Article

Full Access

AQP2-Induced Acceleration of Renal Cell Proliferation Involves the Activation of a Regulatory Volume Increase Mechanism Dependent on NHE2

Corresponding Author

Valeria Rivarola

[email protected]

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Correspondence to: Dr. Valeria Rivarola, PhD, Facultad de Medicina, Departamento de Ciencias Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Paraguay 2155 piso 7 (C1121ABG), Buenos Aires, Argentina. E-mail: [email protected]

Search for more papers by this author

Gisela Di Giusto,

Gisela Di Giusto

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

María José Christensen,

María José Christensen

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

Paula Ford,

Paula Ford

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

Claudia Capurro,

Claudia Capurro

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

Valeria Rivarola,

Corresponding Author

Valeria Rivarola

[email protected]

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

Gisela Di Giusto,

Gisela Di Giusto

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

María José Christensen,

María José Christensen

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

Paula Ford,

Paula Ford

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

Claudia Capurro,

Claudia Capurro

Facultad de Medicina, Departamento de Ciencia Fisiológicas, Laboratorio de Biomembranas, IFIBIO Houssay, CONICET-UBA, Universidad de Buenos Aires, Buenos Aires, Argentina

Search for more papers by this author

First published: 18 May 2016

https://doi.org/10.1002/jcb.25602

Citations: 6

Share a link

Email
Wechat
Bluesky

ABSTRACT

We have previously shown in renal cells that expression of the water channel Aquaporin 2 (AQP2) increases the rate of cell proliferation by shortening the transit time through the S and G₂/M phases of the cell cycle. This acceleration is due, at least in part, to a down-regulation of regulatory volume decrease (RVD) mechanisms when volume needs to be increased in order to proceed into the S phase. We hypothesize that in order to increase cell volume, RVD mechanisms may be overtaken by regulatory volume increase mechanisms (RVI). In this study, we investigated if the isoform 2 of the Na⁺/H⁺ exchanger (NHE2), the main ion transporter involved in RVI responses, contributed to the AQP2-increased renal cell proliferation. Three cortical collecting duct cell lines were used: WT-RCCD₁ (not expressing AQPs), AQP2-RCCD₁ (transfected with AQP2), and mpkCCD_c14 (with inducible AQP2 expression). We here demonstrate, for the first time, that both NHE2 protein activity and expression were increased in AQP2-expressing cells. NHE2 inhibition decreased cell proliferation and delayed cell cycle progression by slowing S and G₂/M phases only if AQP2 was expressed. Finally, we observed that only in AQP2-expressing cells a NHE2-dependent RVI response was activated in the S phase. These observations suggest that the AQP2-increased proliferation involves the activation of a regulatory volume increase mechanism dependent on NHE2. Therefore, we propose that the accelerated proliferation of AQP2-expressing cells requires a coordinated modulation of the RVD/RVI activity that contributes to cell volume changes during cell cycle progression. J. Cell. Biochem. 118: 967–978, 2017. © 2016 Wiley Periodicals, Inc.

The Na⁺/H⁺ exchanger (NHE) plays an important role in a remarkable assortment of physiological processes, ranging from intracellular pH (pH_i) homeostasis and epithelial salt transport to cellular volume regulation and cell proliferation [Orlowski and Grinstein, 2011; Donowitz et al., 2013; Fuster and Alexander, 2014]. Some of these events, such as cell proliferation, are closely associated with changes in cell volume [Lang et al., 2000, 2005]. In fact, previous reports indicate that factors that affect cell volume also have an effect on mechanisms that control cellular proliferation [Rouzaire-Dubois and Dubois, 1998; Kunzelmann, 2005]. It is well accepted that during progression through the cell cycle, cellular volume increases as a result of the accumulation of diverse substrates, protein synthesis, and DNA duplication [Kunzelmann, 2005]. This cell volume increase may be achieved by inhibition of regulatory volume decrease mechanisms (RVD) and/or by activation of regulatory volume increase (RVI) mechanisms. In line with this proposal, several reports demonstrate that, in many cell types, RVI mechanisms are up regulated during cell growth [Lang et al., 1991, 1998b; Ritter and Wöll, 1996] and that RVD capacity is actively modulated throughout the cell cycle [Chen et al., 2002; Wang et al., 2002; Di Giusto et al., 2012]. Other reports, in tumor ras-oncogene-expressing cells, suggest that both RVD and RVI mechanisms are necessary for cell cycle progression [Ritter and Wöll, 1996; Lang et al., 1998a]. Therefore, evidence indicates that cellular proliferation requires mechanisms for cell volume regulation, and thus the movement of ions and water. However, little is known regarding the sequence of events leading from cell volume changes to cell cycle progression.

The water channels Aquaporins (AQPs) have been proposed to play a key role not only in the control of cell volume but also in cell migration, proliferation, and apoptosis [Jablonski et al., 2004; Ford et al., 2005; Verkman, 2008; Flamenco et al., 2009; Rivarola et al., 2010; Shimizu et al., 2014; Galan-Cobo et al., 2015, 2016]. In renal cells, we have demonstrated that AQP2 enhances cell proliferation rate by a coordinate activation of specific RVD mechanisms in the G₁ phase and their subsequent inhibition in the S phase of the cell cycle [Di Giusto et al., 2012]. What still remains unclear is whether activation of RVI mechanisms is also required to increase cell volume.

RVI is mainly accomplished by cellular NaCl uptake (Na⁺/H⁺ and Cl⁻/HCO₃⁻ exchangers acting in parallel) and the obligated water influx that leads to cell swelling [Lang et al., 2007]. Although to date four different NHEs isoforms have already been described in epithelia (NHE1–4), in renal cells NHE2 is the isoform proposed to be involved in cell volume increase. It has been demonstrated that hyperosmolality increases its mRNA expression [Soleimani et al., 1994]. Moreover, our group previously demonstrated that a hypertonic shock activates NHE2 function [Ford et al., 2002]. Bearing in mind these observations and those showing that NHE2 favors cell growth in CHO cells [Kapus et al., 1994], the aim of the present study was to investigate the putative link between AQP2 expression, NHE2 function, and RVI response during cell cycle progression. For this purpose, we used three renal cortical collecting duct cell lines: WT-RCCD₁ that do not express renal aquaporins (AQPs), AQP2-RCCD₁ that constitutively expresses AQP2 in the apical plasma membrane [Capurro et al., 2001; Ford et al., 2005], and mpkCCD_c14 cells that modulate AQP2 expression upon exposure to AVP or its analogues [Hasler et al., 2002]. Our results showed that AQP2-expressing cells have a higher basal pH_i, together with an increased NHE2 expression and increased NHE2 activity. In addition, only in the presence of AQP2, the inhibition of NHE2 reduced cell proliferation by slowing the transit through S and G₂/M phases of the cell cycle. In S phase, an activation of a NHE2-mediated RVI response was observed only in AQP2-expressing cells. These results indicate that NHE2 and AQP2 would work in coordination as part of the cellular device that regulates collecting duct cell cycle progression.

