Increased AQP2 targeting in primary cultured IMCD cells in response to angiotensin II through AT1 receptor

doi:10.1152/ajprenal.00090.2006 292:340-350, 2007. First published Aug 8, 2006;Am J Physiol Renal Physiol

Søren Nielsen and Tae-Hwan Kwon Yu-Jung Lee, In-Kyung Song, Kyung-Jin Jang, Jakob Nielsen, Jørgen Frøkiær,receptor

1cells in response to angiotensin II through ATIncreased AQP2 targeting in primary cultured IMCD

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Increased AQP2 targeting in primary cultured IMCD cells in responseto angiotensin II through AT1 receptor

Yu-Jung Lee,1 In-Kyung Song,1 Kyung-Jin Jang,1 Jakob Nielsen,2

Jørgen Frøkiær,2,3 Søren Nielsen,2 and Tae-Hwan Kwon1,2

1Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook NationalUniversity, Taegu, Korea; 2Water and Salt Research Center, University of Aarhus,and 3Institute of Clinical Medicine, Aarhus University Hospital, Aarhus, Denmark

Submitted 19 March 2006; accepted in final form 31 July 2006

Lee YJ, Song IK, Jang KJ, Nielsen J, Frøkiær J, Nielsen S,Kwon TH. Increased AQP2 targeting in primary cultured IMCD cellsin response to angiotensin II through AT1 receptor. Am J PhysiolRenal Physiol 292: F340–F350, 2007. First published August 8, 2006;doi:10.1152/ajprenal.00090.2006.—Vasopressin and angiotensin II(ANG II) play a major role in renal water and Na� reabsorption. Wepreviously demonstrated that ANG II AT1 receptor blockade de-creases dDAVP-induced water reabsorption and AQP2 levels in rats,suggesting cross talk between these two peptide hormones (Am JPhysiol Renal Physiol 288: F673–F684, 2005). To directly addressthis issue, primary cultured inner medullary collecting duct (IMCD)cells from male Sprague-Dawley rats were treated for 15 min with 1)vehicle, 2) ANG II, 3) ANG II � the AT1 receptor blocker cande-sartan, 4) dDAVP, 5) ANG II � dDAVP, or 6) ANG II � dDAVP �candesartan. Immunofluorescence microscopy revealed that 10�8 MANG II or 10�11 M dDAVP (protocol 1) was associated withincreased AQP2 labeling of the plasma membrane and decreasedcytoplasmic labeling, respectively. cAMP levels increased signifi-cantly in response to 10�8 M ANG II and were potentiated bycotreatment with 10�11 M dDAVP. Consistent with this finding,immunoblotting revealed that this cotreatment significantly increasedexpression of phosphorylated AQP2. ANG II-induced AQP2 targetingwas blocked by 10�5 M candesartan. In protocol 2, treatment with alower concentration of dDAVP (10�12 M) or ANG II (10�9 M) didnot change subcellular AQP2 distribution, whereas 10�12 MdDAVP � 10�9 M ANG II enhanced AQP2 targeting. This effect wasinhibited by cotreatment with 10�5 M candesartan. ANG II-inducedcAMP accumulation and AQP2 targeting were inhibited by inhibitionof PKC activity. In conclusion, ANG II plays a role in the regulationof AQP2 targeting to the plasma membrane in IMCD cells throughAT1 receptor activation and potentiates the effect of dDAVP on AQP2plasma membrane targeting.

aquaporin; cAMP; intracellular trafficking; dDAVP; protein kinase C

RENAL REABSORPTION AND EXCRETION of water and Na� arecritical to the regulation of extracellular fluid volume. Vaso-pressin and angiotensin II (ANG II) have important roles in therenal conservation of water and Na� (13, 26, 25, 38). Vaso-pressin is a peptide hormone that controls body fluid homeosta-sis through the regulation of renal tubular water reabsorption(25, 26). Its main site of action in kidney tubules is thecollecting duct, where vasopressin binds to the vasopressin V2

receptor and stimulates an increase in intracellular cAMPcontent via adenylyl cyclase (2, 26, 48, 49). Subsequently,cAMP activates protein kinase A (PKA), which phosphorylates

various proteins, including aquaporin 2 (AQP2) (7, 12, 39). Inthe collecting duct principal cells, AQP2 is then translocatedfrom intracellular vesicles to the apical plasma membrane,increasing osmotic water permeability (37). Moreover, tran-scriptional regulation of AQP2 in vivo is thought to be a resultof a vasopressin-induced increase in intracellular cAMP levels(11, 17), which is capable of stimulating AQP2 gene transcrip-tion in cultured cells by acting through cAMP response ele-ment and activator protein-1 sites in the AQP2 promoter (18,32). Binding of vasopressin to the V2 receptor is also associ-ated with intracellular Ca2� mobilization from ryanodine-sensitive stores in the inner medullary collecting duct (IMCD)cells, which triggers calmodulin-dependent regulatory pro-cesses within the cell (6).

ANG II is a peptide hormone that regulates renal blood flow,glomerular filtration rate, and aldosterone secretion. Moreover,it is well established that ANG II increases Na� and bicarbon-ate reabsorption in the kidney proximal tubule via the AT1

receptor (14). Recent studies have demonstrated that ANG IIalso has an important effect on the thick ascending limb (TAL)and collecting duct, where ANG II receptor mRNA and proteinare present (15, 47). For example, 1) ANG II stimulates theactivity of the epithelial Na� channel in an isolated corticalcollecting duct (41), and ANG II induces vasopressin V2

receptor mRNA expression in cultured IMCDs (53), 2) ANG IIinfusion in rats increases expression of the Na�/H� exchangerNHE3 and the Na�-K�-2Cl� cotransporter NKCC2 in themedullary TAL (mTAL) (27), and 3) ANG II increases vaso-pressin-stimulated facilitated urea transport in the rat terminalIMCD (21). These findings suggest an important role of ANGII in regulation of urinary concentration capacity.

