVP. The vasopressin V2 receptor messenger RNA in the IMCD is stimulated by ANG II (12). In Chinese hamster ovary cells transfected with AT1a and V2 receptors, ANG II potentiates AVP-dependent cAMP (9) and forskolin potentiates the ANG II-induced increase in intracellular calcium in the cortical TAL (7). The ANG II AT1a receptor blocker losartan has been shown to decrease AVP-mediated cAMP accumulation in the TAL and normalize increased Na-K-2Cl cotransporter (NKCC2) in rats with chronic congestive heart failure (22). This leads to the hypothesis that the actions of ANG II and AVP are synergistic on urinary concentration via cAMP- and calcium-related pathways in the TAL and collecting duct. An in vivo interaction between ANG II and AVP on urinary concentration has been proposed (11). AT1a knockout mice have been reported to exhibit polyuria which is associated with decreases in adenylyl cyclase and decreased AQP2 trafficking to the apical membrane, indicating that AT1a receptor deletion causes polyuria and urine concentration defects by impairing AVP-induced receptor signaling in the inner medulla (14).
Our laboratory has demonstrated that ANG II AT1 receptor blockade in dDAVP-treated rats on a low-salt diet was associated with decreased urine concentration as well as decreased inner medullary AQP2, p-AQP2, and AQP3 expression, suggesting that AT1 receptor activation plays a significant role in regulating aquaporin expression and modulating urine concentration in vivo (23). Therefore, the present cell culture studies in mpkCCDC14 cells investigated the role of ANG II on AQP2 trafficking and expression in renal collecting duct cells, including examining the signaling pathways.
MATERIALS AND METHODS
Cell culture.
mpkCCDcl4 cells were grown in modified DMEM/Ham's F-12, 1:1 vol/vol (60 nM sodium selenate, 5 μg/ml transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine, 10 ng/ml epidermal growth factor, 5 μg/ml insulin, 20 mM d-glucose, 2% fetal calf serum, and 20 mM HEPES, pH 7.4) at 37°C in 5% CO2-95% air. Experiments were performed in confluent cells seeded on semipermeable polycarbonate filters (Transwell, 0.4-μm pore size, 24-mm insert, Corning Costar, Cambridge, MA). Cells were grown in DMEM until confluence (day 6 after seeding) and then in serum-free, hormone-deprived DMEM for another 24 h before use. The medium was changed every 2 days, and all experiments were performed between passages 25 and 35, as described previously (1).
Protein extraction.
After incubation without or with hormones and/or drugs, cells were washed twice with phosphate-buffered saline and then homogenized in 150 μl of ice-cold lysis buffer [(in mM) 2 EGTA, 2 EDTA, 30 NaF, 30 Na4O7P2, 2 Na3VO4, 1 PMSF, 1 4-(2-aminoethyl)benzenesulfonyl fluoride, and 20 Tris·HCl as well as 10 μg/ml leupeptin, 4 μg/ml aprotinin, 0.1% SDS, and 1% Triton X-100, pH 7.4]. Protein concentrations were measured by BCA protein assay (Pierce, Rockford, IL).
Immunoblot analysis.
Samples of membrane fractions were run on 12% acrylamide gels using methods described previously in detail (13). For each gel, an identical gel was run in parallel and subjected to Coomassie staining for confirmation of equal loading of protein. The other gel was subjected to Western blot analysis. 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 primary antibodies (see below) overnight at 4°C. After washing with PBS-T, the blots were incubated with horseradish peroxidase-conjugated secondary antibody (diluted 1:3,000, P448, Dako, Glostrup, Denmark). After a final washing as above, antibody binding was visualized using the enhanced chemiluminescence (ECL) system (Amersham).
Primary antibodies.
For semiquantitative immunoblotting and immunocytochemistry, antibodies to AQP2 have been previously characterized (21, 24). Mouse monoclonal anti-β-actin antibody was obtained from Sigma-Aldrich (St. Louis, MO). Anti-Na-K-ATPase α1-subunit antibody was obtained from Upstate Biotechnology (Lake Placid, NY).
Immunofluorescence studies.
Filters were detached from the holders, and cells were fixed with methanol for 2 min at −20°C, and permeabilized with PBS-Triton 0.3% for 25 min at room temperature and then vacuum permeabilization buffer. Filters were incubated with the rabbit polyclonal anti-rat AQP2 antibody (dilution 1:50) overnight at 4°C. Samples were incubated with a goat anti-rabbit IgG antibody (dilution 1:250, Invitrogen, Carlsbad, CA) for 90 min at room temperature. Specimens examined by confocal laser-scanning microscopy (Carl Zeiss, Thornwood, NY) were viewed in the x-y and x-z planes, and the images were photographed. Apical AQP2 fluorescence intensity was measured using the LSM Image analyzer postacquisition software (Zeiss). The same microscope setting was used for each condition.