MATERIALS AND METHODS

CELL CULTURE

RCCD₁ cells, an epithelial cell line derived from rat renal cortical collecting duct, which maintains very high transepithelial resistance similar to that observed in the native tissue [Blot-Chabaud et al., 1996]. Two types of RCCD₁ cells were used: WT-RCCD₁ cells, which do not express aquaporin water channels [Capurro et al., 2001] and AQP2-RCCD₁ cells, stably transfected with cDNA coding for rat AQP2 [Ford et al., 2005]. WT-RCCD₁ cells were grown in a modified-DMEM (DM) medium (Dulbecco's modified Eagle's medium/Ham's F-12, 1:1 v/v; 14 mM NaHCO₃, 2 mM glutamine; 50 nM dexamethasone; 30 nM sodium selenite; 5 μg/ml insulin; 5 μg/mL transferrin; 10 ng/ml epidermal growth factor; 50 nM triiodothyronine; 100 U/ml penicillin-streptomycin; 20 mM Hepes; pH 7.4) and 2% fetal bovine serum (Invitrogen, San Diego, CA) at 37°C in 5% CO₂/95% air atmosphere [Blot-Chabaud et al., 1996]. AQP2-RCCD₁ cells were maintained in DM medium containing Geneticin (200 μg/ml, Invitrogen) as previously reported [Ford et al., 2005]. All experiments were performed on WT- and AQP2-RCCD₁ cells between the 20th and 40th passages.

The mpkCCD_c14 cells (provided by A. Vandewalle and M. Bens, Institut National de la Santé et de la Recherche Médicale, Paris, France) were grown in flasks (passage 20–30th) in DM supplemented with 2% fetal calf serum (DM: DMEM-Ham's F12 at 1:1 vol/vol, 60 nM sodium selenate, 5 g/ml transferrin, 2 mM glutamine, 50 nM dexamethasone, 1 nM triiodothyronine,10 ng/ml epidermal growth factor, 5 g/ml insulin, 20 mM d-glucose, and 20 mM HEPES, pH 7.4) (4) at 37°C in 5% CO₂–95% air atmosphere [Bens et al., 1999].

CELL CYCLE SYNCHRONIZATION

Cells were synchronized in the S phase of the cell cycle by the double-block technique, using thymidine as an inhibitor of DNA synthesis [Harper, 2005; Di Giusto et al., 2012]. WT-RCCD₁, AQP2-RCCD₁, or mpkCCD_c14 cells in exponential growth were first exposed for 12 h to 2 mM thymidine. Cells were then released in fresh media for another 12 h, later followed by a second thymidine block for another 12 h.

CELL GROWTH MEASURES

For cell number counting of WT- and AQP2-RCCD₁ cells were seeded on 24 polywell at 25 × 10³ cells/ml densities, allowed to attach for 24 h, and then were grown in the different experimental conditions.

For cell number counting of mpkCCD_c14 cells were seeded on 24 polywell at 2 × 10³ cells/ml densities in the presence of vehicle (water) or 100 μM dibutyryl cyclic AMP (dbAMPc), allowed to attach for 24 h, and then were grown in the different experimental conditions.

After incubation, cells were detached by trypsination, resuspended in PBS-trypan blue to exclude non-viable cells, and counted in a Neubauer hemocytometer.

CELL PROLIFERATION AND CELL CYCLE ANALYSIS BY FLOW CYTOMETRY

For dual parameter flow cytometry cells were pulsed with 20 μM of the thymidine analog 5-Bromodeoxyuridine (BrdU, Sigma–Aldrich, St. Louis) for 1 h. After removal of BrdU, cells were allowed to continue cycling in fresh media for a chase period of up to 6 h. Cells were then harvested and fixed in 70% ethanol. BrdU content was analyzed after DNA denaturation with 2N HCl plus 0.1% Triton X-100 at room temperature followed by neutralization with 0.1 M Na₂B₄O₇ buffer, pH 8.5. The cells were labeled using specific anti-BrdU monoclonal antibodies (BD Biosciences, CA) and Cy2-conjugated secondary antibodies (Jackson Immuno, PA) and suspended in the presence of propidium iodide (PI, 50 μg/ml) and RNase A (1 mg/ml) in PBS for up to 4 h at 4°C in the dark. Bivariate histograms of BrdU/DNA content were obtained collecting Cy2 green fluorescence as a measure of BrdU positive cells (BrdU⁺) cells (log scale) and red PI fluorescence as a measure of DNA content (linear scale) for 30,000 events in each sample using a FACS Calibur flow cytometer (Becton-Dickinson, CA). Cell cycle compartments were calculated from bivariate histograms after doublets exclusion (Win MDI 2.8 Joseph Trotter, The Scripps Research Institute, CA). The transit times through the S phase (T_s) and the G₂/M phase (T_G2/M) were calculated as previously described [Begg et al., 1985; Baron and Penit, 1990].

CELL SURFACE BIOTINYLATION

Ninety to ninety-five percent confluent WT-RCCD_1, AQP2-RCCD₁, or mpkCCD_c14 cells grown for 4 days in the presence of vehicle (−AQP2) or 100 μM dbAMPc (+AQP2) were washed three times in cold PBS and cell surface biotinylation was performed with a Cell Surface Protein Isolation kit (Pierce, IL) following the manufacturer's instructions. Briefly, the cells were rinsed with PBS and then biotinylated using EZ-Link Sulfo-NHS-SS-Biotin in PBS for 30 min on ice. After quenching, the cells were harvested and solubilized in Lysis Buffer and incubated for 30 min on ice. After centrifugation at 10,000g for 2 min at 4°C, the supernatant was added to Immobilized NeutrAvidin^TM gel and incubated for 60 min at room temperature. After being washed, the biotinylated proteins were eluted in SDS/PAGE sample buffer containing 50 mM dithiothreitol.

WESTERN BLOT STUDIES

Immunoblotting experiments were carried out as previously described [Rivarola et al., 2010]. Briefly, total cell lysates or cell surface biotinylated membranes were subjected to 7.5% SDS-polyacriylamide minigels electrophoresis using the Tris-Tricine buffer system and then transferred to nitrocellulose membranes (Mini Protean II, Bio-Rad, Hercules, CA). Blots were blocked with 1% BSA and then probed using either rabbit polyclonal anti-NHE2 (dilution 1:300, Alfa Diagnostics International, TX), rabbit polyclonal anti-AQP2 (dilution 1:1000) generated against a peptide corresponding to amino acids 250–271 of rat AQP2, conjugated with keyhole limpet hemocyanin [Shi et al., 2001] or streptavidin-HRP (dilution 1:6000 DakoCytomation, Denmark). After being rinsed with PBS-Tween, membranes were incubated with the appropriate secondary antibody (anti rabbit or anti mouse IgG conjugated to horseradish peroxidase, dilution 1:25,000, Sigma–Aldrich A0545) and visualized using the chemiluminescence method (SuperSignal Substrate, Pierce). For densitometric quantification of biotinylated membranes from WT- and AQP2-RCCD₁ cells or −AQP2 and +AQP2 mpkCCD_c14 cells, samples were run in the same gel. Images were captured on a GBOX (Syngene, MD) and the bands corresponding to AQP2 (29 kDa), NHE2 (100 kDa), or streptavidin-HRP biotinylated protein (150 kDa band) were quantified by a densitometric analysis using Gel-Pro Analyzer software (Media Cybernetics, MD) and expressed as the NHE2/streptavidin ratio.

