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Cytokines and Sodium Induce Protein Kinase A–Dependent Cell-Surface Na,K-ATPase Recruitment via Dissociation of NF-B/IB/Protein Kinase A Catalytic Subunit Complex in Collecting Duct Principal Cells

Manlio Vinciguerra^*,, Udo Hasler^*, David Mordasini^*, Martine Roussel^*, Maria Capovilla, Eric Ogier-Denis, Alain Vandewalle, Pierre-Yves Martin^* and Eric Feraille^*

^* Service of Nephrology, Foundation for Medical Research, Geneva, Switzerland; Dulbecco Telethon Institute, Laboratory of Medical Genetics, S. Orsola-Malpighi Hospital, Bologna, Italy; and INSERM U479 and INSERM U478, Faculty of Medicine Xavier Bichat, Paris, France

Address correspondence to: Dr. Eric Féraille, Service de Néphrologie, Fondation pour Recherches Médicales, 64 Rue de la Roseraie, CH-1211 Geneva 4, Switzerland. Phone: +41-22-382-3837; Fax: +41-22-347-5979; E-mail: eric.feraille{at}medecine.unige.ch

Received for publication April 29, 2005. Accepted for publication May 27, 2005.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Collecting duct (CD) principal cells are exposed to large physiologic variations of apical Na⁺ influx as a result of variations of Na⁺ intake and extrarenal losses. It was shown previously that increasing intracellular [Na⁺] induces recruitment of Na,K-ATPase to the cell surface in a protein kinase A (PKA)-dependent manner in both native and cultured renal CD principal cells. As described previously in response to cytokines in nonrenal cells, PKA activation in response to increased intracellular [Na⁺] was independent of cAMP and required proteasomal activity. With the use of cultured mpkCCD_cL4 cells as a model of CD principal cells, whether cytokines and increased intracellular [Na⁺] share a common signaling pathway leading to cell-surface Na,K-ATPase recruitment was investigated. Results showed that two potent inducers of NF-B, LPS and TNF-, enhance Na⁺ transport and induce cell-surface Na,K-ATPase recruitment in mpkCCD_cL4 cells via cAMP-independent PKA activation. In addition, increased intracellular [Na⁺] after selective plasma membrane permeabilization by a low concentration of the Na⁺ ionophore amphotericin B (1 µg/ml) induced dissociation of the PKA catalytic subunit from p65–NF-B and IB. Moreover, inhibitors of NF-B/IB dissociation prevented both Na⁺-dependent stimulation of PKA activity and cell-surface Na,K-ATPase recruitment. Altogether, these results revealed the presence of a novel Na⁺-dependent intracellular signaling pathway leading to Na,K-ATPase cell-surface recruitment via dissociation of the PKA catalytic subunit from a macromolecular complex that contains NF-B and IB in CD epithelial cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Renal collecting duct (CD) principal cells are subjected to large fluctuations of transcellular Na⁺ flux that arise from variations of luminal Na⁺ delivery and flow rate as well as variation of circulating hormones and paracrine/autocrine factors concentration. In principal cells, Na⁺ enters via apical Na⁺ channels (ENaC) and is extruded by basolateral Na,K-ATPase (1). Na,K-ATPase, which provides the driving force for active Na⁺ and K⁺ transport and secondary active transport of other solutes (2), is under tight multifactorial control (1,3). Intracellular Na⁺ is likely the most important nonhormonal factor regulating Na,K-ATPase activity because any increase or decrease in intracellular [Na⁺] stimulates or reduces, respectively, Na,K-pump activity (4). In addition, we have shown that a rise in intracellular [Na⁺] induced by the Na⁺ ionophore amphotericin B or by hypotonic exposure rapidly increases Na,K-ATPase cell-surface expression independent of de novo transcription and/or protein synthesis in CD principal cells (5,6). This Na,K-ATPase cell-surface recruitment after an increase in intracellular [Na⁺] relies on cAMP-independent protein kinase A (PKA) activation (5), independent of cell volume variation (6), suggesting the existence of an intracellular [Na⁺]-sensing pathway in CD principal cells.

