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Jan 28, 2015 - lation and intracellular trafficking of AQP2 in inner medullary collecting duct cells. Am J Physiol Renal Physiol 308: F737–F748, 2015. First.
Am J Physiol Renal Physiol 308: F737–F748, 2015. First published January 28, 2015; doi:10.1152/ajprenal.00376.2014.

Extracellular pH affects phosphorylation and intracellular trafficking of AQP2 in inner medullary collecting duct cells Hyo-Jung Choi,1,2 Hyun Jun Jung,1 and Tae-Hwan Kwon1,2 1

Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea; and 2BK21 Plus KNU Biomedical Convergence Program, Department of Biomedical Science, Kyungpook National University, Taegu, Korea Submitted 1 July 2014; accepted in final form 21 January 2015

Choi HJ, Jung HJ, Kwon TH. Extracellular pH affects phosphorylation and intracellular trafficking of AQP2 in inner medullary collecting duct cells. Am J Physiol Renal Physiol 308: F737–F748, 2015. First published January 28, 2015; doi:10.1152/ajprenal.00376.2014.—Kidney collecting duct cells are continuously exposed to the changes of extracellular pH (pHe). We aimed to study the effects of altered pHe on desmopressin (dDAVP)-induced phosphorylation (Ser256, Ser261, Ser264, and Ser269) and apical targeting of aquaporin-2 (AQP2) in rat kidney inner medullary collecting duct (IMCD) cells. When freshly prepared IMCD tubule suspensions exposed to HEPES buffer with pH 5.4, 6.4, 7.4, or 8.4 for 1 h were stimulated with dDAVP (10⫺10 M, 3 min), AQP2 phosphorylation at Ser256, Ser264, and Ser269 was significantly attenuated under acidic conditions. Next, IMCD cells primary cultured in transwell chambers were exposed to a transepithelial pH gradient for 1 h (apical pH 6.4, 7.4, or 8.4 vs. basolateral pH 7.4 and vice versa). Immunocytochemistry and cell surface biotinylation assay revealed that exposure to either apical pH 6.4 or basolateral pH 6.4 for 1 h was associated with decreased dDAVP (10⫺9 M, 15 min, basolateral)-induced apical targeting of AQP2 and surface expression of AQP2. Fluorescence resonance energy transfer analysis revealed that the dDAVP (10⫺9 M)-induced increase of PKA activity was significantly attenuated when LLC-PK1 cells were exposed to pHe 6.4 compared with pHe 7.4 and 8.4. In contrast, forskolin (10⫺7 M)-induced PKA activation and dDAVP (10⫺9 M)-induced increases of intracellular Ca2⫹ were not affected. Taken together, dDAVPinduced phosphorylation and apical targeting of AQP2 are attenuated in IMCD cells under acidic pHe, likely via an inhibition of vasopressin V2 receptor-G protein-cAMP-PKA actions. acidosis; collecting duct; fluorescence resonance energy transfer; vasopressin; vasopressin V2 receptor RENAL TUBULAR EPITHELIAL CELLS,

including kidney collecting duct cells, are continuously exposed to changes of the microenvironment, e.g., luminal fluid shear stress, transepithelial osmotic gradients, and changes of luminal or interstitial pH. We previously demonstrated the effects of altered luminal fluid shear stress on the translocation of aquaporin-2 (AQP2) and reorganization of the actin cytoskeleton (F-actin) in kidney inner medullary collecting duct (IMCD) cells (14). Immunocytochemistry demonstrated that exposure to luminal fluid shear stress per se in the microfluidic chamber induced depolymerization of F-actin associated with increased apical targeting of AQP2, even in the absence of vasopressin stimulation. This finding suggests that changes of the microenvironment,

Address for reprint requests and other correspondence: T.-H. Kwon, Dept. of Biochemistry and Cell Biology, School of Medicine, Kyungpook National Univ., Dongin-dong 101, Taegu 700-422, South Korea (e-mail: thkwon@knu. ac.kr). http://www.ajprenal.org

including extracellular pH (pHe), could be importantly involved in the apical targeting of AQP2. AQPs are a family of membrane proteins that transport water molecules through biomembranes (18, 21, 31). AQP2 is the water-selective channel protein that plays a key role in arginine vasopressin (AVP)-mediated water reabsorption in collecting ducts (2, 20, 21, 27, 30, 31, 41). Vasopressin V2 receptor (V2R)-mediated cAMP/PKA signaling has been shown to be a principal signaling pathway for both AQP2 trafficking and protein expression via activation of Gs␣-mediated adenylyl cyclase activity (7, 16, 30, 41, 42). However, despite the well-known effects of AVP stimulation, the concurrent effects of changes in the luminal or interstitial microenvironment in vivo, e.g., pH, on the phosphorylation and apical targeting of AQP2 in collecting duct cells are not well understood. Urine pH can vary between 4.6 and 8.0, depending on the amount of urinary net acid excretion. A recent clinical study (3) demonstrated that almost 60% of patients with stones had a urine pH of ⬍6, a value consistent with that found in the general population. It has been demonstrated that the affinity of vasopressin for V2R was significantly lower under the condition of acidic pH compared with that at neutral pH in LLC-FLAG-V2R cells (44). Moreover, rats with metabolic acidosis had significantly decreased urinary excretion of AQP2, whereas renal AQP2 mRNA and protein expressions were increased (28). In addition, it has been demonstrated that urine alkalinization promoted the excretion of urinary exosomal AQP2 independent of vasopressin stimulation in rats (9). Therefore, we hypothesized that changes in pHe could affect intracellular pH (pHi) of collecting duct cells, resulting in an altered response of AQP2 phosphorylation (Ser256, Ser261, Ser264, and Ser269) and apical targeting of AQP2 to vasopressin stimulation. First, desmopressin (dDAVP)-induced AQP2 phosphorylation (Ser256) was examined in IMCD tubule suspensions exposed to pH 5.4, 6.4, 7.4 and 8.4, respectively. To study the selective effects of apical or basolateral pH on dDAVP-induced intracellular trafficking of AQP2 in collecting duct cells, IMCD cells from rat kidneys were primary cultured in a transwell chamber system. The effects of acidic (pH 6.4), neutral (pH 7.4), or alkaline (pH 8.4) conditions in the apical or basolateral sides of cells were examined on dDAVP-induced intracellular trafficking and cell surface expression of AQP2. Moreover, to further elucidate the underlying signaling mechanisms, fluorescence resonance energy transfer (FRET) analysis was exploited, and time-lapse changes of AVP- or forskolin-induced PKA activity and intracellular Ca2⫹ dynamics were monitored in LLC-PK1 cells exposed to acidic (pH 6.4), neutral (pH 7.4), or alkaline (pH 8.4) microenvironments.