The action of vasopressin and ANG II is mediated byintracellular secondary messengers, which are mainly coupledto the cAMP-PKA and phosphoinositide pathways, respec-tively. Vasopressin induces an increase in intracellular cAMPlevels (10, 23), whereas ANG II induces a rise in intracellularCa2� concentration ([Ca2�]i) by inositol 1,4,5-triphosphate (4)and protein kinase C (PKC) activation by diacylglycerol (20).Importantly, it has previously been demonstrated that ANG IIpotentiates the vasopressin-dependent cAMP accumulation inChinese hamster ovary cells transfected with cDNA of AT1A

and V2 receptors (24). Moreover, forskolin potentiates theANG II-induced increase in [Ca2�]i in an isolated cortical TAL(19). These results suggest cross talk between the signaling

Address for reprint requests and other correspondence: T.-H. Kwon, Dept.of Biochemistry and Cell Biology, School of Medicine, Kyungpook NationalUniv., Dongin-dong 101, Taegu 700-422, Korea (e-mail: [email protected]).

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

Am J Physiol Renal Physiol 292: F340–F350, 2007.First published August 8, 2006; doi:10.1152/ajprenal.00090.2006.

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pathways of vasopressin and ANG II. Consistent with thesefindings, we recently demonstrated that pharmacologicalblockade of the ANG II AT1 receptor in dDAVP-treated ratssubjected to dietary NaCl restriction (to induce high plasmaendogenous ANG II levels) increased urine production, de-creased urine osmolality, and blunted the dDAVP-inducedupregulation of AQP2 and phosphorylated AQP2 (i.e., AQP2phosphorylated in the PKA-phosphorylation consensus siteSer256) expression in the inner medulla (28).

Since regulation of intracellular AQP2 trafficking and os-motic water permeability in the collecting duct principal cellsinvolves intracellular cAMP production (11, 45), we hypothe-sized that these two peptide hormones have additive effects onthe intracellular cAMP accumulation in IMCD cells and,hence, on the stimulation of AQP2 phosphorylation, AQP2trafficking, and the increase in osmotic water permeability ofthe collecting duct cells. To directly address this issue, the aimof the present study was to examine the direct effect of 1)dDAVP or ANG II alone and 2) dDAVP � ANG II in primarycultured IMCD cells. We also examined the effect of dDAVPor ANG II alone in AQP2-transfected Madin-Darby caninekidney (MDCK) cells.

Specifically, we examined 1) the short-term effect ofdDAVP or ANG II on AQP2 targeting to the plasma mem-brane, corresponding changes of cAMP level and phosphory-lated AQP2 expression, 2) the additive effect of ANG II inpotentiating the effect of dDAVP on AQP2 targeting to theplasma membrane, cAMP level, and phosphorylated AQP2expression (i.e., dDAVP � ANG II), 3) whether the effect ofANG II on AQP2 targeting was mediated by the ANG II AT1

receptor, and 4) whether ANG II-induced activation of PKCactivity played a role in the effect of ANG II on AQP2targeting and cAMP levels.

METHODS

Primary Culture of Rat Kidney IMCD Cells: Protocols 1, 2, and 3

Primary cultures enriched in IMCD cells were prepared frompathogen-free male Sprague-Dawley rats (200–270 g body wt;Samtako, Osan, Korea) as described previously (6). The animalprotocols were approved by the Animal Care and Use Committee ofthe Kyungpook National University, Korea. Both kidneys were rap-idly removed from male Sprague-Dawley rats under light enfluraneinhalation anesthesia. The kidneys were placed in cold D-PBS [1,000ml of double-distilled H2O, 0.2 g of KCl, 0.2 g of KH2PO4, 1.15 g ofNa2HPO4, 80 mM urea, and 130 mM NaCl (pH 7.4, 640 mosmol/kgH2O)] and quickly dissected into inner medulla and other parts. Theinner medulla was placed on an ice-cold petri dish, minced, trans-ferred to enzyme solution [10 ml of DMEM-Ham’s F-12 withoutphenol red (catalog no. 21041-025, GIBCO), 20 mg of collagenase B(catalog no. 1088815, Roche, Mannheim, Germany), 7 mg of hyal-uronidase (catalog no. H-3884, Sigma, St. Louis, MO), 80 mM urea,and 130 mM NaCl], and incubated at 37°C under continuous agitation(300 rpm) for 90 min in a humidified incubator under 5% CO2-95%air. Centrifugation of the resulting suspension at 160 g for 1 minyielded a pellet that contained IMCD fragment and IMCD cells. Thepellet was washed in prewarmed culture medium without enzyme[DMEM-Ham’s F-12 without phenol red, 80 mM urea, 130 mM NaCl,10 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin (10,000U/ml), 50 nM hydrocortisone, 5 pM 3,3,5-triiodothyronine, 1 nMsodium selenate, 5 mg/l transferrin, and 10% FBS (pH 7.4, 640mosmol/kgH2O)]. The IMCD cell suspension was then seeded inhuman fibronectin-coated chamber slides (Lab-Tek Chamber Slides

System, catalog no. 177402, NUNC, Roskilde, Denmark) for immu-nocytochemical analysis or in a 24-well chamber (catalog no. 142485,NUNC) for immunoblot analysis and cAMP measurement. For im-munoblot analysis, an IMCD cell suspension of only the lowertwo-thirds of the inner medulla (IM-2 and IM-3) was cultured toexclude the cell component of intercalated cells expressed in IM-1.IMCD cells were fed every 24 h and grown in hypertonic culturemedium (640 mosmol/kgH2O) supplemented with 10% FBS at 37°Cin 5% CO2-95% air for 3 days and then in FBS-free culture mediumfor an additional 1 day before the experiment on day 5.