RNA extraction, analysis, and message quantification.
Cytosolic RNA was isolated from confluent cell cultures using an RNeasy kit (Qiagen, Valencia, CA) as per the manufacturer's protocol. Before quantitative PCR (QPCR), sample RNA concentration and integrity were assessed by UV spectrometry (absorbance at 260 nm). RNA was converted to cDNA using an iScript cDNA synthesis kit (Bio-Rad). QPCR was performed using primer pairs identified and designed using Beacon Designer 7.0 (Premier Biosoft, Palo Alto, CA), mouse AQP2 forward primer 5′-GCCCTGCTCTCTCCATTG-3′ and reverse primer 5′-TCAAACTTGCCAGTGACAAC-3′. QPCR runs were performed using the SYBR green JumpStart Taq Readymix QPCR kit (Sigma) on an I-Cycler (Bio-Rad). QPCR runs were analyzed by agarose gel electrophoresis and melt curve to verify that the correct amplicon is produced. β-Actin RNA was used as an internal control in all QPCRs, and the amount of RNA was calculated by the comparative CT method.
Measurement of cAMP production.
cAMP was extracted with 150 μl of 0.1 N HCl at room temperature for 20 min and measured with an EIA kit (Cayman Chemical, Ann Arbor, MI) according to the manufacturer's instructions. Results were expressed in picomoles per milliliter of cell lysate. Each determination was performed in triplicate.
Statistical methods.
Multiple group comparisons were performed using a one-way ANOVA with posttest according to Newman-Keuls. Values represent means ± SE of three independent sets of experiments.
RESULTS
ANG II increased AQP2 protein levels in dose- and time-dependent manners.
To investigate the effect of ANG II on AQP2 expression and trafficking, we examined protein expression levels of AQP2 in response to different concentrations and different time courses of ANG II in mpkCCDC14 cells. As shown in Fig. 1A, after 24 h of incubation ANG II-induced AQP2 expression was observed at the concentration of 10−10 M, increased in a dose-dependent manner to 10−8 M, and was maintained at high levels of 10−7 and 10−6 M. No difference was observed among 10−8, 10−7, and 10−6 M. AQP2 protein bands were detected as nonglycosylated (ng-AQP2), core-glycosylated (cg-AQP2), and fully glycosylated (g-AQP2) forms. AQP2 expression in basal medium was assigned as onefold.
Fig. 1.
Immunoblots of aquaporin-2 (AQP2) in mpkCCDC14 cells after ANG II treatment. Immunoblot reacted with an anti-AQP2 antibody. Protein bands were detected as nonglycosylated (ng-AQP2), core-glycosylated (cg-AQP2), and fully glygosylated (g-AQP2) forms. A: cells were incubated with different concentrations of ANG II. ANG II-induced AQP2 expression was observed at the concentration of 10−10 to 10−6 M. B: cells were incubated with 10−7 M ANG II for 2, 6, 12, 24, and 48 h. ANG II-induced AQP2 expression was observed 2 h after incubation until 48 h. AQP2 expression in basal medium was assigned as 1-fold. β-Actin and α1-Na-K-ATPase protein expression levels are shown as loading controls, and there was no difference after ANG II treatment.*P < 0.05, **P < 0.01 compared with nontreated cells.
Next, cells were incubated in the continuous presence of 10−7 M ANG II for 2, 6, 12, 24, and 48 h. AQP2 protein levels were increased after ANG II treatment for 2–48 h (Fig. 1B). There was no significant difference among 6, 12, 24, and 48 h.
This induction of AQP2 protein was shown when ANG II was added only to the basolateral membrane side, but not the apical side. Furthermore, there was no AQP2 induction in cells cultured on a plastic dish (data not shown).
β-Actin and α1-Na-K-ATPase protein expression levels are shown in Fig. 1, and there was no change after ANG II treatment.
ANG II increased AQP2 labeling density in cortical collecting duct cells.