QUANTIFICATION OF PLASMA MEMBRANE NHE2 BY IMMUNOFLUORESCENCE STUDIES

To quantify NHE2 proteins at the plasma membrane (PM) in RCCD₁ cells, immunofluorescence studies were performed. WT- or AQP2-RCCD₁ cells were seeded on coverslips and allowed to grow. Then, monolayers were incubated with the specific plasma membrane marker Alexa 488-conjugated wheat germ agglutinin (WGA; Molecular Probes—Thermo Fisher Scientific, MA) for 30 min at 4°C to label the surface glycoproteins. Cells were then fixed in 3% paraformaldehyde for 30 min and then permeabilized with 0.2% Triton X-100 at room temperature. Samples were sequentially incubated with polyclonal anti-rat NHE2 (dilution 1:50, Santa Cruz Biotechnology sc-30167) ON at 4°C and secondary antibodies (rabbit anti-Cy3 conjugate, dilution 1:100, Jackson Immuno 111-165-003) for 2 h at room temperature. Then, nuclei were stained with Hoechst (5 μg/ml) for 1 min. Coverslips were mounted with Vectashield mounting medium. Images were captured using epifluorescence on an Olympus FluoView FV1000 confocal microscope and digitalized.

For quantification of NHE2 at the plasma membrane, we create a mask of the plasma membrane as previously described [Janecki et al., 2000]. Briefly, we used Image-J software as follows: WGA images were binarized so that the signal from WGA was ascribed the value of “1” and the remainder of the image was ascribed the value of “0.” The Boolean logical operation “AND” was then performed on the corresponding images, representing signals from NHE2-Cy3 and from WGA (binary mask). This resulted in generation of a new image in which only the NHE2-Cy3 fluorescent signal corresponding to the PM was present. The intensity of NHE2-Cy3 fluorescence corresponding to the PM was quantified by densitometry using Image-J software and then analyzed using the formula:

$urn:x-wiley:14381656:media:jcb25602:jcb25602-math-0001$

where n stands for the number of optical sections required to scan the entire cell and IF_PM (n) stands for integrated fluorescence intensity of the plasma membrane within a given optical section. Plasma membrane NHE2 fluorescence was finally normalized to the number of cells per field.

INTRACELLULAR pH STUDIES

RCCD₁ monolayers, grown on permeable filters, were inserted between two lucite frames, separating two fluid compartments when diagonally placed in a quartz cuvette with control solution (Table I) as previously reported [Ford et al., 2002]. Free access both to the apical and basolateral baths was possible. Measurements were made with a computerized and thermoregulated (37°C) spectrofluorometer (Jasco 770, MD). For measurements, the cell monolayer was placed forming a 45° angle with the exploring beam. Fluorescence emission was monitored at 535 nm, with excitation wavelengths of 439 and 510 nm. For intracellular pH (pH_i) measurements, cells were loaded with 8 μM of 2′,7′bis(2-carboxyethyl)-5-(and–6) carboxyfluorescein acetoxymethyl ester (BCECF/AM, Molecular Probes) during 60 min at 37°C, both from the apical and basolateral baths. The ratio of the BCECF fluorescence emitted from dye-loaded cells was calibrated, in terms of pH, by incubating the cells in “high K⁺ solution” (140 mM KCl, 4.6 mM NaCl, 1 mM MgCl₂, 2 mM CaCl₂, 10 mM Hepes, 5 mM glucose) and then by permeabilizing the cells with 5 μM nigericin to balance extracellular pH (pH_o) with pH_i. Then, pH-bathing solution was stepped between 6.6 and 8.5. The 510/439 ratio was linear over this pH range (r = 0.99, n = 6).

Table I. Composition of Solutions Used for Determination of pH_i Recovery

Solution	Control	NH₄Cl	0 Na⁺
NaCl	147	127	–
Hepes	30	30	30
KCl	3	3	3
CaCl₂	1.8	1.8	1.8
MgSO₄	1	1	1
KH₂PO₄	1	1	1
K₂SO₄	1	1	1
NH₄Cl	–	20	–
TMA	–	–	147
Glucose	5.6	5.6	5.6

All concentrations are given in mM; pH was adjusted to 7.4 at 37°C.

In some experiments, NHE activity was monitored evaluating pH_i recovery after an acid load by the NH₄Cl pre-pulse technique as previously described [Ford et al., 2002]. The cells were exposed to control solution. Then, to a 20 mM NH₄Cl pulse (NH₄Cl in Table I), followed by Na⁺ removal (0 Na⁺ in Table I). Then, Na⁺ was restituted to the bath and Na⁺-dependent pH_i recovery mechanisms were evaluated. Then, acid fluxes were calculated as

$urn:x-wiley:14381656:media:jcb25602:jcb25602-math-0002$

where J_H⁺ is the H⁺ flux, dpH/dt is the rate of recovery from the acid load, and β_i is the intracellular buffering power.

Intracellular H⁺ buffering power (β_i) was estimated using application of external permeant weak bases as described by Roos and Boron [1981]. In the present work, we used application and removal of NH₄Cl. This agent freely permeates the cell membrane in its uncharged form but exist mainly in ionized form once inside the cell. The ionization of a weak base absorbs H⁺ ions. NH₄Cl can, therefore, be used to induce changes of pH_i which can then be used to estimate β_i following:

$urn:x-wiley:14381656:media:jcb25602:jcb25602-math-0003$

where [H⁺]_i (mM) is the concentration of alkali (per liter of cytoplasm) introduced into the cell and ΔpH_i is the resulting change of pH_i. [H⁺]_I is assumed to equal the intracellular concentration of NH₄⁺ ions at the moment of their removal from the external solution. [NH₄⁺]_i at the moment of removal is given by

$urn:x-wiley:14381656:media:jcb25602:jcb25602-math-0004$

where [NH₄⁺]₀ is calculated from the total concentration of external NH₄Cl ([NH₄Cl]) and its pK using a rearrangement of the Henderson–Hasselbalch equation:

$urn:x-wiley:14381656:media:jcb25602:jcb25602-math-0005$

MEASUREMENT OF REGULATORY VOLUME INCREASE ACTIVITY

To determine the regulatory volume increase (RVI) activity WT-RCCD₁, AQP2-RCCD₁, or mpkCCD_c14 cells (stimulated or not with 100 μM dbAMPc) were seeded on culture flasks, either allowed to grow freely or synchronized. Cells were harvested by trypsinization with Trypsin/EDTA, washed in an isosmotic solution, and then incubated for up to 10 min in the desired solution. Cells were incubated either in an isosmotic solution (311 ± 5 mOsM) containing (in mM): 90 NaCl, 10 NaHCO₃, 5 KCl, 1 CaCl₂, 0.8 MgSO₄, 1 MgCl₂, 100 mannitol, 20 Hepes, 5 glucose, or in a hypertonic solution (419 ± 5 mOsM) prepared by mannitol addition to obtain the desired osmolality, while maintaining the ionic strength. Solutions osmolality were routinely measured in a pressure vapor osmometer (Vapro; Wescor, Logan, UT). All solutions were titrated to pH 7.40 using Tris (Sigma–Aldrich) and bubbled with atmospheric air.

Cell volume was estimated from suspended cells using a Scepter™ handheld automated cell counter (Millipore, MA) fitted with a 60 µm Scepter sensor and ScepterPro software. The Scepter uses a Coulter principle to generate a histogram of cell-volume distribution ranging from 0 to 24 pL [Winterberg et al., 2012; Xiao et al., 2012].

RVI at t = 10–20 min, associated with the volumetric response of cells exposed to hypertonic medium, was calculated using the following equation:

$urn:x-wiley:14381656:media:jcb25602:jcb25602-math-0006$

where V₀ is the initial cell volume, V is the volume at time = 10–20 min. (V/V₀)_min is the minimal value of V/V₀ attained during hypertonic shrinkage and (V/V₀)₁₀ represents the value of V/V₀ observed at time = 10–20 min. RVI thus denotes the magnitude of volume regulation at time = 10–20 min with 100% RVI indicating complete volume regulation and 0% indicating no volume regulation.

STATISTICS

Values were reported as mean ± SEM, and n is the number of experiments. Student's t-test for unpaired data was used. P < 0.05 was considered a significant difference.

RESULTS

NHE2 ACTIVITY AND EXPRESSION ARE INCREASED IN THE PRESENCE OF AQP2

The first set of experiments examined the consequences of AQP2 expression on NHE activity in RCCD₁ cells. Intracellular pH (pH_i) measurements were performed in monolayers grown on permeable filters using an $urn:x-wiley:14381656:media:jcb25602:jcb25602-math-0007$ -free medium (Control, Table I) to minimize bicarbonate-transporting systems. Figure 1A shows that in AQP2-expressing cells, basal pH_i was higher than in WT cells. We then investigated whether this alkalinization could be due to a higher NHE activity. Since we have previously reported that WT-RCCD₁ cells expresses two isoforms of the Na⁺/H⁺ exchanger (NHE1 and NHE2) [Ford et al., 2002], we used the highly specific inhibitor HOE-694 to identify the isoforms. The inhibitor HOE-694 at low doses (1 μM) inhibits NHE1, but not NHE2, while at higher doses (15 μM) block both isoforms [Counillon et al., 1993; Counillon and Pouyssegur, 2000]. Figure 1B shows that independently of AQP2 expression 1 μM HOE-694 did not affect basal pH_i, indicating that NHE1 does not contribute to basal pH_i. In contrast, 15 μM HOE-694 induced an acidification significantly higher in AQP2-expressing cells than in WT cells. These results suggest that the higher basal pH_i observed in AQP2-RCCD₁ cells could be explained by an increased NHE2 activity. We further evaluated this possibility analyzing the difference in the NHE1 and NHE2 activity between WT and AQP2-expressing cells by the NH₄Cl pre-pulse technique [Boron and De Weer, 1976]. Figure 1C and D shows time-course experiments of pH_i evolution, using the NH₄Cl pre-pulse technique, in control conditions and in the presence of 1 μM or 15 μM HOE-694. It can be noted that both cell lines acidify to the same extent when NH₄Cl is washout (ΔpH_i WT: −0.33 ± 0.15 vs. AQP2: −0.39 ± 0.08. ns, n = 10). In addition, the intracellular Buffer power (ß_i) was not affected by the expression of AQP2 (ß_i, mM pH⁻¹, WT: 8.27 ± 2.29 vs. AQP2: 7.61 ± 2. NS, n = 10). NHE1 activity was estimated as the difference between the H⁺-fluxes of experiments performed in the absence or presence of 1 μM HOE-694 and NHE2 activity as the difference between experiments performed in the presence of 1 μM and 15 μM HOE-694. It can be seen in Figure 1E that NHE1 activity was similar in both cell lines. However, in AQP2-expressing cells, NHE2 activity was significantly higher than in WT cells.

Details are in the caption following the image — **Figure 1**
Open in figure viewer PowerPoint

Analysis of Na⁺/H⁺ exchanger activity in WT- and AQP2-RCCD₁ cells. (A) Basal intracellular pH (pH_i). (B) Contribution of Na⁺/H⁺ exchanger isoforms to basal pH_i. (C and D) Time course of pH_i regulation after an acid load and contribution of Na⁺/H⁺ exchanger isoforms to pH_i recovery in WT- (C) and AQP2-RCCD₁ cells (D). (E) Contribution of Na⁺/H⁺ exchanger isoforms to the proton fluxes (J_H+) after an acid load. Values are mean ± SEM of 5–7 experiments. ^#P < 0.05, ^##P < 0.01 AQP2-RCCD₁ as compared to WT-RCCD₁ cells.

To investigate whether the increase in NHE2 activity of AQP2-expressing cells was parallel to a change in protein expression, immunoblots experiments were carried out on WT- and AQP2-RCCD₁ cell surface biotinylated membranes. Figure 2A illustrates that NHE2 was expressed in both cell lines; however, densitometric analysis showed that the ratio of NHE2/Streptavidin protein was higher in AQP2-expressing cells. We also performed immunoblots experiments using another cortical collecting duct cell model: mpkCCD_c14 cell line. In the absence of stimulation, mpkCCD_c14 cells do not express plasma membrane AQP2 but upon addition of vasopressin, or its analogues, cells translocate AQP2 to the apical membrane AQP2 [Hasler et al., 2002]. Figure 2B shows that +AQP2-mpkCCD_c14 cells not only express plasma membrane AQP2 but also increase NHE2 protein expression.