Experimental Protocols for Primary Cultured IMCD Cells

Protocol 1: higher dose of dDAVP and ANG II. IMCD cells weretreated with 1) vehicle (FBS-free culture medium), 2) dDAVP (10�11

M), 3) ANG II (10�8 M), 4) dDAVP (10�11 M) � ANG II (10�8 M),5) ANG II (10�8 M) � AT1 receptor blocker (candesartan, 10�5 M;a gift from AstraZeneca Pharmaceutical), and 6) dDAVP (10�11

M) � ANG II (10�8 M) � candesartan (10�5 M) for 15 min.Protocol 2: lower dose of dDAVP and ANG II. IMCD cells were

treated with 1) vehicle (FBS-free culture medium), 2) dDAVP (10�12

M), 3) ANG II (10�9 M), 4) dDAVP (10�12 M) � ANG II (10�9 M),5) ANG II (10�9 M) � candesartan (10�5 M), and 6) dDAVP (10�12

M) � ANG II (10�9 M) � candesartan (10�5 M) for 15 min.Protocol 3: ANG II with or without PKC inhibitor. IMCD cells

were treated with 1) vehicle (FBS-free culture medium), 2) vehicle(FBS-free culture medium) � the PKC inhibitor staurosporine (cata-log no. S6942, Sigma; 10�7 M), 3) ANG II (10�8 M), and 4) ANG II(10�8 M) � staurosporine (10�7 M) for 15 min. The cells subjectedto PKC inhibition were preincubated with staurosporine (10�7 M) for10 min and then incubated with vehicle or ANG II for another 15 minin the presence of staurosporine.

Primary Culture of Rat Kidney IMCD Cells in Isosmotic orHyperosmotic Culture Medium Without Urea

To avoid the potential effects of urea or hyperosmolality on theshort-term AQP2 targeting in the primary cultured IMCD cells (8, 16,50), control experiments were done with primary cultured IMCD cellsgrown in hyperosmotic (640 mosmol/kgH2O) and isosomotic (300mosmol/kgH2O) culture media, neither of which contained urea. Theosmolality of the culture medium [DMEM-Ham’s F-12 without phe-nol red, 10 mM HEPES, 2 mM L-glutamine, penicillin-streptomycin(10,000 U/ml), 50 nM hydrocortisone, 5 pM 3,3,5-triiodothyronine, 1nM sodium selenate, 5 mg/l transferrin, and NaCl (pH 7.4)] wasadjusted by changing NaCl concentration and determined by freezing-point depression (Osmomat 030-D, Gonotec, Berlin, Germany).IMCD cells were fed every 24 h and cultured in medium that wassupplemented with 10% FBS at 37°C in 5% CO2-95% air for 3 daysand then in FBS-free culture medium for additional 1 day before theexperiment on day 5. IMCD cells cultured in hyperosmotic culturemedium without urea (640 mosmol/kgH2O) were treated with 1)vehicle, 2) dDAVP (10�11 M), or 3) ANG II (10�8 M) for 15 min onday 5.

Immunocytochemistry of Rat Kidney IMCD Cells

IMCD cells were grown to confluence in each chamber for 4 daysand treated on day 5 after seeding according to the experimentalprotocols. After treatment, cells in each chamber were washed twicein PBS and fixed with 4% paraformaldehyde in PBS (pH 7.4) for 30min at room temperature. After the cells were washed twice in PBS,they were permeabilized with 0.3% Triton X-100 in PBS at roomtemperature for 15 min. The cells were incubated with anti-AQP2antibody (36) in PBS overnight at 4°C, washed in PBS, and incubatedwith goat anti-rabbit IgG Alexa Fluor 488 secondary antibody (Mo-lecular Probes, Eugene, OR) for 1.5 h at room temperature. Cells werewashed and mounted with a hydrophilic mounting medium containing

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antifading reagent (catalog no. P36930, Molecular Probes). Lightmicroscopy was carried out with a light microscope (Axioplan2imaging, Zeiss, Jena, Germany) or an inverted microscope (modelDM IRB, Leica Microsystems, Wetzlar, Germany) equipped with aCoolSNAP HQ camera (Photometrics, Tucson, AZ).

cAMP Measurement in Primary Cultured IMCD Cells

IMCD cells were cultured to confluence in a 24-well chamber for4 days after they were seeded and treated on day 5 according to theprotocols. All measurements was carried out in the presence of 1 mM3-isobutyl-1-methylxanthine (IBMX; Sigma) to inhibit cyclic nucle-otide phosphodiesterases. After 10 min of preincubation with 1 mMIBMX, dDAVP, ANG II, or dDAVP � ANG II was added for anadditional 15 min in the continued presence of IBMX. cAMP contentof the samples was measured using a competitive enzyme immuno-assay kit (Cayman Chemical, Ann Arbor, MI), and the results wereexpressed in picomoles per milliliter of cell lysate (2 � 105 cellsseeded per chamber). Each determination was performed in triplicate.

For the measurement of cAMP levels in response to PKC inhibitortreatment (protocol 3), we repeated the determination twice. Eachdetermination was performed in triplicate; thus the results werepooled and expressed as a fraction of the control level (vehicle-treatedgroup).