The PKA holoenzyme is a heterotetramer that consists of two catalytic (PKAc) subunits associated with two regulatory subunits (7–9). Classically, the dissociation of the holoenzyme is induced by binding of cAMP to regulatory subunits, which alleviates autoinhibitory contacts and releases active PKAc. Recently, an alternative cAMP-independent mechanism of PKA activation was described. In response to cytokines, free active PKAc is released upon the dissociation of a PKAc/IB/p65–NF-B complex and proteasomal degradation of IB (10). p65–NF-B belongs to the Rel family of transcription factors that regulate the expression of genes involved in immune and inflammatory response, i.e., cytokines, chemokines, and cell adhesion molecules (11). Prototypically, p65–NF-B is retained by IB (, , , and isoforms) in its inactive form in the cytoplasm of unstimulated cells (11). In response to various stimuli, IB, the best characterized of all IB isoforms, is phosphorylated on Ser-32 and Ser-36 residues by IB kinase (IKK) and is subsequently rapidly ubiquitinated and degraded by the 26S proteasome (12–14). A similar process is involved in the degradation of IB (15).

Interactions between the NF-B signaling pathway and renal transepithelial Na⁺ transport have been highlighted recently following the demonstration of IKK binding to ENaC (16). Moreover, coexpression of ENaC and IKK in Xenopus oocytes resulted in increased ENaC-mediated Na⁺ current. In this study, we sought to examine whether LPS and TNF-, two potent activators of NF-B, stimulate transepithelial Na⁺ transport via cAMP-independent PKA activation and, conversely, whether increasing intracellular [Na⁺] activates PKA through dissociation of an endogenous PKAc/IB/p65–NF-B complex. We used cultured mouse principal CD mpkCCD_cL4 cells (17), which form a tight epithelium when grown on permeable filters and remain highly differentiated because they retain ENaC and aquaporin-2 expression as well as aldosterone- and vasopressin-regulated Na⁺ transport (17–20).

	Materials and Methods

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Cell Culture
The mpkCCD_cL4 cells (passages 23 to 32) were grown to confluence on polycarbonate filters (Transwell; Costar, Cambridge, MA) for 6 to 8 d in defined medium supplemented with 2% (vol/vol) FCS and then in serum- and hormone-deprived medium 24 h before use (5). For experiments, cells were preincubated for 1 h at 37°C in isotonic low Na⁺ (110 mM choline chloride and 30 mM NaCl) or high Na⁺ (140 mM NaCl) solution, supplemented or not with drugs, and were incubated further in the same incubation solutions supplemented with 1 µg/ml of the Na⁺ ionophore amphotericin B, as described (5). In some experiments, cells were preincubated for 1 h at 37°C in isotonic incubation solution (140 mM NaCl) and incubated further in the same incubation solution or in hypotonic (200 mOsm/L) incubation solution (90 mM NaCl), as described (6).

PKA and IKK Activity Assay
Cells were scraped off and homogenized in a kinase extraction buffer (25 mM Tris-HCl [pH 7.4], 0.5 mM EDTA, 0.5 mM EGTA, 10 mM -mercaptoethanol, 1 µg/ml leupeptin, and 1 µg/ml aprotinin). PKA activity then was measured using the SignaTECT cAMP-Dependent Protein Kinase (PKA) Assay System (Promega, Madison, WI) according to the manufacturer’s instructions. For measurements of IKK activity, incubation was performed for 10 min at 37°C in a total volume of 25 µl: 5 µl of 5x reaction buffer (40 mM MOPS and 1 mM EDTA), 5 µl of IKK substrate peptide (Upstate Cell Signaling, Norcross, GA), 5 µl of cell lysate, 10 µl of [-³²P]ATP (3000 Ci/mmol) solution. After incubation, 20-µl aliquots were added to the center of 2 x 2-cm P81 paper squares (Upstate Cell Signaling), washed three times with 0.75% phosphoric acid and once with acetone, and transferred into scintillation counting vials. Results were expressed as pmol ATP/min per µg protein.

Measurement of Intracellular cAMP Content
After incubation under various experimental conditions, confluent mpkCCD_cL4 cells that were grown on filters were scraped off the filter and homogenized in a buffer that contained 50 mM Tris and 4 mM EDTA (pH 7.4). Cellular cAMP concentration then was measured using the Cyclic AMP (³H) system (Amersham-Pharmacia Biotech, Little Chalfont, UK) according to the manufacturer’s instructions. Results were expressed as a pmol cAMP/µg protein per h.