1931-857X/15 Copyright © 2015 the American Physiological Society

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MATERIALS AND METHODS

Primary culture of IMCD cells from rat kidneys. The animal protocols were approved by the Animal Care and Use Committee of Kyungpook National University (KNU 2012-10). Primary cultures enriched in IMCD cells were prepared from pathogen-free male Sprague-Dawley rats (200 –250 g, Charles River, Seongnam, Korea), as previously described in detail (6, 23, 24). Briefly, rats were anesthetized under enflurane inhalation, and kidneys were rapidly removed. After the IMCD cell suspension had been isolated, cells were seeded on either collagen-coated 35-mm glass-base dishes (Asahi Techno Glass, Tokyo, Japan) for pHi measurements or semipermeable filters of the transwell system (0.4-␮m pore size, Transwell Permeable Supports, catalog no. 3450, Corning) for cell surface biotinylation assays. IMCD cells were fed every 48 h and grown in hypertonic culture medium (640 mosmol/kgH2O) supplemented with 10% FBS at 37°C in a 5% CO2-95% air atmosphere for 3 days and then in FBS-free culture medium for an additional 1 day before the experiment on day 5. The culture medium was DMEM-F-12 without phenol red, containing 80 mM urea, 130 mM NaCl, 10 mM HEPES, 2 mM L-glutamine, 10,000 U/ml penicillin-streptomycin, 50 nM hydrocortisone, 5 pM 3,3,5-triiodo-thyronine, 1 nM sodium selenate, 5 mg/l transferrin, and 10% FBS (pH 7.4, 640 mosmol/kgH2O). IMCD tubule suspensions. Fresh IMCD tubules were prepared from rat kidneys as previously described (5, 35). Rats were anesthetized under enflurane inhalation. The kidney inner medulla was dissected, minced, and digested in IMCD suspension buffer (250 mM sucrose and 10 mM triethanolamine, pH 7.4) containing collagenase B (3 mg/ml) and hyaluronidase (2 mg/ml). The IMCD suspension was then continuously agitated in a warm water bath at 37°C for 90 min. Thereafter, the IMCD tubule suspension was centrifuged at 60 g for 30 s to separate the IMCD-enriched fraction (pellet) and non-IMCD fraction (supernatant). The IMCD-enriched fraction was collected and washed twice with ice-cold IMCD suspension buffer. For the experiments, IMCD tubule suspensions were exposed to HEPES-buffered saline solution (162.5 mM NaCl, 2.5 mM Na2HPO4, 4 mM KCl, 25 mM HEPES, 2 mM CaCl2, 1.2 mM MgSO4, and 5.5 mM glucose) with different pH (pH 5.4, 6.4, 7.4, or 8.4) for 1 h, which was established by adding HCl (for pH 5.4) or NaOH (for pH 6.4, 7.4, and 8.4) (43). dDAVP stimulation (10⫺10 M) was done during the last 3 min (Fig. 1A). Semiquantitative immunoblot analysis. SDS-PAGE was performed on 12% polyacrylamide gels, as previously described (34). Primary

dDAVP 10-10 M, 3 min

50

AQP2

37 25 50

p-AQP2 (S256)

37 25

β-actin

50

pH 5.4

pH 6.4

pH 7.4

B pH 8.4 Normalized Band Density

(kDa)

(S256/Total AQP2)

A

antibodies used were anti-AQP2 (H7661AP, 1:2,000) (15, 23), antiphosphorylated (p-)AQP2 at Ser256 (K0307AP, 1:3,000) (34), anti-pAQP2 at Ser261, Ser264, and Ser269 (Phosphosolutions, 1:1,000), and anti-␤-actin (Sigma, 1:100,000). Immunofluorescence microscopy of primary cultured IMCD cells. IMCD cells were grown to confluence in each transwell chamber (0.4-␮m pore size, Transwell Permeable Supports, catalog no. 3460, Corning) for 4 days. On day 5, IMCD cells were subjected to a transepithelial pH gradient for 1 h (pH 6.4, 7.4, or 8.4 at the apical side vs. pH 7.4 at the basolateral side and vice versa) at 37°C. dDAVP stimulation (10⫺9 M, 15 min, basolateral side only) was done during the last 15 min, and cells were then fixed with 3% paraformaldehyde in PBS (pH 7.4) for 20 min at room temperature. After fixation, cells were washed twice in PBS and permeabilized with 0.3% Triton X-100 in PBS at room temperature for 15 min. Cells were washed and then labeled with anti-AQP2 (H7661AP, 1:200) antibody, and nuclei were stained with 4=,6-diamidino-2-phenylindole. AQP2 localization was determined using laser confocal microscopy (Zeiss LSM 5 EXCITER, Jena, Germany), and a ⫻63 (numerical aperture: 1.4) objective lens (Zeiss) was used. Digital images were collected and analyzed using the Zeiss Aim Image Examiner program. Cell surface biotinylation assays. IMCD cells were primary cultured for 4 days on semipermeable filters of the transwell system (0.4-␮m pore size, Transwell Permeable Supports, catalog no. 3450, Corning). On day 5, they were exposed to a transepithelial pH gradient for 1 h (apical pH 6.4, 7.4, or 8.4 vs. basolateral pH 7.4 and vice versa) at 37°C. Cells were then washed three times with ice-cold 10 mM PBS containing 1 mM CaCl2 and 0.1 mM MgCl2 (PBS-CM; pH 7.5) and incubated at 4°C for 45 min in ice-cold biotinylation buffer (10 mM triethanolamine, 2 mM CaCl2, and 125 mM NaCl, pH 8.9) containing 1 mg/ml sulfosuccinimidyl 2-(biotinamido)-ethyl-1,3dithiopropionate (sulfo-NHS-SS-biotin, Thermo Scientific, Rockford, IL) on the apical side. Cells were washed once with quenching buffer (50 mM Tris·HCl in PBS-CM, pH 8.0) followed by two washes with PBS-CM. Lysis buffer [1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 50 mM Tris·HCl (pH 7.5), 1.15 mM PMSF, 0.4 ␮g/ml leupeptin, 0.1 mg/ml Pefabloc, 0.1 ␮M okadaic acid, 1 mM Na3VO4, and 25 mM NaF] was added, and lysates were sonicated with 2 ⫻ 6 pulses at 20% of amplitude. Lysates were centrifuged at 10,000 g for 5 min at 4°C. The supernatant was transferred to columns where Neutravidin agarose resin (200 ␮l) had been previously loaded (Thermo Scientific) and incubated for 60 min at room temperature

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

#+ #+

*+ *# pH 5.4 pH 6.4 pH 7.4 pH 8.4 (n=6) (n=6) (n=6) (n=6)