Semiquantitative Immunoblotting of Phosphorylated AQP2

After incubation, IMCD cells were homogenized in RIPA buffer[10 mM Tris �HCl, 0.15 M NaCl, 1% NP-40, 1% Na-deoxycholate,0.5% SDS, 0.02% sodium azide, and 1 mM EDTA (pH 7.4)]. Thetotal protein concentration was measured using the bicinchoninic acidprotein assay reagent kit (Pierce, Rockford, IL). All samples wereadjusted with isolation solution to the identical final protein concen-trations, solubilized at 65°C for 15 min in Laemmli sample buffer, andthen stored at 4°C. For confirmation of equal loading of protein, aninitial gel was stained with Coomassie blue dye as described previ-ously (28, 36). SDS-PAGE was performed on 12% polyacrylamidegels. The proteins were transferred from the gel electrophoretically(Mini Protean II, Bio-Rad) to nitrocellulose membranes (Hybond ECLRPN3032D, Amersham Pharmacia Biotech, Little Chalfont, UK).After transfer, the blots were blocked with 5% milk in PBS-T [80 mMNa2HPO4, 20 mM NaH2PO4, 100 mM NaCl, and 0.1% Tween 20 (pH7.5)] for 1 h and incubated overnight at 4°C with antibodies directedagainst phosphorylated AQP2 (7). The sites of antibody-antigenreaction were visualized with horseradish peroxidase-conjugated sec-ondary antibodies (1:3,000 dilution; catalog no. P448, Dako, Glostrup,Denmark) with an enhanced chemiluminescence (ECL) system andexposed to photographic film (Hyperfilm ECL, RPN3103K, Amer-sham Pharmacia Biotech). For quantitation of the band densities (29-and 35- to 50-kDa phosphorylated AQP2 bands), the films werescanned and the densitometry values were normalized to facilitatecomparisons. For immunoblot analysis, in each group, four differentprotein samples were obtained from IMCD cells cultured in fourdifferent culture well chambers. For each immunoblotting gel, twodifferent protein samples from each group were loaded. The measuredband densities (i.e., from 4 different protein samples in each group)were pooled and expressed as a fraction of the control level (vehicle-treated group).

AQP2-Transfected MDCK Cells

pCDNAI/Neo with rat cDNA encoding AQP2 (tagged with aCOOH-terminal c-myc epitope) was generously provided by Dr. D.Brown (30). MDCK cells were grown in DMEM supplemented with10% (vol/vol) FBS at 37°C in 5% CO2. MDCK cells were then seededin chamber slides (Lab-Tek Chamber Slides System) and transientlytransfected using Lipofectamine reagent (catalog no. 18324-020, In-vitrogen). Cells were kept in serum-free medium containing 10 �M

indomethacin overnight before experiments to inhibit endogenousprostaglandin production and to prevent cAMP production and ana-lyzed 48 h after transfection (35). Cells were treated with vehicle or10�8 M dDAVP or 10�7 M ANG II for 15 min. The experiment wasrepeated three times. Cells were fixed in 4% paraformaldehyde for 10min, rinsed twice in PBS, and blocked for 15 min in blocking/permeabilization solution (PBS containing 0.1% BSA and 0.3%Triton X-100). Cells were incubated with anti-c-myc antibody (catalogno. C-3956, Sigma) diluted in blocking solution overnight at 4°C,washed three times in PBS, and incubated with the secondary anti-body Alexa Fluor 488 goat anti-rabbit IgG (Molecular Probes; 1:400dilution) for 1 h at room temperature. Cells were analyzed using aninverted microscope (model DM IRB, Leica Microsystems) equippedwith a laser confocal scanning unit (model CSU-10, Yokokawa,Tokyo, Japan), an Ar-Kr laser (Melles Griot, Irvine, CA), and aCoolSNAP HQ camera. Fluorescent images were acquired, and semi-quantitative measurement of average pixel intensities in the plasmamembrane and cytoplasmic AQP2 labeling was done by using Meta-morph software (Molecular Devices, Sunnyvale, CA). The averagepixel intensities of the plasma membrane AQP2 labeling were ex-pressed as a fraction of that of cytoplasmic AQP2 labeling.

Presentation of Data and Statistical Analyses

Values are means � SE. Data were analyzed by one-way ANOVAfollowed by Tukey’s honestly significant different multiple-compari-sons test. Multiple-comparisons tests were applied only when asignificant difference was determined by ANOVA (P � 0.05). P �0.05 was considered statistically significant.

RESULTS

Increased AQP2 Targeting to the Plasma Membrane inResponse to Short-Term Treatment With dDAVP or ANG II:Protocol 1

AQP2 immunofluorescent labeling was largely dispersedalong the cytoplasm of primary cultured IMCD cells in theabsence of dDAVP or ANG II stimulation (Fig. 1A). Incontrast, treatment with 10�11 M dDAVP for 15 min wasassociated with an increase in AQP2 immunofluorescent label-ing of the plasma membrane and a decrease in labeling in thecytoplasm (Fig. 1C), indicating that dDAVP increased AQP2targeting to the plasma membrane in IMCD cells. Treatmentwith 10�8 M ANG II for 15 min was also associated withenhanced AQP2 labeling of the plasma membrane, indicatingthat ANG II per se directly stimulated AQP2 targeting to theplasma membrane of IMCD cells (Fig. 1E). Pretreatment with10�5 M candesartan, an ANG II AT1 receptor blocker, inhib-ited AQP2 targeting to the plasma membrane induced by 10�8

M ANG II (Fig. 1F), demonstrating that the ANG II-inducedAQP2 targeting was mediated by the ANG II AT1 receptor.Treatment with 10�11 M dDAVP � 10�8 M ANG II was alsoassociated with strong AQP2 labeling of the plasma membrane(Fig. 1B), similar to the effect of 10�11 M dDAVP (Fig. 1C) or10�8 M ANG II (Fig. 1E), and the effect was not completelyinhibited by pretreatment with 10�5 M candesartan (Fig. 1D).