Cell membrane expression of NHE2 in RCCD₁ cells was confirmed by immunofluorescence experiments. Since we were interested in quantifying plasma membrane-associated NHE2, WGA, a plasma membrane marker, was added in the immunofluorescence experiments. We then created a mask to analyze NHE2 immunofluorescence associated with plasma membrane as described in Methods section [Janecki et al., 2000]. Figure 2C illustrates that in AQP2-expressing cells, the plasma membrane-associated NHE2 stain was more intense. Signal quantification confirmed higher levels of plasma membrane-associated NHE2 immunofluorescence in AQP2-expressing cells as compared to WT cells (Fig. 2D).

Altogether, these results suggest that the basal alkalinization observed in AQP2-expressing cells is probably due to the higher membrane protein NHE2 expression that leads to the higher NHE2 activity of these cells.

NHE2 ACTIVITY CONTRIBUTES TO CELL CYCLE PROGRESSION IN AQP2-EXPRESSING CELLS

We then investigated whether NHE2 increased activity in AQP2-expressing cells could be implicated in the enhanced cell proliferation observed in these cells. In order to study only NHE2 activity, cells were grown under conditions where NHE1 is inhibited. For this, we used two experimental conditions, one, cells grown in the presence of 1 μM HOE-694, with NHE2 being the only active Na⁺/H⁺ isoform (+NHE2) and the other, cells grown in the presence of 15 μM HOE-694, to also inhibit NHE2 (−NHE2). Our results showed that inhibition of NHE2 activity did not affect WT-RCCD₁ cell growth but did significantly reduce the number of AQP2-RCCD₁ cells to a level similar to that observed in WT cells (Fig. 3A). Further studies assessed BrdU incorporation by flow cytometry analysis in order to evaluate the proliferation of growing cells (Fig. 3B). Results showed that the percentage of BrdU⁺ cells was significantly reduced in the absence of NHE2 activity only in cells expressing AQP2.

We also evaluated the time course of growing cells using the mpkCCD_c14 cells. Our results showed that inhibition of NHE2 activity did not affect non stimulated mpkCCD_c14 cells (-AQP2) growth, but did significantly reduce the number of cell growth in mpkCCD_c14 expressing AQP2 (Fig. 3C). These results confirm that independently of the cortical collecting duct cell model AQP2 expression per se plays a role in the increase of the rate of cell growth.

We then determined whether cell growth disparities could be due to differences in cell cycle progression. To evaluate the progression of RCCD₁ cell through the cell cycle we performed BrdU pulse-chase experiments. Figure 4A and C shows that independently of NHE2 activity immediately after BrdU pulse most BrdU⁺ cells are in the S phase in WT- and AQP2-RCCD₁ cells. Figure 4B and D shows that after 5 h chase in fresh medium, the percentage of cells in S-phase decreased and cells reached the subsequent phases. However, WT cells have significantly fewer BrdU⁺ cells in G1 phase and higher BrdU⁺ cells in G₂/M phase than AQP2-expressing cells. When NHE2 was inhibited (−NHE2) in WT cells, the proportion of BrdU⁺ cells in each phase of the cell cycle did not change. However, in AQP2-expressing cells, after NHE2 inhibition the proportion of BrdU⁺ cells in G₁ phase decreased.

We then used the BrdU pulse-chase results to calculate the duration of the S phase (T_s) and the duration of the G₂/M phase (T_G2/M) of the cell cycle. It can be observed in Figure 5A and B that when NHE2 is active (+NHE2), T_s and T_G2/M times were both shorter in AQP2-expressing cells, but they increased to levels similar to WT cells when NHE2 activity was blocked. Together these results demonstrate that NHE2 has an important role in AQP2-induced acceleration of cell proliferation.

NHE2-DEPENDENT RVI RESPONSE IS ACTIVATED DURING THE S PHASE OF THE CELL CYCLE IN AQP2-EXPRESSING CELLS

We then investigated whether the increased NHE2 activity and the higher cell proliferation rate observed in the presence of AQP2 were linked to changes in the capacity of cell volume regulation during cell cycle progression. RVI activity was measured as cell volume recovery in response to a hypertonic gradient (ΔOsM = +100 mOsM) in synchronized (most cells at S phase) and unsynchronized cells (most cells at G₁ phase). Figure 6 shows the time course of cell volume in WT- and AQP2-RCCD₁ cells during the hypertonic challenge. It can be noted that, independently of AQP2 expression, in G₁ phase cells shrank but failed to recover cell volume (Fig. 6A). In contrast, at S phase, only cells expressing AQP2 were able to recover cell volume (Fig. 6B). Figure 6C illustrates the percentage of cell volume recovery 10–20 min after the hypertonic shock (%RVI). It can be observed that RVI machinery was inactive during G₁ phase in both cell lines, but only AQP2-expressing cells activated the RVI during S phase. We then performed experiments on mpkCCD_c14 cells expressing or not AQP2. Figure 6D shows that only in the S phase of AQP2-expressing mpkCCD₁₄, cells are able to activate the RVI machinery. Finally, we investigated whether this activation was dependent on NHE2 activity.

Our results in AQP2-expressing cells showed that the blockage of NHE2 activity with HOE-694 15 μM inhibited S phase-associated RVI (%RVI in S: +NHE2 = 34.08 ± 8.92 vs. −NHE2 = 1.93 ± 1.40, P < 0.01 n = 15). For mpkCCD_c14 cells grown expressing AQP2, the blockage of NHE2 activity with HOE-694 15 μM also inhibited S phase-associated RVI (%RVI in S: +NHE2 = 15.33 ± 2.90 vs. −NHE2 = 0 ± 0.01, P < 0.01 n = 6). Altogether, these results demonstrate that the RVI machinery activated during S phase in AQP2 expressing cells involves NHE2 activity.

DISCUSSION

Although the Na⁺/H⁺ exchanger has long been implicated in the control of cell proliferation, the specific mechanisms are not yet fully elucidated. To date, most studies have focused in the participation of NHE1 isoform, having a permissive role rather than an obligatory signaling role per se. In fact, it appears that NHE1-induced changes in pH_i may contribute to modulation of cell proliferation [Putney et al., 2002; Fuster and Alexander, 2014]. However, some lines of evidence indicate that under certain conditions NHE2 is also able to support proliferation [Kapus et al., 1994]. Moreover, it has been described that growth factors modulate NHE2 expression and activity through specific domains [Levine et al., 1993; Nath et al., 1999; Xu et al., 2001]. Since cell cycle progression is dependent on an increase in cell volume, in part mediated via NHEs, and AQPs are involved in cell volume regulation [Ford et al., 2005; Galizia et al., 2008], in this study, we analyzed if NHE2 could influence renal cell proliferation focusing on its relationship with AQP2 expression and cell volume regulation.