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Fig. 1. Semiquantitative immunoblot analysis of aquaporin-2 (AQP2) and phosphorylated (p-)AQP2 (Ser256) in inner medullary collecting duct (IMCD) tubule suspensions. A: IMCD tubule suspensions were incubated for 1 h in HEPES-buffered saline solution with different pH (pH 5.4, 6.4, 7.4, or 8.4). IMCD tubule suspensions were stimulated with desmopressin (dDAVP; 10⫺10 M) for the last 3 min. Immunoblots of IMCD tubule suspensions reacted with antibodies against AQP2 and p-AQP2 (deglycosylated ⬃29 kDa and glycosylated ⬃35–50 kDa). B: densitometric analysis. *P ⬍ 0.05 compared with dDAVP-treated IMCD tubule suspensions exposed to pH 7.4; #P ⬍ 0.05 compared with dDAVP-treated IMCD tubule suspensions exposed pH 6.4; ⫹P ⬍ 0.05 compared with dDAVP-treated IMCD tubule suspensions exposed pH 5.4. n is the number of IMCD tubule suspension preparations from independent experiments. AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

EXTRACELLULAR pH AFFECTS dDAVP-INDUCED AQP2 TRAFFICKING

with end-over-end mixing. After being washed four times with PBS containing protease inhibitors, 1⫻ sample buffer containing DTT was added to the column, and samples were incubated for 60 min at room temperature. Samples were heated at 65°C for 10 min. pHi measurements. pHi was measured using the pH-sensitive fluorescent dye BCECF-AM (Molecular Probes). BCECF-AM is converted to the membrane-impermeant fluorescent dye BCECF by intracellular esterases. Primary cultured IMCD cells from rat kidneys were seeded and cultured on collagen-coated 35-mm glass-base dishes (Asahi Techno Glass) in hypertonic culture media (pH 7.4 and 640 mosmol/kgH2O) for 4 days. On day 5, cells were incubated with nonserum IMCD culture media (DMEM-F-12 without phenol red, 80 mM urea, 130 mM NaCl, and 10,000 U/ml penicillin-streptomycin) containing 2 ␮M BCECF-AM at 37°C in 5% CO2-95% air for 30 min. Cells were washed twice in PBS and changed to nonserum IMCD culture media (pH 7.4) for 10 min in a 37°C incubator before experiments. IMCD cells were mounted in the chamber on the temperature-controlled stage of Nikon Ti-E inverted microscope (Nikon, Tokyo, Japan). Cells were continuously perfused with Na⫹free solution with different pH values (pH 6.4, 7.4, and 8.4, see below) for 60 min by a six-channel system (VC-2, Warner Instruments, Hamden, CT), and the perfusate was collected by the peristaltic pump (EYELA MP3, Tokyo, Japan). pHi was measured using the dual-excitation fluorescence ratio method. The fluorescence was monitored at 530 nm with excitation wavelengths of 440 nm (a wavelength at which the fluorescence is relatively pH insensitive) and 490 nm (a wavelength at which the fluorescence is very pH sensitive). pHi was time lapse obtained by the Nikon Ti-E inverted microscope equipped with a Cascade 512B (EMCCD) camera (Roper Scientific, Trenton, NJ) and excitation and emission filter wheels (MAC5000, Ludl Electronic Products, Hawthorne, NY). All systems were controlled by MetaMorph software (Universal Imaging, Downingtown, PA). Fluorescent images were acquired sequentially through cyan fluorescent protein (CFP) filter channels. Images were acquired using the 4 ⫻ 4 binning mode and 200-ms exposure time. A ⫻60 (numerical aperture: 1.4) objective lens (Nikon) was used for measurements. The 490-to-440-nm ratio was created by MetaMorph software. Data were obtained from five independent experiments in both groups, respectively. pHi was calculated by the nigericin calibration technique (3). IMCD cells were exposed to Na⫹-free solution (0 mM Na⫹, 105 mM ⫹ 2⫹ K⫹, 0 mM NH⫹ ,0 4 , 49 mM N-methyl-D-glucamine , 5 mM Mg mM Ca2⫹, 6.3 mM Cl⫺, 106.4 mM aspartate⫺, 0.4 mM H2PO⫺ 4 , 1.6 ⫺ 2⫺ ⫺ mM HPO2⫺ 4 , 17.8 mM HEPES , 0 mM HCO3 , 5 mM SO4 , 10 mM 2⫺ EGTA , 5.5 mM glucose, 5 mM alanine, and 14.4 mM HEPES) with different pH values (6.4, 7.4, and 8.4) by adding KOH containing 10 ␮M of the K⫹/H⫹ exchanger nigericin. When the K⫹ concentration was equal between intracellular and extracellular compartments, pHi and pHe should be the same with nigericin (490-to-440-nm ratio: 1.0, pH 7.0) (3). Therefore, data were normalized to make the ratio at pH 7.0 equal to unity. Raw data of intensity at each excitation wavelength were corrected for background-corrected fluorescence intensity before calculation of the ratios. The ratios of backgroundcorrected emission intensities (490-to-440-nm ratio) were transformed into pH values as pH ⫽ log {[Rn ⫺ Rn(min)]/[Rn(max) ⫺ Rn]} ⫹ pKa, where Rn is the background-substracted ratio normalized to pH 7.0, Rn(min) and Rn(max) are minimum and maximum obtainable values for the normalized ratio, and pKa is the ⫺log of the dissociation constant of the fluorophore (1, 3). FRET imaging for PKA activity. LLC-PK1 cells stably transfected with AKAR3EV (PKA FRET-based indicator) were cultured on collagen-coated 35-mm glass-base dishes (Asahi Techno Glass). The plasmid for AKAR3EV was kindly provided by Prof. M. Matsuda (Kyoto University, Kyoto, Japan). AKAR3EV was composed of FHA1 (ligand domain), yellow fluorescent protein (YFP), the sensor domain of PKA (LRRATLVD), and CFP (17). In this probe design, the sensor domain changes its conformation upon perception of the