To support the finding that dDAVP- or ANG II-stimulatedAQP2 targeting to the plasma membrane was specific, weconducted two control experiments to show that the effects areindependent of the urea (80 mM) in culture medium and thehyperosmolality (640 mosmol/kgH2O) of the culture medium(Fig. 2). First, urea may affect PKC-mediated signaling (8),which in turn may affect regulation of AQP2 targeting. Wetherefore examined the effect of dDAVP and ANG II without

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urea in the culture medium (Fig. 2). AQP2 immunofluorescentlabeling in IMCD cells grown in culture medium without ureawas dispersed in the cytoplasm in the absence of dDAVP orANG II stimulation (Fig. 2B). Treatment for 15 min with 10�11

M dDAVP (Fig. 2C) or 10�8 M ANG II (Fig. 2D) wasassociated with enhanced AQP2 targeting to the plasma mem-brane, which indicates that the dDAVP- or ANG II-stimulatedAQP2 targeting (Figs. 1 and 3) was not dependent on urea.Second, hyperosmolality can regulate AQP2 in a vasopressin-independent manner (50). Because the hyperosmolar (640mosmol/kgH2O) culture medium used in the first study mighthave affected the results, we examined AQP2 labeling inprimary cultured IMCD cells grown for 4 days in isosmolar(300 mosmol/kgH2O) culture medium without urea or in hy-perosmolar (640 mosmol/kgH2O) culture medium withouturea. AQP2 labeling was much weaker in IMCD cells culturedin isosmolar medium (Fig. 2A) than in those cultured inhyperosmolar medium (Fig. 2B). This is consistent with theprevious finding that the expression of AQP2 (nonglycosylatedform) was increased by hyperosmolality (50). However, im-

munolabeling was mainly seen in the cytoplasm in both con-ditions (Fig. 2, A and B), indicating that hyperosmolity of cellculture medium per se is unlikely to play a major role in theshorter-term AQP2 targeting.

Additive Effect of dDAVP and ANG II on AQP2 Targeting tothe Plasma Membrane: Protocol 2

Next we tested the effects of lower doses of dDAVP andANG II (protocol 2) to examine whether dDAVP and ANG IIhave an additive effect on AQP2 targeting. In the absence ofdDAVP or ANG II stimulation, AQP2 labeling was dispersedalong the cytoplasm of primary cultured IMCD cells (Fig. 3A),as in protocol 1 (Fig. 1A). In protocol 2, however, little AQP2targeting to the plasma membrane was observed in response to15 min of treatment with 10�12 M dDAVP or 10�9 M ANG II(Fig. 3, C and E). In contrast, 15 min of treatment with 10�12

M dDAVP � 10�9 M ANG II was associated with enhancedAQP2 targeting to the plasma membrane (Fig. 3B), which wasblocked by cotreatment with the ANG II AT1 receptor blocker

Fig. 1. Immunofluorescence microscopy ofaquaporin 2 (AQP2) in primary cultured in-ner medullary collecting duct (IMCD) cells(protocol 1). A: AQP2 labeling exclusivelylocalized at the cytoplasm in response tovehicle. C and E: increase in AQP2 targetingto the plasma membrane in response todDAVP (C) or ANG II (E). F: blockade ofANG II-induced AQP2 targeting by cotreat-ment with candesartan. B and D: abundantAQP2 targeting to the plasma membrane inresponse to dDAVP � ANG II and no inhi-bition by ANG II AT1 receptor blockade.




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candesartan (10�5 M; Fig. 3D). This finding strongly suggeststhat dDAVP and ANG II have an additive effect on AQP2targeting to the plasma membrane of IMCD cells and that theANG II effect is mediated by the ANG II AT1 receptor.

cAMP Production Was Potentiated in Response to dDAVP �ANG II Compared With dDAVP or ANG II

To examine whether the enhanced AQP2 targeting to theplasma membrane in response to dDAVP or ANG II ordDAVP � ANG II was associated with an increase in cAMPproduction, we measured cAMP levels in primary culturedIMCD cells. In a preliminary study to test the validity of cAMPmeasurement in this study, we examined the effects of 15 minof treatment with different concentrations of dDAVP (10�11,10�9, and 10�7 M) and forskolin (10�4 M) on cAMP levels inprimary cultured IMCD cells. Consistent with previous results(9), cAMP levels increased in response to dDAVP treatment ina dose-dependent manner (Fig. 4). Moreover, forskolin mark-edly increased cAMP levels in primary cultured IMCD cells(Fig. 4).

In protocol 1, cAMP levels were unchanged in response to15 min of treatment with 10�11 M dDAVP, whereas levelsincreased in response to 10�8 M ANG II (Fig. 5A). Impor-tantly, the cAMP increase after ANG II was potentiated by10�11 M dDAVP � 10�8 M ANG II (Fig. 5A), suggesting anadditive effect. In protocol 2, the changes in cAMP levels weresimilar to those in protocol 1 (Fig. 5B). cAMP levels wereunchanged by 10�12 M dDAVP but increased by 10�9 M ANGII (Fig. 5B). The additive effect of 10�12 M dDAVP � 10�9 MANG II on cAMP levels compared with the effect of 10�9 MANG II alone did not reach statistical significance in protocol2 (Fig. 5B; P � 0.06).