We here demonstrate, for the first time, that AQP2-expressing cells have both higher pH_i and higher protein levels of NHE2 than WT-RCCD₁ cells. As we have previously demonstrated that in these cells, NHE2 is mainly involved in steady-state pH_i regulation [Ford et al., 2002], the increase in basal pH_i in AQP2 expressing cells could be explained by the increased NHE2 protein abundance that leads to a rise in NHE2 activity. We also show that the increased NHE2 expression in the presence of AQP2 seems to be characteristic of CCD cells since stimulating AQP2 expression in another CCD cell line, the mpkCCD_c14 cells, NHE2 protein abundance also enhances. As it is well-known that in many systems, increases in pH_i may accelerate cell proliferation by stimulating the synthesis of protein, RNA, and DNA [Schreiber, 2005], it is likely that a higher basal pH_i observed in AQP2 cells could act as a facilitator to initiate cell cycle. Our data support this hypothesis since AQP2-expressing cells presented a NHE2-sensitive increased proliferation and inhibition of NHE2 activity slowed down the progress through the S and G₂/M phase of the cell cycle.

However, an increase in pH_i is not the only factor that contributed to accelerate proliferation since we have previously shown that the accelerated proliferation rate seen in AQP2-expressing cells depends, at least in part, on an increase in cell volume and the capacity for RVD changes during the S phase [Rivarola et al., 2010; Di Giusto et al., 2012]. We here demonstrated that while there was no RVI activity in cells in G₁ phase, in S phase, AQP2-expressing cells had an NHE2-mediated RVI activity. The participation of NHE2 in RVI is in line with previous reports in the collecting duct showing that hyperosmolality increases NHE2 mRNA expression and that transcriptional regulation of the NHE2 gene is mediated by two osmotic response elements [Soleimani et al., 1994; Bai et al., 1999]. Moreover, our previous studies indicated that NHE2 was activated by tonicity [Ford et al., 2002].

What could be the interplay between AQP2 and NHE2 to contribute to cell proliferation? It has recently been described that AQP2 promotes cell migration and epithelial morphogenesis through interaction with β-integrins that are connected to the actin cytoskeleton [Tamma et al., 2011; Chen et al., 2012]. It has also been shown that both AQP2 and NHE2 proteins can interact with cytoskeleton through binding to cytoskeletal protein α-spectrin [Chow et al., 1999; Noda et al., 2005]. So it is likely that the increased NHE2 activity and the NHE2 associated-RVI observed in the presence of the water channel AQP2 may be related to the fact that both proteins may form part of an interactome that process proliferative cues. In line with this, it has recently been described that AQP1 and AQP3 overexpression accelerates the cell proliferation because affects the expression of key proteins for the cell cycle progression and contributes to cell volume changes needed for this process [Galan-Cobo et al., 2015, 2016]. So, it cannot be discarded that along with volume changes other proteins related to proliferation may also be modified in AQP2-expressing cells. Further experimental efforts are needed to address this complex interplay between AQP2 and RVI activity at each phase of the cell cycle.

In conclusion, our data show that, in collecting duct cells, the water channel AQP2 plays an important and novel role in regulating cell proliferation through modulating the activation of the RVI machinery during S phase, a mechanism that in turn depends on NHE2 activity. Clarification of the mechanisms governing cell volume control by AQP2 would be very important in learning about correct cellular function as well as about diseases associated with failure in AQP2 expression and alterations in cell proliferation as in the case of Insipidus Nephrogenic Diabetes induced by chronic lithium treatment.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Juerguen Puenter (Aventis Pharma Deutschland GmbH, Germany) who kindly provided us with the HOE-694 compound. This work was supported by grants from Fondo Nacional para la Ciencia y la Tecnología, Argentina, Grant Number: PICT 12-0750; Universidad de Buenos Aires, Argentina, Grant Numbers: UBACYT 20020100100648 and UBACYT 20020130100697; Consejo Nacional de Ciencia y Tecnología, Argentina, Grant Number: PIP 0088 and PIP 0296.