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PKA signal (17). This sensitized sensor domain interacts with the ligand domain, thereby inducing a global change of the biosensor conformation and a concomitant increase of FRET efficiency from the donor to the acceptor. With this AKAR3EV probe, the activity of PKA was time lapse obtained by the Nikon Ti-E inverted microscope equipped with a Cascade 512B (EMCCD) camera (Roper Scientific) and excitation and emission filter wheels (MAC5000, Ludl Electronic Products). All systems were controlled by MetaMorph software (Universal Imaging). Fluorescent images were acquired every 15 s sequentially through CFP and FRET filter channels. Images were acquired using the 4 ⫻ 4 binning mode and 200-ms exposure time. The FRET-to-CFP ratio was created with MetaMorph software, and the value was normalized to 1 at the starting time point in both groups. Data were obtained from five independent experiments in both groups, respectively. FRET imaging for intracellular Ca2⫹ dynamics. Two days after the transient transfection of YC3.60 (Ca2⫹ FRET-based indicator) (29), LLC-PK1 cells in DMEM (phenol red free) were subjected to timelapse imaging. LLC-PK1 cells transiently transfected with YC3.60 were cultured on collagen-coated 35-mm glass-base dishes (Asahi Techno Glass). The plasmid for YC3.60 was also provided by Prof. M. Matsuda (Kyoto University). YC3.60 was composed of calmodulin (CaM), enhanced YFP, a glycylglycine linker, the CaM-binding peptide of myosin light chain kinase (M13), and enhanced CFP (29). Ca2⫹ binding to the CaM moiety initiates an intramolecular interaction between CaM and M13 domains, which causes the chimeric protein to shift from a spread form to a more compact form, therefore increasing the efficiency of FRET from CFP to YFP (29). With this YC3.60 indicator, changes of intracellular Ca2⫹ levels were time lapse obtained by the Nikon Ti-E inverted microscope. Images were acquired every 15 s using the 4 ⫻ 4 binning mode and 200-ms exposure time. The FRET-to-CFP ratio was created with MetaMorph software, and the value was normalized to 1 at the starting time point in all groups. Data were obtained from eight independent experiments in both groups, respectively. Statistical analyses. Values are presented as means ⫾ SE. Data were analyzed by one-way ANOVA followed by Bonferroni’s multiple-comparison test. Multiple-comparisons tests were only applied when a significant difference was determined by ANOVA. P values of ⬍0.05 were considered as significant. RESULTS

Effects of pHe on dDAVP-induced AQP2 phosphorylation in IMCD tubule suspensions. To examine whether changes of pHe affect dDAVP-induced AQP2 phosphorylation in IMCD cells, freshly prepared IMCD tubule suspensions were incubated for 1 h in HEPES-buffered saline solution with different pH (pH 5.4, 6.4, 7.4, or 8.4). First, AQP2 phosphorylation was examined with specific antibodies against p-AQP2 at Ser256 (PKA phosphorylation consensus site), and the p-AQP2-to-AQP2 ratio was examined in IMCD tubule suspensions treated with either vehicle or dDAVP (10⫺10 M, 3 min; Fig. 1, A and B). Semiquantitative immunoblot analysis revealed that dDAVPinduced AQP2 phosphorylation (Ser256) was significantly decreased when tubule suspensions were exposed to pH 5.4 and 6.4 compared with pH 7.4 and 8.4 (Fig. 1B). Moreover, the decrease of AQP2 phosphorylation was more prominent when tubule suspensions were exposed to more acidic conditions (pH 5.4) compared with pH 6.4 (Fig. 1B), suggesting that acidic pHe decreases dDAVP-induced phosphorylation of AQP2 in IMCD cells and that lower pH values have even more prominent effects. Based on this finding, three pH levels, i.e., pH 6.4, 7.4, and 8.4, were chosen for further experiments, as urine pH can vary between 4.6 and 8.0 in humans.

AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

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EXTRACELLULAR pH AFFECTS dDAVP-INDUCED AQP2 TRAFFICKING

Next, the effects of dDAVP at pH 6.4, 7.4, and 8.4 were compared with vehicle-treated control at pH 6.4, 7.4, and 8.4, respectively, in IMCD tubule suspensions (Fig. 2). Changes of dDAVP-induced AQP2 phosphorylation at Ser256, Ser261, Ser264, and Ser269 in IMCD tubule suspensions were examined by semiquantitative immunoblot analysis. At pH 7.4, AQP2 was significantly phosphorylated at Ser256, Ser264, and Ser269 after short-term dDAVP stimulation (10⫺10 M, 3 min), whereas AQP2 phosphorylation at Ser261 was markedly decreased (Fig. 2, A and D–G). AQP2 phosphorylation at Ser256 was increased after dDAVP stimulation (10⫺10 M, 3 min) under the alkaline condition (pH 8.4), whereas Ser261 phosphorylation was decreased (Fig. 2, C–E). In contrast, Ser264 and Ser269 were not phosphorylated after short-term dDAVP stimulation (10⫺10 M, 3 min) under pH 8.4 (Fig. 2, C, F, and

(kDa)

S261

S264

S269

β-actin

D 5.0 4.0

*

dDAVP (n=3)

C

pH 6.4

(kDa)

Vehicle (n=3)

dDAVP (n=3)

pH 8.4

(kDa)

50 37

50

50

37

37

25

25

25

50 37

50 37

50

25

25

25

50

50

50

37

37

37

25

25

25

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50

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25

50 37

50 37

37

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25

25

50 37

50 37

50 37

3.0 2.0 1.0 0.0 pH 7.4 pH 6.4 pH 8.4 (n=3) (n=3) (n=3)

F 1.5

*

1.0 0.5

dDAVP (n=3)

50

E

*

Vehicle (n=3)

37

*

0.0 pH 7.4 pH 6.4 pH 8.4 (n=3) (n=3) (n=3)

S264/Total AQP2

S256

Vehicle (n=3)

S261/Total AQP2

AQP2

S256/Total AQP2

B

pH 7.4

2.0

G

*

1.5 1.0

*

0.5 0.0 pH 7.4 pH 6.4 pH 8.4 (n=3) (n=3) (n=3)

S269/Total AQP2

A

G), possibly due to slower phosphorylation than the corresponding increase in Ser256 phosphorylation, which has been demonstrated in a previous study (10). In contrast, at pH 6.4, Ser256 and Ser269 of AQP2 were not significantly phosphorylated, and phosphorylation at Ser264 of AQP2 was even decreased after dDAVP stimulation (10⫺10 M, 3 min; Fig. 2, B, D, F, and G). Moreover, the effects of dDAVP stimulation (10⫺10 M, 3 min) on AQP2 phosphorylation were compared among IMCD tubule suspensions exposed to pH 6.4, 7.4, or 8.4 for 1 h, respectively (Fig. 3, A–F). When compared with dDAVPinduced AQP2 phosphorylation at pH 7.4, AQP2 phosphorylation at Ser256 (76 ⫾ 3%, P ⬍ 0.05), Ser264 (44 ⫾ 2%, P ⬍ 0.05), and Ser269 (26 ⫾ 4%, P ⬍ 0.05) was significantly decreased under the acidic condition (pH 6.4; Fig. 3, B, C, E,

2.0

*

1.5 1.0 0.5 0.0 pH 7.4 pH 6.4 pH 8.4 (n=3) (n=3) (n=3)

Fig. 2. Semiquantitative immunoblot analysis of AQP2 and p-AQP2 (Ser256, Ser261, Ser264, and Ser269) in IMCD tubule suspensions. A–C: IMCD tubule suspensions were incubated for 1 h in HEPES-buffered saline solution with different pH [pH 6.4 (B), 7.4 (A), or 8.4 (C)]. IMCD tubule suspensions were stimulated with either vehicle or dDAVP (10⫺10 M) for the last 3 min. Immunoblots of IMCD tubule suspensions reacted with antibodies against AQP2 and p-AQP2 (deglycosylated ⬃29 kDa and glycosylated ⬃35–50 kDa). D–G: densitometric analysis. *P ⬍ 0.05 compared with vehicle-treated IMCD tubule suspensions. n is the number of IMCD tubule suspension preparations from independent experiments. AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