Phosphorylated AQP2 Expression Was Increased inResponse to dDAVP � ANG II

Since cAMP activates PKA, which phosphorylates variousproteins, including AQP2, we examined the changes in expres-sion of phosphorylated AQP2 (phosphorylated in the PKAphosphorylation consensus site Ser256). Treatment with 10�11

M dDAVP � 10�8 M ANG II significantly increased phos-phorylated AQP2 expression to 135 � 4% of vehicle-treatedcontrol levels (100 � 3%, P � 0.05; Fig. 6). Treatment with10�11 M dDAVP or 10�8 M ANG II did not significantlychange phosphorylated AQP2 expression (97 � 1% and 117 �2% of vehicle-treated control levels, respectively, P � notsignificant). This result suggests an additive effect ofdDAVP � ANG II on AQP2 phosphorylation, consistent withthe additive effect on cAMP production (Fig. 5) and AQP2targeting to the plasma membrane (Fig. 3B). The increasedexpression of phosphorylated AQP2 by dDAVP � ANG II wasattenuated by the ANG II AT1 receptor blocker candesartan(114 � 1% of vehicle-treated control levels, P � not signifi-cant; Fig. 6). Again, this suggests that the effect of ANG IIon AQP2 phosphorylation is mediated by the ANG II AT1

receptor.

AQP2 Targeting to the Plasma Membrane Was Increased inResponse to dDAVP or ANG II in AQP2-c-myc-TransfectedMDCK Cells

To confirm the results observed in primary cultured IMCDcells, we tested the direct effect of dDAVP or ANG II onsubcellular AQP2 localization in MDCK cells that were tran-siently transfected with AQP2. In nontreated MDCK cells,AQP2 was mainly localized intracellularly [Fig. 7A; semiquan-titative measurement of average pixel intensity of AQP2 label-

Fig. 2. Immunofluorescence microscopy ofAQP2 in primary cultured IMCD cells. A:weak AQP2 labeling exclusively localized atthe cytoplasm in response to vehicle treat-ment in cells grown in isosmotic culturemedium without urea. B: stronger AQP2 la-beling mainly localized at the cytoplasm inresponse to vehicle treatment in cells grownin hyperosmolar culture medium withouturea. C and D: increase in AQP2 targeting tothe plasma membrane in response to dDAVP(C) or ANG II (D) in cells grown in hyper-osmolar culture medium without urea.




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ing in the plasma membrane of 30 randomly selected trans-fected cells: 14 � 2% of the cytoplasmic intensity (Fig. 7D)].AQP2 targeting to the plasma membrane was clearly observedin cells treated for 15 min with 10�8 M dDAVP [Fig. 7B;

average pixel intensity of AQP2 labeling in the plasma mem-brane of 30 randomly selected transfected cells: 31 � 3% ofthe cytoplasmic intensity (Fig. 7E)]. Stimulation with 10�7 MANG II also resulted in translocation of AQP2 to the plasmamembrane [Fig. 7C; average pixel intensity of AQP2 labelingin the plasma membrane of 30 randomly selected transfectedcells: 24 � 2% of the cytoplasmic intensity (Fig. 7F)], sup-porting the results obtained in the primary cultured IMCDcells.

Treatment With PKC Inhibitor Decreased ANG II-InducedAQP2 Targeting to the Plasma Membrane and cAMPProduction in Primary Cultured IMCD Cells

PKC is a component of the signal transduction pathway ofANG II. Since there is evidence demonstrating that PKC playsa role in stimulation of the receptor-coupled adenylate cyclasein some cell systems (24, 33, 43), we examined the effect of thePKC inhibitor staurosporine on ANG II-induced AQP2 target-ing and cAMP production in primary cultured IMCD cells.

Fig. 3. Immunofluorescence microscopy ofAQP2 in primary cultured IMCD cells (pro-tocol 2). A: AQP2 labeling exclusively local-ized at the cytoplasm in response to vehicletreatment. C and E: no or little AQP2 target-ing to the plasma membrane in response todDAVP (C) or ANG II (E). B and D: in-crease in AQP2 targeting to the plasmamembrane in response to dDAVP � ANG IIand inhibition by ANG II AT1 receptorblockade.

Fig. 4. cAMP in primary cultured IMCD cells. cAMP (pmol/ml cell lysate)was dose dependently increased in response to dDAVP. Forskolin significantlyincreased cAMP in primary cultured IMCD cells (2 � 105 cells seeded perchamber). *P � 0.05 vs. vehicle, �P � 0.05 vs. 10�11 or 10�9 M dDAVP.




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Consistent with the results shown in Fig. 1, treatment with10�8 M ANG II for 15 min was associated with AQP2targeting to the plasma membrane in IMCD cells (Fig. 8A). Incontrast, 10�8 M ANG II � 10�7 M staurosporine was asso-ciated with decreased AQP2 targeting (Fig. 8B). Moreover,cAMP levels increased in response to 10�8 M ANG II,whereas cAMP levels did not increase in response to 10�8 MANG II � 10�7 M staurosporine (Fig. 9).

DISCUSSION

In the present study, we demonstrated that 1) short-termtreatment with dDAVP or ANG II had a stimulatory effect onAQP2 targeting to the plasma membrane in primary culturedIMCD cells and AQP2-transfected MDCK cells; 2) dDAVP �ANG II potentiated cAMP production, phosphorylated AQP2expression, and AQP2 targeting in primary cultured IMCDcells compared with dDAVP or ANG II alone; 3) the effect ofANG II on phosphorylated AQP2 expression and AQP2 tar-geting was inhibited by ANG II AT1 receptor blockade, sug-gesting that the ANG II effect was mediated by the ANG IIAT1 receptor; and 4) ANG II � PKC inhibitor decreased ANGII-induced cAMP production and AQP2 targeting in primarycultured IMCD cells, suggesting that ANG II-induced activa-tion of PKC, at least partly, played a role in this process.

dDAVP or ANG II Has a Stimulatory Effect on AQP2Targeting to the Plasma Membrane

Regulation of the water permeability of the apical plasmamembrane in the collecting duct principal cells is critical forregulation of renal water reabsorption and body water balance.In the collecting duct, AQP2 is expressed in the apical plasma

membrane and subapical vesicles in the collecting duct prin-cipal cells and is the chief target for regulation of the osmoticwater permeability in response to vasopressin by short-termregulated translocation of intracellular AQP2-bearing vesiclesto the apical plasma membrane (37) and by long-term regula-tion of the AQP2 expression (31). This process allows produc-tion of concentrated urine and is essential for water balanceregulation.