REFERENCES

Bai L, Collins JF, Muller YL, Xu H, Kiela PR, Ghishan FK. 1999. Characterization of cis-elements required for osmotic response of rat Na⁺/H⁺exchanger-2 (NHE-2) gene. Am J Physiol 277: R1112–R1119.
CAS PubMed Web of Science® Google Scholar
Baron C, Penit C. 1990. Study of the thymocyte cell cycle by bivariate analysis of incorporated bromodeoxyuridine and DNA content. Eur J Immunol 20: 1231–1236.
10.1002/eji.1830200606
PubMed Web of Science® Google Scholar
Begg AC, McNally NJ, Shrieve DC, Karcher H. 1985. A method to measure the duration of DNA synthesis and the potential doubling time from a single sample. Cytometry 6: 620–626.
10.1002/cyto.990060618
CAS PubMed Web of Science® Google Scholar
Bens M, Vallet V, Cluzeaud F, Pascual-Letallec L, Kahn A, Rafestin-Oblin ME, Rossier BC, Vandewalle A. 1999. Corticosteroid-dependent sodium transport in a novel immortalized mouse collecting duct principal cell line. J Am Soc Nephrol 10: 923–934.
CAS PubMed Web of Science® Google Scholar
Blot-Chabaud M, Laplace M, Cluzeaud F, Capurro C, Cassingena R, Vandewalle A, Farman N, Bonvalet JP. 1996. Characteristics of a rat cortical collecting duct cell line that maintains high transepithelial resistance. Kidney Int 50: 367–376.
10.1038/ki.1996.325
CAS PubMed Web of Science® Google Scholar
Boron WF, De Weer P. 1976. Intracellular pH transients in squid giant axons caused by CO2, NH3, and metabolic inhibitors. J Gen Physiol 67: 91–112.
10.1085/jgp.67.1.91
CAS PubMed Web of Science® Google Scholar
Capurro C, Rivarola V, Kierbel A, Escoubet B, Farman N, Blot-Chabaud M, Parisi M. 2001. Vasopressin regulates water flow in a rat cortical collecting duct cell line not containing known aquaporins. J Membr Biol 179: 63–70.
10.1007/s002320010037
CAS PubMed Web of Science® Google Scholar
Chen L, Wang L, Zhu L, Nie S, Zhang J, Zhong P, Cai B, Luo H, Jacob TJ. 2002. Cell cycle-dependent expression of volume-activated chloride currents in nasopharyngeal carcinoma cells. Am J Physiol Cell Physiol 283: C1313–C1323.
10.1152/ajpcell.00182.2002
CAS PubMed Web of Science® Google Scholar
Chen Y, Rice W, Gu Z, Li J, Huang J, Brenner MB, Van Hoek A, Xiong J, Gundersen GG, Norman JC, Hsu VW, Fenton RA, Brown D, Lu HA. 2012. Aquaporin 2 promotes cell migration and epithelial morphogenesis. J Am Soc Nephrol 23: 1506–1517.
10.1681/ASN.2012010079
CAS PubMed Web of Science® Google Scholar
Chow CW, Woodside M, Demaurex N, Yu FH, Plant P, Rotin D, Grinstein S, Orlowski J. 1999. Proline-rich motifs of the Na+/H+ exchanger 2 isoform. Binding of Src homology domain 3 and role in apical targeting in epithelia. J Biol Chem 274: 10481–10488.
10.1074/jbc.274.15.10481
CAS PubMed Web of Science® Google Scholar
Counillon L, Pouyssegur J. 2000. The expanding family of eucaryotic Na⁺/H⁺ exchangers. J Biol Chem 275: 1–4.
10.1074/jbc.275.1.1
CAS PubMed Web of Science® Google Scholar
Counillon L, Scholz W, Lang HJ, Pouyssegur J. 1993. Pharmacological characterization of stably transfected Na⁺/H⁺ antiporter isoforms using amiloride analogs and a new inhibitor exhibiting anti-ischemic properties. Mol Pharmacol 44: 1041–1045.
10.1016/S0026-895X(25)13266-5
CAS PubMed Web of Science® Google Scholar
Di Giusto G, Flamenco P, Rivarola V, Fernandez J, Melamud L, Ford P, Capurro C. 2012. Aquaporin 2-increased renal cell proliferation is associated with cell volume regulation. J Cell Biochem 113: 3721–3729.
10.1002/jcb.24246
CAS PubMed Web of Science® Google Scholar
Donowitz M, Ming Tse C, Fuster D. 2013. SLC9/NHE gene family, a plasma membrane and organellar family of Na⁺/H⁺ exchangers. Mol Aspects Med 34: 236–251.
10.1016/j.mam.2012.05.001
CAS PubMed Web of Science® Google Scholar
Flamenco P, Galizia L, Rivarola V, Fernandez J, Ford P, Capurro C. 2009. Role of AQP2 during apoptosis in cortical collecting duct cells. Biol Cell 101: 237–250.
10.1042/BC20080050
CAS PubMed Web of Science® Google Scholar
Ford P, Rivarola V, Kierbel A, Chara O, Blot-Chabaud M, Farman N, Parisi M, Capurro C. 2002. Differential role of Na⁺/H⁺ exchange isoforms NHE-1 and NHE-2 in a rat cortical collecting duct cell line. J Membr Biol 190: 117–125.
10.1007/s00232-002-1030-8
CAS PubMed Web of Science® Google Scholar
Ford P, Rivarola V, Chara O, Blot-Chabaud M, Cluzeaud F, Farman N, Parisi M, Capurro C. 2005. Volume regulation in cortical collecting duct cells: Role of AQP2. Biol Cell 97: 687–697.
10.1042/BC20040116
CAS PubMed Web of Science® Google Scholar
Fuster DG, Alexander RT. 2014. Traditional and emerging roles for the SLC9 Na⁺/H⁺ exchangers. Pflugers Arch 466: 61–76.
10.1007/s00424-013-1408-8
CAS PubMed Web of Science® Google Scholar
Galan-Cobo A, Ramirez-Lorca R, Serna A, Echevarria M. 2015. Overexpression of AQP3 modifies the cell cycle and the proliferation rate of mammalian cells in culture. PLoS ONE 10: e0137692.
10.1371/journal.pone.0137692
PubMed Web of Science® Google Scholar
Galan-Cobo A, Ramirez-Lorca R, Toledo-Aral JJ, Echevarria M. 2016. Aquaporin-1 plays important role in proliferation by affecting cell cycle progression. J Cell Physiol 231: 243–256.
10.1002/jcp.25078
CAS PubMed Web of Science® Google Scholar
Galizia L, Flamenco MP, Rivarola V, Capurro C, Ford P. 2008. Role of AQP2 in activation of calcium entry by hypotonicity: Implications in cell volume regulation. Am J Physiol Renal Physiol 294: F582–F590.
10.1152/ajprenal.00427.2007
CAS PubMed Web of Science® Google Scholar
Harper JV. 2005. Synchronization of cell populations in G1/S and G2/M phases of the cell cycle. Methods Mol Biol 296: 157–166.
CAS PubMed Google Scholar
Hasler U, Mordasini D, Bens M, Bianchi M, Cluzeaud F, Rousselot M, Vandewalle A, Feraille E, Martin PY. 2002. Long term regulation of aquaporin-2 expression in vasopressin-responsive renal collecting duct principal cells. J Biol Chem 277: 10379–10386.
10.1074/jbc.M111880200
CAS PubMed Web of Science® Google Scholar
Jablonski EM, Webb AN, McConnell NA, Riley MC, Hughes FM, Jr. 2004. Plasma membrane aquaporin activity can affect the rate of apoptosis but is inhibited after apoptotic volume decrease. Am J Physiol Cell Physiol 286: C975–C985.
10.1152/ajpcell.00180.2003
CAS PubMed Web of Science® Google Scholar
Janecki AJ, Janecki M, Akhter S, Donowitz M. 2000. Quantitation of plasma membrane expression of a fusion protein of Na/H exchanger NHE3 and green fluorescence protein (GFP) in living PS120 fibroblasts. J Histochem Cytochem 48: 1479–1492.
10.1177/002215540004801105
CAS PubMed Web of Science® Google Scholar
Kapus A, Grinstein S, Wasan S, Kandasamy R, Orlowski J. 1994. Functional characterization of three isoforms of the Na⁺/H⁺ exchanger stably expressed in Chinese hamster ovary cells. ATP dependence, osmotic sensitivity, and role in cell proliferation. J Biol Chem 269: 23544–23552.