We examined the labeling staining of AQP2 in mpkCCDC14 cells after short-term ANG II treatment. Confluent cells were grown in six-well Transwell filters (24-mm diameter inserts) and imaged with the polyclonal AQP2 antibody and an Alexa 569-conjugated secondary antibody by a Zeiss LSM 510 confocal laser-scanning microscope. Cells grown under 10−7 M ANG II treatment demonstrated increased staining density of AQP2 at 30 min and 1, 2, 4, and 6 h (Fig. 2A). Z-stack view demonstrated that AQP2 staining specifically increased at both apical and lateral membranes of the cells after ANG II treatment from 30 min to 6 h (Fig. 2B). Quantization of fluorescence intensity demonstrated that apical plasma membrane density of AQP2 significantly increased within the first 30 min after exposure of cells to ANG II, reaching maximum intensity at 6 h (8.3-fold increase, P < 0.001, Fig. 2C). This indicated that short-term ANG II treatment increased AQP2 expression and apical trafficking in the collecting duct mpkCCDC14 cells.
Fig. 2.
Immunofluorescence against AQP2 after ANG treatment in mpkCCDC14 cells. Cells acutely exposed to ANG II (10−7 M) for 30 min, 1, 2, 4, and 6 h. A: results showed that AQP2 localization (green) was increased in the apical plasma membrane in cells 30 min after ANG II treatment until 6 h. B: line Z-scan analysis confirmed that AQP2 labeling density was located and increased in the apical plasma membrane after ANG II treatment. C: quantization of AQP2 fluorescence intensity in the apical membrane domain demonstrated significant apical expression from 30 min to 6 h after exposure of cells to ANG II.
ANG II increased AQP2 mRNA levels.
Cells were treated with ANG II (10−7 M) for 0.5, 2, 6, 12, 24 h. Messenger RNA (mRNA) was isolated, and quantitative real-time PCR was performed. QPCR analysis showed that compared with baseline, ANG II (10−7 M) induced AQP2 mRNA accumulation in a time-dependent manner, but the highest expression of mRNA appeared at 0.5 h and 2 h (Fig. 3).
Fig. 3.
Expression of AQP2 mRNA in mpkCCDC14 cells was increased by ANG II (10−7 M) at 0.5, 1, 2, 6, 12, and 24 h. *P < 0.05, **P < 0.01 compared with nontreated cells.
ANG II increased AQP2 expression via PKC, PKA, and calmodulin signaling pathways.
It is well known that vasopressin stimulates AQP2 expression via the cAMP-PKA pathway. In the current study, the PKA and PKC signaling pathways were examined when mpkCCDC14 cells were treated with ANG II. Cells were pretreated with or without the PKC inhibitor [3-[1-[3-(amidinothiol) propyl-1 H-indoyl-3-yl] maleimide methane sulfonate (Ro 31–8220; 5 × 10−6 M), and the PKA inhibitor N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide (H89; 10−5 M) for 30 min and then incubated without or with ANG II (10−7 M) in the presence of inhibitors for 4 h. PKC inhibitor Ro 31–8220 and the PKA inhibitor H89 blocked ANG II-induced AQP2 expression, respectively, indicating that ANG II-induced AQP2 expression in mpkCCDC14 cells is mediated by both PKA and PKC pathways (Fig. 4).
Fig. 4.
Effect of PKC and PKA inhibition on ANG II-induced AQP2 expression. mpkCCDC14 cells were preincubated for 30 min in the absence or presence of PKC inhibitor (Ro 31–8220; 5 × 10−6 M) or PKA inhibitor (H89; 10−5 M) and then incubated with vehicle and ANG II (10−9 M) in the presence of inhibitors for 4 h. ANG II increased AQP2 protein expression. PKC or PKA inhibitors blocked ANG II-induced AQP2 expression. *P < 0.05 vs. controls.
Cells were pretreated with or without the calmodulin inhibitor W-7 (25 μM) for 30 min and then incubated with or without ANG II (10−9 M) and/or dDAVP (10−10 M) in the presence of inhibitor for 4 h. W-7 blocked ANG II-induced AQP2 expression, indicating a calmodulin-dependent pathway in ANG II-induced AQP2 expression in mpkCCDC14 cells (Fig. 5). Together with previous studies that W-7 blocked AVP-induced AQP2 protein expression (3), these results suggested that both dDAVP and ANG II could regulate AQP2 expression via a calmodulin-dependent pathway.
Fig. 5.