We demonstrated that acute dDAVP treatment enhancedAQP2 targeting to the plasma membrane of primary culturedIMCD cells. This finding is consistent with a number ofprevious in vitro and in vivo studies (6, 7, 22, 26, 37, 38, 44,51, 54). It is well known that increased AQP2 labeling densityof the apical plasma membrane in response to vasopressincorrelates closely with the increased osmotic water permeabil-ity of the isolated perfused IMCD (37). Moreover, vasopressinor dDAVP treatment of vasopressin-deficient Brattleboro ratswas associated with a marked increase in AQP2 labelingintensity in the apical plasma membrane of the collecting ductprincipal cells and an increase in urinary concentration (7, 54).Interestingly, we demonstrated that short-term treatment withANG II was also associated with increased AQP2 targeting tothe plasma membrane in IMCD cells, which was inhibited by

Fig. 5. cAMP in primary cultured IMCD cells in protocols 1 (A) and 2 (B). A:no change in cAMP in response to dDAVP and increase in response to ANGII. Increase in cAMP levels was potentiated by dDAVP � ANG II. B: changein cAMP levels similar to that in protocol 1. *P � 0.05 vs. vehicle. �P � 0.05vs. ANG II. #P � 0.05 vs. dDAVP.

Fig. 6. Semiquantitative immunoblotting of phosphorylated AQP2 (p-AQP2)in primary cultured IMCD cells (protocol 1). A: immunoblot reacted withanti-phosphorylated AQP2 revealed 29- and 35- to 50-kDa phosphorylatedAQP2 bands. B: densitometric analysis. For immunoblot analysis, in eachgroup, 4 different protein samples were obtained from IMCD cells cultured in4 different culture well chambers. Phosphorylated AQP2 expression (29- and35- to 50-kDa bands) was significantly increased in response to 10�11 MdDAVP � 10�8 M ANG II (d � A) compared with vehicle-treated controls,whereas it was unchanged in response to 10�11 M dDAVP or 10�8 M ANG IIalone. Increase in phosphorylated AQP2 expression in d � A group wasinhibited by ANG II AT1 receptor blocker candesartan (d � A � C). *P �0.05 vs. all other groups.




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cotreatment with the ANG II AT1 receptor blocker candesar-tan. This suggests that short-term treatment with ANG II could,at least partly, play a role in the activation of signal transduc-tion pathways leading to AQP2 recruitment to the plasmamembrane and that this was mediated by ANG II AT1 receptoractivation.

The signal transduction pathways for AQP2 targeting to theplasma membrane have been investigated in a number ofprevious studies (25, 38). cAMP levels in the collecting ductprincipal cells are increased in response to vasopressin bindingto the V2 receptor (2, 48, 49), and, subsequently, cAMPactivates PKA, which phosphorylates various proteins, includ-ing AQP2 (7, 12, 39, 48, 49). Consistent with this, we dem-onstrated that cAMP levels increased in response to dDAVP ina dose-dependent manner in primary cultured IMCD cells.cAMP levels were also significantly increased by ANG II, and,

importantly, dDAVP � ANG II had an additive effect oncAMP production, phosphorylated AQP2 expression, andAQP2 targeting in primary cultured IMCD cells.

We demonstrated enhanced AQP2 targeting to the plasmamembrane and increased cAMP levels in response to ANG IIwith no significant changes in phosphorylated AQP2 expres-sion (Fig. 6). Since intracellular cAMP accumulation in IMCDcells is known to activate PKA, which subsequently increasesAQP2 phosphorylation and AQP2 trafficking, an increase inphosphorylated AQP2 expression would be expected. How-ever, at the time point used no significant changes wereobserved, but it cannot be excluded that AQP2 phosphorylationmay have been increased at some time during the stimulation.Moreover, it remains possible that only a minor fraction ofAQP2 is subject to phosphorylation and at a level wherefractional changes escape detection.

Fig. 7. Subcellular localization of AQP2 inAQP2-transfected Madin-Darby canine kid-ney (MDCK) cells. A: AQP2 exclusivelylocalized intracellularly in vehicle-treatedcells. B: AQP2 labeling at the plasma mem-brane (arrows) in dDAVP-treated cells. C:translocation of AQP2 to the plasma mem-brane (arrows) in ANG II-treated cells. D–F:semiquantitative measurement of averagepixel intensities in the plasma membrane(PM) and cytoplasmic (C) AQP2 labeling inAQP2-transfected MDCK cells in responseto vehicle (D), dDAVP (E), and ANG II (F).Average pixel intensities of the plasma mem-brane AQP2 labeling are expressed as a frac-tion of that of cytoplasmic AQP2 labeling.




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dDAVP � ANG II Potentiated cAMP Production,Phosphorylated AQP2 Expression, and AQP2 Targeting

The actions of the peptide hormones vasopressin and ANGII are mediated by intracellular secondary messengers. Vaso-pressin induces an increase in intracellular cAMP levels,whereas ANG II induces a rise in [Ca2�]i by inositol 1,4,5-triphosphate (4) and PKC activation by diacylglycerol (20).Our findings revealed that ANG II had an additive effect on thedDAVP-induced cAMP production, phosphorylated AQP2 ex-pression, and AQP2 targeting. This suggests cross talk betweenthe intracellular signaling pathways of these two peptide hor-mones, consistent with our previous findings that ANG II AT1

receptor blockade decreased dDAVP-induced water reabsorp-tion and AQP2 levels in rat kidney (28).