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EXTRACELLULAR pH AFFECTS dDAVP-INDUCED AQP2 TRAFFICKING

dDAVP (10-10 M)

A Extracellular pH: 6.4, 7.4, 8.4

57

0 dDAVP 10-10 M

50 37 25 50

p-AQP2 (S256)

37

pH 6.4

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pH 8.4

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1.5 1.0 0.5 0.0

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37 25 50 37 25

β-actin

50 37

S264/Total AQP2

50

p-AQP2 (S269)

*

*

1.0 0.5 0.0 pH 7.4 pH 6.4 pH 8.4 (n=6) (n=6) (n=6)

37 25

p-AQP2 (S264)

1.5

pH 7.4 pH 6.4 pH 8.4 (n=6) (n=6) (n=6)

50

p-AQP2 (S261)

*

F

1.5 1.0

* 0.5

S269/Total AQP2

AQP2

pH 7.4

C

S261/Total AQP2

(kDa)

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B

60 (min)

0.0

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*

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pH 7.4 pH 6.4 pH 8.4 (n=6) (n=6) (n=6)

pH 7.4 pH 6.4 pH 8.4 (n=6) (n=6) (n=6)

Fig. 3. Semiquantitative immunoblot analysis of AQP2 and p-AQP2 (Ser256, Ser261, Ser264, and Ser269) in IMCD tubule suspensions. A: study design for dDAVP stimulation. IMCD tubule suspensions were incubated for 1 h in HEPES-buffered saline solution with different pH (pH 6.4, 7.4, or 8.4). IMCD tubule suspensions were stimulated with dDAVP (10⫺10 M) for the last 3 min. B: immunoblots of IMCD tubule suspensions reacted with antibodies against AQP2 and p-AQP2 (deglycosylated ⬃29 kDa and glycosylated ⬃35–50 kDa). C–F: densitometric analysis. *P ⬍ 0.05 compared with dDAVP-stimulated IMCD tubule suspensions exposed to pH 7.4. n is the number of IMCD tubule suspension preparations from independent experiments.

and F), whereas Ser261 was more phosphorylated (122 ⫾ 6%, P ⬍ 0.05; Fig. 3, B and D). Under the alkaline condition (pH 8.4), however, Ser256 and Ser264 of AQP2 were phosphorylated similar to the condition of pH 7.4 (Fig. 3, B, C, and E). In contrast, increased phosphorylation of Ser261 (133 ⫾ 3%, P ⬍ 0.05; Fig. 3, B and D) and decreased phosphorylation of Ser269 (50 ⫾ 3%, P ⬍ 0.05, Fig. 3, B and F) were observed. Changes of pHi in primary cultured IMCD cells from rat kidneys. Changes of pHi of IMCD cells were monitored when cells were exposed to Na⫹-free solution containing nigericin with different pH values (pH 6.4, 7.4, or 8.4) for 60 min, respectively. Changes of pHi were measured in IMCD cells where BCECF-AM was applied. As shown in Fig. 4A, changes of the 490-to-440-nm ratio were time lapse monitored when IMCD cells were exposed to Na⫹-free solution of pH 6.4, 7.4, and 8.4 containing 10 ␮M nigericin and high K⫹ (105 mM). The background-corrected fluorescence intensity was then normalized to 1 when pHi was 7.0 (Fig. 4B). Within 3 min of exposure to pHe 6.4 or 8.4, pHi was rapidly changed (Fig. 4C). At 60 min, pHi became 6.1, 7.4, or 8.2 when cells were

exposed to pHe 6.4, 7.4, or 8.4, respectively (n ⫽ 5 cells/group; Fig. 4C). Decreased dDAVP-induced apical translocation of AQP2 in IMCD cells when cells were exposed to acidic pH. It is conceivable that cellular pH is more sensitive to basolateral pH in IMCD cells as the apical membrane is tight. To examine whether pH changes at the apical side (i.e., apical pH) or basolateral side (i.e., basolateral pH) of IMCD cells affect dDAVP (basolateral)-induced AQP2 expression in the apical plasma membrane, cell surface biotinylation assays were performed in IMCD cells cultured in transwell chambers. IMCD cells were exposed to a transepithelial pH gradient for 1 h (pH 6.4, 7.4, or 8.4 at the apical side vs. pH 7.4 at the basolateral side and vice versa). Changes of actual pH values in culture media in both compartments were directly measured after 1 h by a pH meter (Mettler-Toledo) and are shown in Table 1. In the presence of dDAVP stimulation (10⫺9 M, 15 min, basolateral side only), cell surface biotinylation assays demonstrated that the biotinylated AQP2-to-total AQP2 ratio was significantly decreased when IMCD cells were exposed to

AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

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7.4

6.4

8.4

7.4

6.4

8.4

B

7.4

2.0 1.5 1.0 0.5 0.0

1.5 1.0 0.5 0.0

0

C

2.0

490/440 nm Ratio

490/440 nm Ratio

A

5

10 15 20 Time (min)

25

30

6

7

8

9

pHi

pHi

10 8

pH 6.4

6

pH 7.4

4

pH 8.4

2

Extracellular pH

pH 7.4 (n=5)

pH 6.4 (n=5)

pH 8.4 (n=5)

Intracellular pH

7.4 ± 0.01

6.1 ± 0.01

8.2 ± 0.01

0

0

10

20

30

40

50

60

Time (min) Fig. 4. Changes of intracellular pH (pHi) in primary cultured IMCD cells where BCECF-AM was applied. A: time course of changes in the fluorescence excitation 490-to-440-nm ratio during exposure to Na⫹-free solution of pH 6.4, 7.4, and 8.4 containing 10 ␮M nigericin and high K⫹ (105 mM). B: dependence of the normalized fluorescence excitation ratio on pHi. C: changes of pHi in IMCD cells exposed to extracellular pH (pHe) 6.4, 7.4, or 8.4 for 60 min.

apical compartment pH 6.4 for 1 h (60 ⫾ 6% of dDAVPtreated IMCD cells exposed to apical pH 7.4, P ⬍ 0.05; Fig. 5). Moreover, the biotinylated AQP2-to-total AQP2 ratio was more decreased when IMCD cells were exposed to basolateral pH 6.4 for 1 h (48 ⫾ 5% of dDAVP-treated IMCD cells exposed to basolateral pH 7.4, P ⬍ 0.05; Fig. 6). In contrast, under the alkaline condition (pH 8.4) in either the apical or basolateral side of cells, the biotinylated AQP2-to-total AQP2 ratio after dDAVP stimulation (10⫺9 M, 15 min, basolateral side only) was similar to that observed under pH 7.4 (Figs. 5 and 6). Laser scanning confocal microscopic examination of AQP2 labeling was done in IMCD cells cultured in transwell chamTable 1. Changes of pH values in culture media after a 1-h transepithelial pH gradient on inner medullary collecting duct cells in transwell chambers Apical pH 6.4, 7.4, or 8.4 Versus Basolateral pH 7.4