Effect of calmodulin inhibitor on ANG II and/or dDAVP-induced AQP2 expression. mpkCCDC14 cells were preincubated for 30 min in the absence or presence of calmodulin inhibitor W-7 (25 μM) and then incubated with vehicle, or ANG II (10−9 M) and/or dDAVP (10−10 M) in the presence of inhibitors for 4 h. Either ANG II or dDAVP or both increased AQP2 protein expression. *P < 0.05, **P < 0.01, ***P < 0.001 vs. controls. W-7 blocked ANG II-induced AQP2 expression, aP < 0.01. W-7 blocked dDAVP-induced AQP2 expression, bP < 0.05. W-7 blocked ANG II+dDAVP-induced AQP2 expression, cP < 0.01.
dDAVP and ANG II increased AQP2 expression via V2 and AT1 receptors.
mpkCCDC14 cells were treated with ANG II (10−9 M), dDAVP (10−10 M), or cotreatment with both ANG II and dDAVP with or without the V2 receptor antagonist SR121463B or AT1 receptor antagonist losartan for 16 h. SR121463B or losartan was preincubated for 30 min. AQP2 protein expression levels (Figs. 6 and 7) and cAMP levels (Table 1) were examined in these studies.
Fig. 6.
Effect of vasopressin V2 receptor and AT1a receptor antagonists on ANG II-induced AQP2 expression. mpkCCDC14 cells were preincubated for 30 min in the absence or presence of V2R antagonist SR121463B at different concentrations of 10−10, 10−9, and 10−8 M or AT1 receptor antagonist losartan (3 × 10−8, 3 × 10−7, and 3 × 10−6 M) and then incubated with vehicle, or ANG II (10−9 M) in the presence of inhibitors for 16 h. ANG II increased AQP2 protein expression. ***P < 0.001 vs. controls. SR121463B and losartan blocked ANG II-induced AQP2 expression in a dose-dependent manner. ***P < 0.001, **P < 0.01 vs. control. aP < 0.05, bP < 0.001 vs. ANG II.
Fig. 7.
Effect of vasopressin V2 receptor and AT1a receptor antagonists on dDAVP- and dDAVP+ANG II-induced AQP2 expression. mpkCCDC14 cells were preincubated for 30 min in the absence or presence of V2R antagonist SR121463B (10−8 M) or AT1 receptor antagonist losartan (5 × 10−6 M) and then incubated with vehicle, ANG II (10−9 M), and/or dDAVP (10−10 M) in the presence of inhibitors for 16 h. dDAVP and ANG II+dDAVP increased AQP2 protein expression. ***P < 0.001 vs. controls. SR121463B blocked dDAVP-induced AQP2 expression. aP < 0.001 vs. dDAVP and dDAVP+ANG II-induced AQP2 expression. bP < 0.001 vs. ANG II+dDAVP. Losartan partially blocked dDAVP-induced AQP2 expression (aP < 0.001 vs. dDAVP, cP < 0.05) vs. SR+dDAVP and dDAVP+ANG II-induced AQP2 expression (bP < 0.001 vs. ANG II+dDAVP). ***P < 0.001 vs. control. dP < 0.01 vs. SR+ANG II+dDAVP.
Table 1.
Intracellular cAMP levels in mpkCCDC14 cells after ANG II (10−9 M), dDAVP (10−10 M), and ANG II+dDAVP treatment with or without V2 receptor antagonist SR121463B (10−8 M) and AT1 receptor antagonist losartan (5 × 10−6 M)
| cAMP, pg/ml | Control | ANG II | dDAVP | ANG II+dDAVP |
|---|---|---|---|---|
| − | 29.34 ± 0.31 | 34.55 ± 1.35a | 39.39 ± 0.7a | 45.6 ± 4.98b |
| +SR121463B | 31.17 ± 1.83 | 22.47 ± 0.63c | 25.58 ± 2.22d | 22.55 ± 1.69e |
| +Losartan | 27.35 ± 1.39 | 26.67 ± 1.5c | 31.47 ± 3.03d | 34.79 ± 2.14e |
Values are means ± SE.
P < 0.05,
P < 0.001 compared with controls.
P < 0.05 compared with ANG II treatment without antagonist.
P < 0.05 compared with dDAVP treatment without antagonist.
P < 0.05 compared with ANG II+dDAVP treatment without antagonist.