How does short-term treatment with ANG II stimulatecAMP accumulation, AQP2 phosphorylation, and AQP2 tar-geting in primary cultured IMCD cells? It is known thatincreases in [Ca2�]i and in cAMP levels are required for AQP2targeting and the accompanying increase in osmotic waterpermeability (6, 55). The role of Ca2� in the cross talk betweenANG II and vasopressin was investigated in a previous study(24) using Chinese hamster ovary cells transfected with cDNAof the AT1A receptor and the V2 receptor. This study demon-strated that chelation of intracellular Ca2� lowered cAMPaccumulation by vasopressin and markedly decreased potenti-ation of vasopressin-induced cAMP accumulation by ANG II(22). On the other hand, the same study also demonstrated thatincreased [Ca2�]i induced by thapsigargin comparable to that

induced by ANG II did not potentiate the vasopressin-depen-dent cAMP accumulation, indicating that the increase in[Ca2�]i does not likely play a major role in the cross talk.Consistent with this, Lorenz et al. (29) demonstrated thatAQP2 shuttling is evoked neither by a vasopressin-dependentincrease of [Ca2�] nor by a vasopressin-independent increaseof [Ca2�] in primary cultured IMCD cells from rat kidney,although clamping of [Ca2�]i below resting levels inhibitsAQP2 exocytosis. However, there is a discrepancy between theresults from primary cultured IMCD cells (29) and isolatedperfused IMCD tubules (6) with regard to the role of [Ca2�]i invasopressin-induced AQP2 trafficking. Further studies are re-quired to clarify this discrepancy, which may be related toaltered expression levels of vasopressin receptors and/or AQP2or other elements in the AQP2 trafficking system.

Another transduction pathway of ANG II is PKC. In thisstudy, we demonstrated that cAMP levels were significantlyincreased in response to short-term ANG II treatment, whereasthe levels were not increased in response to ANG II treatmentin the presence of the PKC inhibitor staurosporine. Moreover,immunocytochemistry demonstrated that AQP2 targeting tothe plasma membrane was stimulated by ANG II alone,whereas it was attenuated in response to ANG II � staurospor-ine. These findings suggest that activation of PKC could, atleast in part, play a role in stimulation of adenylate cyclase andAQP2 targeting in primary cultured IMCD cells. It has beenshown that PKC activation can inhibit or enhance cAMPaccumulation in different cell types (3, 34, 43, 46). Theinhibition is frequently caused by phosphorylation and inacti-vation of receptors linked to adenylate cyclase (34, 46). Incontrast, several studies also demonstrated an enhancement ofcAMP accumulation by PKC (1, 43, 52). For example, in asystem using Swiss 3T3 cells, which provided a useful modelto elucidate the early signals and molecular events capable ofinitiating a mitogenic response, Rozengurt et al. (43) demon-strated that PKC activation by biologically active phorbolesters markedly enhanced cAMP accumulation induced byforskolin and that this was prevented by downregulation ofPKC. The modulatory effect of ANG II on adenylate cyclasecould involve phosphorylation of different components of thesystem, including Gs and/or Gi protein, and indeed, severalstudies suggested that PKC activation of Gs protein may playa role in the stimulation of adenylate cyclase and increase ofcAMP levels (40, 43). Another possibility is that the stimula-

Fig. 8. Immunofluorescent microscopy ofAQP2 in primary cultured IMCD cells (pro-tocol 3). A: enhanced AQP2 targeting to theplasma membrane in ANG II-treated cells.B: decreased AQP2 targeting to the plasmamembrane in ANG II � PKC inhibitor (stau-rosporine)-treated cells.

Fig. 9. cAMP in primary cultured IMCD cells (protocol 3). cAMP wasunchanged in response to vehicle or vehicle � PKC inhibitor staurosporine(stau). ANG II increased cAMP, but effect of ANG II � staurosporine wassimilar to that of vehicle. *P � 0.05 vs. vehicle.




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tory effect of PKC on cAMP accumulation could be mediatedby interaction with the Gi protein (42), and future studies arewarranted to elucidate these possibilities in IMCD cells.

ANG II-mediated intracellular signaling is not restricted to[Ca2�] and PKC: ANG II also activates ERK, JNK, and STAT.In particular, ERK, p38 kinase, and phosphatidylinositol 3�-kinase (PI3-kinase) were shown to regulate AQP2 (5). Impor-tantly, Bustamante et al. (5) demonstrated that vasopressin-induced AQP2 expression was potentiated by insulin inmpkCCD(cl4) cells and that the insulin-induced stimulation ofAQP2 expression was decreased by inhibition of ERK, p38kinase, and PI3-kinase activities, whereas inhibition of PKCactivity had no effect. Further studies are warranted to examinewhether ANG II plays a role in the long-term regulation ofAQP2 by increasing AQP2 gene transcription by activation ofMAP kinase and PI3-kinase, as demonstrated by insulin (5).

In summary, we demonstrated that short-term ANG II treat-ment of primary cultured IMCD cells could play a role in theregulation of AQP2 targeting to the plasma membrane throughAT1 receptor activation. This could potentiate the effect ofdDAVP on cAMP accumulation, AQP2 phosphorylation, andAQP2 plasma membrane targeting.

GRANTS

This study was supported by Regional Technology Innovation Program ofthe MOCIE Grant RTI04-01-01, the Brain Korea 21 Project in 2006, theKorean Society of Electrolyte and Blood Pressure Research, the DanishNational Research Foundation, the Karen Elise Jensen Foundation, and theDanish Medical Research Council.

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Increased AQP2 targeting in primary cultured IMCD cells in response to angiotensin II through AT1 receptor