Apical compartment Initial pH (n ⫽ 6) pH values at 1 h (n ⫽ 6) Basolateral compartment Initial pH (n ⫽ 6) pH values at 1 h (n ⫽ 6)

6.4 7.1 ⫾ 0.02

7.4 7.5 ⫾ 0.05

8.4 7.9 ⫾ 0.13

7.4 7.5 ⫾ 0.03

7.4 7.5 ⫾ 0.03

7.4 7.6 ⫾ 0.04

Apical pH 7.4 Versus Basolateral pH 6.4, 7.4, or 8.4

Apical compartment Initial pH (n ⫽ 6) pH values at 1 h (n ⫽ 6) Basolateral compartment Initial pH (n ⫽ 6) pH values at 1 h (n ⫽ 6)

7.4 7.6 ⫾ 0.02

7.4 7.7 ⫾ 0.05

7.4 7.8 ⫾ 0.02

6.4 7.3 ⫾ 0.02

7.4 7.7 ⫾ 0.02

8.4 8.1 ⫾ 0.02

bers. Consistent with the findings of cell surface biotinylation assays, dDAVP stimulation (10⫺9 M, 15 min, basolateral side only) induced less AQP2 labeling in the apical plasma membrane when cells were exposed to apical or basolateral compartment pH 6.4 for 1 h (Fig. 7, B and E) compared with exposure to apical compartment pH 7.4 and 8.4 for 1 h (Fig. 7, A and C) or basolateral compartment pH 7.4 and 8.4 for 1 h (Fig. 7, D and F). When cells were exposed to apical or basolateral compartment pH 6.4 for 1 h, intracellular AQP2 labeling was more prominent (Fig. 7, B and E). FRET analysis for PKA activity. The cAMP/PKA signaling pathway is a principal signaling pathway for both AQP2 trafficking and protein expression. We used a PKA FRETbased indicator (AKAR3EV) (17) to investigate the effects of pHe on dDAVP-induced PKA activity. Since IMCD cells were resistant to transfection of the PKA FRET-based indicator, LLC-PK1 cells, which are the proximal tubular origin from pig kidneys and express V2R (36), were used for FRET analysis. With this probe, time-lapse imaging was taken to study the changes in PKA activity in stably transfected LLC-PK1 cells expressing AKAR3EV during exposure to different pHe in the presence of 10⫺9 M dDAVP stimulation. Abundant expression of AKAR3EV and a dDAVP-induced increase in FRET levels were observed in cells (Fig. 8A). When cells were stimulated with a high concentration of dDAVP (10⫺8 M), FRET levels were increased irrespective of pHe changes (Fig. 8, A and B). However, when cells were treated with a lower concentration of dDAVP (10⫺9 M), increased FRET levels, i.e., increased PKA activity, were only seen in cells exposed to pHe 7.4 and 8.4 (Fig. 8, A and C) but not during exposure to pHe 6.4. In contrast, increased FRET levels were seen in cells stimulated with forskolin (both 10⫺6 and 10⫺7 M), independent of pHe

AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

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A

dDAVP (10-9 M)

Apical pH: 6.4, 7.4, 8.4

0

dDAVP (10-9 M) (kDa)

pH 7.4

pH 6.4

C Biotinylated AQP2/Total AQP2

B

pH 8.4

50

Total AQP2

60 (min)

45

37 25 50

Biotinylated 37 AQP2

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

*

pH 7.4 (n=6)

25

pH 6.4 (n=6)

pH 8.4 (n=6)

Fig. 5. Cell surface biotinylation assays for AQP2 expression in the apical plasma membrane. A: study design for dDAVP stimulation. Primary cultured IMCD cells were exposed to a transepithelial pH gradient for 1 h (pH 6.4, 7.4, or 8.4 at the apical side vs. pH 7.4 at the basolateral side) and treated with dDAVP (10⫺9 M, basolateral side, 15 min). B and C: immunoblot analysis of total AQP2 and biotinylated AQP2. Cell surface biotinylation assays examined the effects of changes in apical pH on AQP2 expression at the apical plasma membrane of IMCD cells. *P ⬍ 0.05 compared with dDAVP-treated IMCD cells exposed to apical pH 7.4. n is the number of preparations of IMCD cell lysates.

changes (Fig. 9, A and B), suggesting that acidic pH could attenuate dDAVP-induced PKA activity by affecting vasopressin binding to the V2R and/or Gs␣-mediated adenylyl cyclase activity.

Consistent with this, the effect of pHe on forskolin-induced AQP2 phosphorylation (Ser256) was examined. In contrast to the observed changes of dDAVP-induced AQP2 phosphorylation (Figs. 1–3), semiquantitative immunoblot analysis revealed

A

dDAVP (10-9 M)

Basolateral pH: 6.4, 7.4, 8.4

0

60 (min)

45

dDAVP (10-9 M) (kDa) 50

Total AQP2 37 25 50

Biotinylated 37 AQP2 25

pH 7.4

pH 6.4

pH 8.4

C Biotinylated AQP2/Total AQP2

B

1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

* pH 7.4 (n=6)

pH 6.4 (n=6)

pH 8.4 (n=6)

Fig. 6. Cell surface biotinylation assays for AQP2 expression in the apical plasma membrane. A: study design for dDAVP stimulation. Primary cultured IMCD cells were exposed to a transepithelial pH gradient for 1 h (pH 7.4 at the apical side vs. pH 6.4, 7.4, or 8.4 at the basolateral side) and treated with dDAVP (10⫺9 M, basolateral side, 15 min). B and C: immunoblot analysis of total AQP2 and biotinylated AQP2. Cell surface biotinylation assays examined the effects of changes in basolateral pH on AQP2 expression at the apical plasma membrane of IMCD cells. *P ⬍ 0.05 compared with dDAVP-treated IMCD cells exposed to basolateral pH 7.4. n is the number of preparations of IMCD cell lysates. AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