10.1016/S0021-9258(17)31550-8
CAS PubMed Web of Science® Google Scholar
Kunzelmann K. 2005. Ion channels and cancer. J Membr Biol 205: 159–173.
10.1007/s00232-005-0781-4
CAS PubMed Web of Science® Google Scholar
Lang F, Friedrich F, Kahn E, Woll E, Hammerer M, Waldegger S, Maly K, Grunicke H. 1991. Bradykinin-induced oscillations of cell membrane potential in cells expressing the Ha-ras oncogene. J Biol Chem 266: 4938–4942.
CAS PubMed Web of Science® Google Scholar
Lang F, Busch GL, Ritter M, Volkl H, Waldegger S, Gulbins E, Haussinger D. 1998a. Functional significance of cell volume regulatory mechanisms. Physiol Rev 78: 247–306.
10.1152/physrev.1998.78.1.247
CAS PubMed Web of Science® Google Scholar
Lang F, Lepple-Wienhues A, Paulmichl M, Szabó I, Siemen D, Gulbins E. 1998b. Ion channels, cell volume, and apoptotic cell death. Cell Physiol Biochem 8: 285–292.
10.1159/000016290
CAS PubMed Web of Science® Google Scholar
Lang F, Ritter M, Gamper N, Huber S, Fillon S, Tanneur V, Lepple-Wienhues A, Szabo I, Gulbins E. 2000. Cell volume in the regulation of cell proliferation and apoptotic cell death. Cell Physiol Biochem 10: 417–428.
10.1159/000016367
CAS PubMed Web of Science® Google Scholar
Lang F, Foller M, Lang KS, Lang PA, Ritter M, Gulbins E, Vereninov A, Huber SM. 2005. Ion channels in cell proliferation and apoptotic cell death. J Membr Biol 205: 147–157.
10.1007/s00232-005-0780-5
CAS PubMed Web of Science® Google Scholar
Lang F, Foller M, Lang K, Lang P, Ritter M, Vereninov A, Szabo I, Huber SM, Gulbins E. 2007. Cell volume regulatory ion channels in cell proliferation and cell death. Methods Enzymol 428: 209–225.
10.1016/S0076-6879(07)28011-5
PubMed Web of Science® Google Scholar
Levine SA, Montrose MH, Tse CM, Donowitz M. 1993. Kinetics and regulation of three cloned mammalian Na⁺/H⁺ exchangers stably expressed in a fibroblast cell line. J Biol Chem 268: 25527–25535.
10.1016/S0021-9258(19)74423-8
CAS PubMed Web of Science® Google Scholar
Nath SK, Kambadur R, Yun CH, Donowitz M, Tse CM. 1999. NHE2 contains subdomains in the COOH terminus for growth factor and protein kinase regulation. Am J Physiol 276: C873–C882.
10.1152/ajpcell.1999.276.4.C873
CAS PubMed Web of Science® Google Scholar
Noda Y, Horikawa S, Katayama Y, Sasaki S. 2005. Identification of a multiprotein “motor” complex binding to water channel aquaporin-2. Biochem Biophys Res Commun 330: 1041–1047.
10.1016/j.bbrc.2005.03.079
CAS PubMed Web of Science® Google Scholar
Orlowski J, Grinstein S. 2011. Na⁺/H⁺ exchangers. Compr Physiol 1: 2083–2100.
10.1002/j.2040-4603.2011.tb00390.x
PubMed Web of Science® Google Scholar
Putney LK, Denker SP, Barber DL. 2002. The changing face of the Na⁺/H⁺ exchanger, NHE1: Structure, regulation, and cellular actions. Annu Rev Pharmacol Toxicol 42: 527–552.
10.1146/annurev.pharmtox.42.092001.143801
CAS PubMed Web of Science® Google Scholar
Ritter M, Wöll E. 1996. Modification of cellular ion transport by the Ha-ras oncogene: Steps towards malignant transformation. Cell Physiol Biochem 6: 245–270.
10.1159/000154827
CAS Web of Science® Google Scholar
Rivarola V, Flamenco P, Melamud L, Galizia L, Ford P, Capurro C. 2010. Adaptation to alkalosis induces cell cycle delay and apoptosis in cortical collecting duct cells: Role of Aquaporin-2. J Cell Physiol 224: 405–413.
10.1002/jcp.22136
CAS PubMed Web of Science® Google Scholar
Roos A, Boron WF. 1981. Intracellular pH. Physiol Rev 61: 296–434.
10.1152/physrev.1981.61.2.296
CAS PubMed Web of Science® Google Scholar
Rouzaire-Dubois B, Dubois JM. 1998. K⁺ channel block-induced mammalian neuroblastoma cell swelling: A possible mechanism to influence proliferation. J Physiol 510(Pt 1): 93–102.
10.1111/j.1469-7793.1998.093bz.x
CAS PubMed Web of Science® Google Scholar
Schreiber R. 2005. Ca²⁺ signaling, intracellular pH and cell volume in cell proliferation. J Membr Biol 205: 129–137.
10.1007/s00232-005-0778-z
CAS PubMed Web of Science® Google Scholar
Shi H, Levy-Holzman R, Cluzeaud F, Farman N, Garty H. 2001. Membrane topology and immunolocalization of CHIF in kidney and intestine. Am J Physiol Renal Physiol 280: F505–F512.
10.1152/ajprenal.2001.280.3.F505
CAS PubMed Web of Science® Google Scholar
Shimizu H, Shiozaki A, Ichikawa D, Fujiwara H, Konishi H, Ishii H, Komatsu S, Kubota T, Okamoto K, Kishimoto M, Otsuji E. 2014. The expression and role of Aquaporin 5 in esophageal squamous cell carcinoma. J Gastroenterol 49: 655–666.
10.1007/s00535-013-0827-9
CAS PubMed Web of Science® Google Scholar
Soleimani M, Singh G, Bizal GL, Gullans SR, McAteer JA. 1994. Na⁺/H⁺ exchanger isoforms NHE-2 and NHE-1 in inner medullary collecting duct cells. Expression, functional localization, and differential regulation. J Biol Chem 269: 27973–27978.
10.1016/S0021-9258(18)46882-2
CAS PubMed Web of Science® Google Scholar
Tamma G, Lasorsa D, Ranieri M, Mastrofrancesco L, Valenti G, Svelto M. 2011. Integrin signaling modulates AQP2 trafficking via Arg-Gly-Asp (RGD) motif. Cell Physiol Biochem 27: 739–748.
10.1159/000330082
CAS PubMed Web of Science® Google Scholar
Verkman AS. 2008. Mammalian aquaporins: Diverse physiological roles and potential clinical significance. Expert Rev Mol Med 10: e13.
10.1017/S1462399408000690
CAS PubMed Google Scholar
Wang L, Chen L, Zhu L, Rawle M, Nie S, Zhang J, Ping Z, Kangrong C, Jacob TJ. 2002. Regulatory volume decrease is actively modulated during the cell cycle. J Cell Physiol 193: 110–119.
10.1002/jcp.10156
CAS PubMed Web of Science® Google Scholar
Winterberg M, Rajendran E, Baumeister S, Bietz S, Kirk K, Lingelbach K. 2012. Chemical activation of a high-affinity glutamate transporter in human erythrocytes and its implications for malaria-parasite-induced glutamate uptake. Blood 119: 3604–3612.
10.1182/blood-2011-10-386003
CAS PubMed Web of Science® Google Scholar
Xiao F, Waldrop SL, Khimji AK, Kilic G. 2012. Pannexin1 contributes to pathophysiological ATP release in lipoapoptosis induced by saturated free fatty acids in liver cells. Am J Physiol Cell Physiol 303: C1034–C1044.
10.1152/ajpcell.00175.2012
CAS PubMed Web of Science® Google Scholar
Xu H, Collins JF, Bai L, Kiela PR, Lynch RM, Ghishan FK. 2001. Epidermal growth factor regulation of rat NHE2 gene expression. Am J Physiol Cell Physiol 281: C504–C513.
10.1152/ajpcell.2001.281.2.C504
CAS PubMed Web of Science® Google Scholar

Citing Literature

Volume118, Issue5

May 2017

Pages 967-978

AQP2-Induced Acceleration of Renal Cell Proliferation Involves the Activation of a Regulatory Volume Increase Mechanism Dependent on NHE2

ABSTRACT