As shown in Fig. 6, the V2 receptor antagonist (SR121463B: 10−10, 10−9, and 10−8 M) or the AT1 receptor blocker (losartan: 3 × 10−8, 3 × 10−7, 3 × 10−6 M) blocked the effect of ANG II on increased AQP2 expression in a dose-dependence manner, indicating both V2 and AT1 receptor pathways are involved in ANG II-induced AQP2 expression. dDAVP-induced AQP2 expression was fully blocked by the V2 receptor antagonist (10−8 M). Interestingly, there was also a decrease in the dDAVP-mediated increase in AQP2 expression with the AT1 receptor blocker (3 × 10−6 M) (Fig. 7). The largest increase in AQP2 expression was found with the cotreatment of ANG II and dDAVP, an effect which was decreased by the V2 receptor antagonist and less so by the AT1 receptor antagonist. As shown in Table 1, we found parallel effects on cAMP as occurred in AQP2 with ANG II, dDAVP, or both with and without the V2 receptor antagonist and AT1 receptor antagonist.
DISCUSSION
The renin-angiotensin system and the nonosmotic release of AVP are increased in important disease states of arterial underfilling, including cardiac failure and cirrhosis (16–18). Moreover, plasma renin activity and AVP-mediated hyponatremia are important risk factors for mortality in patients with severe heart failure and liver disease. Recent experimental evidence suggests that in addition to the interaction between angiotensin AT1 and AVP V1 receptors on systemic and renal vasculature, there may be an important interaction between ANG II and AVP in the regulation of the AQP2 water channel (11, 14, 17–19, 22). Experimental studies in sodium-restricted animals with associated renin-angiotensin system stimulation were shown to have diminished AVP-mediated maximal urine concentration in the presence of ANG II AT1 receptor blocker (11, 23). These effects were associated with a decrease in AQP2 water channels in vivo and in collecting duct cells.
The current study in collecting duct cells was undertaken to investigate further the molecular pathways whereby AVP and ANG II interact on AQP2 water channels. The molecular pathway whereby AVP exerts short-term and long-term regulation in these cells was examined. The mpkCCDc14 cells have been used to examine various aspects of AQP2 protein expression (6). The V2 receptor antagonist was shown to block the effect of the V2 agonist dDAVP to increase AQP2 expression. This blocking effect was associated with the failure of dDAVP to stimulate cAMP, the secondary messenger of AVP. It is also known that cAMP increased PKA, which phosphorylates AQP2, an effect critical for the trafficking of this water channel to the apical membrane. An inhibition of PKA was shown to block the effects of AVP on AQP2 (10). Of interest, calcium and calmodulin are also involved in the effect of AVP on AQP2 (3). In this regard, the calmodulin inhibitor W7 was shown in the present study to block the effect of dDAVP on AQP2 expression.
It was important to demonstrate these effects of dDAVP on AQP2 expression to better examine the pathways whereby ANG II affects AQP2 and interacts with dDAVP. ANG II was found to stimulate AQP2 protein trafficking and expression, effects that were blocked by the AT1 receptor inhibitor losartan. This effect of ANG II was dose related and time dependent. With confocal laser-scanning microscopy, AQP2 staining increased at both apical and lateral cell membranes after ANG II treatment for 30 min to 6 h. Further studies examined the effect of ANG II on transcription and demonstrated AQP2 mRNA accumulation in a time-dependent manner. Similar to dDAVP, the effect of ANG II on AQP2 was found to involve the cAMP, PKA, and calmodulin pathways, since inhibitors of these pathways decreased the effects of ANG II as well as the combination of ANG II and AVP on AQP2 expression. Consonant with the known effect of ANG II to stimulate PKC, the PKC inhibitor in the present study was shown block the effect of ANG II on AQP2. Of interest in this regard, ANG II has been shown to facilitate the effect of AVP to stimulate urea permeability in the IMCD by activating PKC (8). While these in vitro studies advance the understanding the molecular pathways whereby ANG II modulates the effect of dDAVP on AQP2, the involvement of other pathways, such as ERK, JNK, and STAT, certainly cannot be excluded.
Last, there appeared to be cross talk between the V2 vasopressin receptor and AT1 angiotensin receptor, since the effect of ANG II to increase AQP2 could be blocked by a V2 receptor antagonist or an angiotensin AT1 receptor blocker. Moreover, the effect of dDAVP on AQP2 could be attenuated, but not totally blocked, by the AT1 receptor blocker. While the primary effect of vasopressin and ANG II on AQP2 trafficking and expression may be the cAMP-PKA pathway, the calcium-calmodulin pathway appears also to be involved. The potential cross talk between the AT1a and V2 receptors is novel (Fig. 8) but is certainly in need of future study.
Fig. 8.
Potential pathway for interaction between ANG II and AVP in the modulation of urinary concentration.
In summary, vasopressin-modulated AQP2 trafficking and expression can be enhanced by ANG II and involves both the cAMP-PKA and calcium-calmodulin signaling pathways.
GRANTS
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-19928.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
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