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pH 7.4

pH 8.4

pH 6.4

B

C

D

E

F

Changes of basolateral pH

Changes of apical pH

A

Fig. 7. Immunocytochemistry of AQP2 in primary cultured IMCD cells on the transwell chamber. Bottom: primary cultured IMCD cells were exposed to a transepithelial pH gradient for 1 h (A–C: pH 6.4, 7.4, or 8.4 at the apical side vs. pH 7.4 at the basolateral side; D–F: pH 7.4 at the apical side vs. pH 6.4, 7.4, or 8.4 at the basolateral side) and treated with dDAVP (10⫺9 M, basolateral side, 15 min). dDAVP stimulation induced more AQP2 expression in the apical plasma membrane when cells were exposed to apical or basolateral compartment pH 7.4 or 8.4 for 1 h compared with apical or basolateral compartment pH 6.4 for 1 h, respectively. In contrast, intracellular AQP2 labeling was more prominent when cells were exposed to apical or basolateral compartment pH 6.4 for 1 h. Top: x–z images.

that the forskolin (10⫺6 M, 3 min)-induced p-AQP2-to-AQP2 ratio was similar irrespective of pHe 6.4, 7.4, or 8.4 (Fig. 10). FRET analysis for intracellular Ca2⫹ levels. To examine whether dDAVP-induced intracellular Ca2⫹ dynamics are affected by changes of pHe, analysis of FRET levels using YC3.60 as an indicator of intracellular Ca2⫹ (29) was performed. LLC-PK1 cells transiently transfected with YC3.60 were analyzed for FRET responses after dDAVP stimulation with changes of pHe (Fig. 11A). Intracellular Ca2⫹ levels were increased after dDAVP stimulation (10⫺8 or 10⫺9 M, 25 min), which were not affected by changes of pHe (Fig. 11, B and C). DISCUSSION

In the present study, we examined the effects of pHe on dDAVP-induced phosphorylation (Ser256, Ser261, Ser264, and Ser269) and apical targeting of AQP2 in IMCD tubule suspensions and primary cultured IMCD cells from rat kidneys.

AQP2 phosphorylation after short-term dDAVP stimulation was attenuated when tubule suspensions were exposed to acidic pH (pH 5.4 and 6.4) compared with that induced under conditions of neutral pH (pH 7.4) and alkaline pH (pH 8.4). Moreover, IMCD cells exposed to a transepithelial pH gradient in the transwell chamber demonstrated that exposure to either apical or basolateral pH 6.4 significantly decreased dDAVP (basolateral side)-induced apical expression of AQP2 compared with that under exposure to pH 7.4 and 8.4. Importantly, FRET analysis was done to study the effects of pHe on dDAVP- and forskolin-induced intracellular PKA activity and Ca2⫹ levels. dDAVP-induced PKA activity was significantly attenuated when LLC-PK1 cells were exposed to acidic pH compared with neutral or alkaline pH, whereas forskolininduced PKA activation was not affected. The dDAVP-induced increase of intracellular Ca2⫹ was not affected by changes of pHe.

AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

EXTRACELLULAR pH AFFECTS dDAVP-INDUCED AQP2 TRAFFICKING

A

- 5 min

0 min

F745

15 min

5 min

10-8 M dDAVP pH 7.4

10-9 M dDAVP pH 7.4 Fig. 8. Fluorescence resonance energy transfer (FRET) analysis for PKA activity in dDAVPstimulated LLC-PK1 cells exposed to different pHe. A: time-lapse live cell FRET imaging analysis in LLC-PK1 cells expressing PKA FRETbased indicator (AKAR3EV). B and C: LLCPK1 cells were exposed to different pH (pH 6.4, 7.4, or 8.4) for 1 h and stimulated by dDAVP (10⫺8 or 10⫺9 M) for the last 15 min. FRET intensity was analyzed every 15 s during 20 min. The values of FRET ratio were calculated from 5 (B) or 11 (C) independent experiments. Red line, exposure to pHe 6.4; black line, exposure to pHe 7.4; green line, exposure to pHe 8.4. *P ⬍ 0.05 compared with dDAVP-treated LLC-PK1 cells exposed to pH 7.4 and 8.4.

10-9 M dDAVP pH 6.4

10-9 M dDAVP pH 8.4

C

dDAVP (10-8 M) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

dDAVP (10-9 M)

Normalized FRET/CFP ratio

Normalized FRET/CFP ratio

B

0

5

10 15 Time (min)

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

20

*

0

5

10 15 Time (min)

Previous studies demonstrated that pH regulates the hydroosmotic action of AVP in the toad urinary bladder (4, 33) and rabbit kidney collecting ducts (26). In the isolated perfused rabbit cortical collecting tubule, when bath pH values were decreased, vasopressin-induced changes in mean hydraulic conductance was decreased, whereas reduced luminal pH did not have an effect on vasopressin-induced hydraulic conductance (26). In particular, in cultured cells of rat kidney papillary collecting ducts, Ishikawa et al. (13) demonstrated that acidic pH reduced AVP-

1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

0

5

10 15 Time (min)

and forskolin-induced cAMP production and AVP-mediated Ca2⫹ mobilization. Acidic pH (pH 6.8) markedly suppressed the receptor binding of [3H]AVP in papillary collecting duct cells, resulting in a reduction of AVP-induced activation of adenylate cyclase (13). This finding was further supported by a recent study (44) demonstrating that V2Rs displayed a significantly reduced affinity for vasopressin under acidic conditions in LLC-FLAGV2R cells, whereas an increase of osmolality had no effect on V2R binding to vasopressin.

B

Forskolin (10-6 M)

Normalized FRET/CFP ratio

Normalized FRET/CFP ratio

A

20

20

Forskolin (10-7 M) 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

0

5

10 15 Time (min)

20

Fig. 9. FRET analysis for PKA activity in forskolin-stimulated LLC-PK1 cells exposed to different pHe. A and B: time-lapse live cell FRET imaging analysis in LLC-PK1 cells expressing PKA FRET-based indicator (AKAR3EV). LLC-PK1 cells were exposed to different pH (pH 6.4, 7.4, or 8.4) for 1 h and stimulated by forskolin (10⫺6 or 10⫺7 M) for the last 15 min. FRET intensity was analyzed every 15 s during 20 min. Values of the FRET ratio were calculated from five independent experiments. Red line, exposure to pHe 6.4; black line, exposure to pHe 7.4; green line, exposure to pHe 8.4.

AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

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A

Forskolin (10-6 M) Extracellular pH: 6.4, 7.4, 8.4

B

C

Forskolin (10-6 M)

(kDa) pH 7.4

pH 6.4

pH 8.4

50

AQP2

37 25 50

p-AQP2 (S256)

37

1.2 1.0 0.8 0.6 0.4 0.2 0.0

pH 7.4 (n=6)

25

β-actin

pH 8.4 (n=6)

37

ability (20, 21, 31). It has been demonstrated that AQP2 is phosphorylated at four serine sites (Ser256, Ser261, Ser264, and Ser269) in the COOH-terminal tail of rat AQP2 (12). Ser256 is part of a consensus motif (RRQS) for phosphorylation by PKA (8). Previous studies (10, 11) have demonstrated that AQP2 phosphorylation at Ser256, Ser264, and Ser269 was increased in IMCD tubule suspensions after short-term dDAVP stimulation, whereas Ser261 showed a decrease in phosphorylation in response to vasopressin or cAMP. Interestingly, phosphorylation at Ser269 was dependent on the prior phosphorylation of Ser256 (10). Therefore, our results revealing decreased phosphorylation of AQP2 (Ser256, Ser264, and Ser269) in IMCD tubule suspensions and apical translocation of AQP2 in IMCD cells when they were stimulated with dDAVP (10⫺10 M, 3 min) under conditions of acidic pH were consistent with previous findings demonstrating the decrease of AVP-induced cAMP production and receptor binding of [3H]AVP under conditions of acidic

A

dDAVP stimulation (10-8 M or 10-9 M)

Extracellular pH: 6.4, 7.4, 8.4

0

70 Time (min)

45

B

C

dDAVP (10-8 M)

dDAVP (10-9 M) 2.5

Normalized FRET/CFP ratio

2.5 Normalized FRET/CFP ratio

pH 6.4 (n=6)

50

Transcellular water reabsorption across renal tubular epithelial cells is dependent on the expression of AQPs (32). Vasopressin plays a key role in water reabsorption in the kidney connecting tubule and collecting ducts, where AQP2, the vasopressin-regulated water channel protein, is regulated by intracellular trafficking for short-term regulation and by altered protein abundance for long-term adaptation of water balance. The most important mediator of both trafficking and protein abundance is vasopressin for the regulation of renal water reabsorption and body water homeostasis. Vasopressin-induced AQP2 trafficking is mediated by the binding of vasopressin to V2Rs, resulting in Gs protein-mediated stimulation of adenlylate cyclase, resulting in increased cAMP levels. The increased concentration of cAMP causes activation of PKA, which subsequently phosphorylates AQP2 and regulatory proteins and stimulates AQP2-expressing vesicles to be fused with the apical plasma membrane, increasing osmotic water perme-

Fig. 11. FRET analysis for intracellular Ca2⫹ levels. A: study design for dDAVP stimulation in LLC-PK1 cells expressing YC3.60 (an indicator of intracellular Ca2⫹) exposed to different extracellular pH. B and C: LLC-PK1 cells were exposed to different pH (pH 6.4, 7.4, or 8.4) for 70 min and stimulated by dDAVP (10⫺8 or 10⫺9 M) for the last 25 min. FRET intensity was analyzed every 15 s during 30 min. Values of the FRET ratio were calculated from five independent experiments. Red line, exposure to pHe 6.4; black line, exposure to pHe 7.4; green line, exposure to pHe 8.4.

60 (min)

57

0

p-AQP2/Total AQP2

Fig. 10. Semiquantitative immunoblot analysis of AQP and p-AQP2 (Ser256) in IMCD tubule suspensions. A: study design for forskolin stimulation. IMCD tubule suspensions were incubated for 1 h in HEPES-buffered saline solution with different pH (pH 6.4, 7.4, or 8.4). IMCD tubule suspensions were stimulated with forskolin (10⫺6 M) for the last 3 min. B and C: immunoblot analysis of IMCD tubule suspensions reacted with antibodies against AQP2 and p-AQP2 (deglycosylated ⬃29 kDa and glycosylated ⬃35–50 kDa). n is the number of IMCD tubule suspension preparations from independent experiments.

2.0 1.5 1.0 0.5

2.0 1.5 1.0 0.5 0.0

0.0 0

5

10

15 20 Time (min)

25

30

0

AJP-Renal Physiol • doi:10.1152/ajprenal.00376.2014 • www.ajprenal.org

5

10

15 20 Time (min)

25

30

EXTRACELLULAR pH AFFECTS dDAVP-INDUCED AQP2 TRAFFICKING

pH in papillary collecting duct cells (13). This finding was further supported by FRET analysis. dDAVP (10⫺9 M)-induced PKA activity was significantly attenuated when LLCPK1 cells were exposed to acidic pH compared with neutral or alkaline pH, whereas forskolin-induced PKA activation was not affected. The results indicate that acidic pH decreases dDAVP-induced phosphorylation and apical targeting of AQP2 in IMCD cells via an inhibition of V2R-G proteincAMP-PKA actions. In contrast, one could reconsider the results in relation to normal urine pH values in human and rodents. Under normal conditions, urine pH in human is ⬍6 in a majority of the general population (22, 39), and in different strains of rodents, urine pH stays at or just above 6.5 and mostly below 7.5 (37). Based on this, one can assume that pH 6.4 used in this study could represent normal conditions, whereas pH 7.4 and 8.4 are higher than this. If it is so, then what is actually reported in this study is that an “elevation of urine pH could improve AQP2 phosphorylation and intracellular trafficking” rather than “acidic pH affects phosphorylation and intracellular trafficking of AQP2.” Based on the results, we could assume that urine alkalinization by administration of NaHCO3 might be, at least in part, effective in concentrating urine in the condition of nephrogenic diabetes insipidus. Moreover, since alkaline pH in urine may stimulate stone formation, which will be aggravated by concentrated urine with increased apical expression of AQP2, this might be pathophysiologically relevant. In primary cultured IMCD cells, vasopressin stimulation induced an increase of intracellular Ca2⫹ concentration to 350 – 400 nM (25). Vasopressin causes an increase in intracellular Ca2⫹ mobilization from ryanodine-sensitive intracellular Ca2⫹ stores, which is thought to play an important role in CaM-mediated translocation of AQP2-expressing vesicles (6, 31). Consistent with this, FRET analysis in the present study demonstrated that dDAVP (10⫺8 and 10⫺9 M) induced an increase of intracellular Ca2⫹ levels in IMCD cells. Ca2⫹ oscillation was not detected, since the FRET images were taken every 15 s for 25 min. However, the dDAVP-induced increase of intracellular Ca2⫹ levels was not affected by changes of pHe. This was in contrast to changes of dDAVP (10⫺9 M)-induced PKA activity, which need to be further elucidated. In addition to the decreased affinity of the V2R to vasopressin in conditions of acidic pH, systemic metabolic acidosis is also known to be associated with decreased expression of V2R mRNA in collecting ducts from rat kidneys (38). This is consistent with the observed vasopressin resistance in chronic renal failure commonly complicated with metabolic acidosis, demonstrating significantly decreased V2R mRNA levels (40) and AQP2 protein abundance (19). In summary, the present study revealed that dDAVP-induced phosphorylation (Ser256, Ser264, and Ser269) and apical targeting of AQP2 were significantly attenuated in IMCD cells under acidic pHe. This is likely via an inhibition of V2R-G protein-cAMP-PKA actions. GRANTS This work was supported by the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning, Korea (Grants 2010-0019393, 2013R1A1A2007266, and 2014R1A5A2009242).

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