Electrophysiologic Studies
For experiments, confluent mpkCCD_cL4 cells that were grown on Snapwell filters (0.4-µm pore size, 12-mm diameter; Corning Costar, Cambridge, MA) were placed in serum- and hormone-deprived medium 24 h before use and then transferred to a Ussing chamber. Short-circuit current (Isc) was measured under voltage-clamp (0 mV) using dual silver-silver chloride electrodes connected to a VCC MC6 voltage clamp apparatus (Physiologic Instruments, San Diego, CA). Cells were equilibrated at 37°C for 30 min in serum- and hormone-deprived culture medium and bubbled with 5% CO₂. After equilibration, cells were treated with 10 ng/ml LPS or 10 ng/ml TNF- for 30 min before addition of the PKA inhibitor H89 (5 x 10^–5 M). In some experiments, cells were preincubated with 5 x 10^–5 H89 or 10^–6 amiloride for 30 min before treatment. At the end of experiments, 10^–6 M amiloride was added to the apical side of the chamber to measure the amiloride-resistant Isc. The amiloride-sensitive Isc reflecting ENaC-mediated Na⁺ transport was calculated as the total Isc minus the amiloride-resistant Isc. By convention, positive Isc corresponded to a flow of positive charges from the apical to the basal solution. Results were expressed as µA/cm².

Measurement of Cell Surface Na,K-ATPase
Cell-surface proteins were labeled using EZ-Link sulfossuccinimidobiotin (Pierce, Rockford, IL) and precipitated by streptavidin-agarose beads (Immunopure immobilized streptavidin; Pierce) as described (21). After resuspension in Laemmli’s buffer (22), samples were processed for 7% SDS-PAGE and proteins were electrotransferred to polyvinylidene difluoride membranes (Immobilon-P; Millipore, Waters, MA). The Na,K-ATPase -subunit was revealed by Western blotting with a polyclonal antibody (dilution 1:10000) raised against the rat holoenzyme (23). Antigen-antibody complexes were detected by enhanced chemiluminescence (ECL; Amersham). The protein bands were quantified using a video densitometer and the ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Immunoprecipitation
After cell lysis in homogenizing buffer (HB; 2 mM EDTA, 2 mM EGTA, 40 µg/ml leupeptin, 2 µg/ml aprotinin, 30 mM NaF, 30 mM Na pyrophosphate, 1 mM PMSF, 1 mM AEBSF, 20 mM Tris HCl [pH 7.4], 2 mM sodium orthovanadate, and 0.5% saponin), 100 or 750 µg of protein was incubated overnight at 4°C with saturating amounts of anti-IB, anti-IB, or anti-PKAc antibodies (Santa Cruz Biotechnology, Santa Cruz, CA). After 1 h of incubation with protein A–Sepharose beads, five washing steps were performed in HB. Precipitated proteins were separated by 7% SDS-PAGE and p65–NF-B was revealed by Western blotting with a mAb (dilution 1:5000; Santa Cruz Biotechnology).

Pull-Down Experiments
Recombinant human PKAc was biotinylated by incubation for 2 h at 4°C with saturating amounts of EZ-Link Sulfo-NHS-Biotin. Biotinylated PKAc was subsequently bound to streptavidin agarose beads by incubation for 2 h at 4°C on a rotating platform. Free streptavidin binding sites then were saturated by incubation for an additional hour with a 10-mg/ml biotin solution. After two washes with TBS, cells were lysed in HB buffer (see above) and 500 µg of protein was incubated overnight at 4°C with streptavidin-bound PKAc. Streptavidin beads then were washed four times with HB buffer, and precipitated proteins were separated by 10% SDS-PAGE. p65–NF-B and IB were revealed by Western blotting using specific mAb (dilution 1:5000) and polyclonal antibodies (dilution 1:5000), respectively.

Statistical Analyses
Results are given as means ± SEM from (n) independent experiments. Each experiment was performed on cultured cells from the same passage. Statistical analysis was done using the Mann-Whitney U test or the Kruskal-Wallis test for comparison of two or more than two groups, respectively. P < 0.05 was considered significant.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
NF-B Activators Increase PKA Activity in a cAMP-Independent Manner
We showed previously that increased Na⁺ influx activates PKAc independent of cAMP in renal CD cells (5). NF-B activating cytokines were shown to induce a cAMP-independent and proteasomal-dependent PKA activation via dissociation of a PKAc/IB/p65–NF-B complex in nonrenal cells (10). To assess whether such a signaling pathway is also present in CD principal cells, we measured the effects of well-known NF-B activators TNF- and LPS on PKA activity and on cellular cAMP content in cultured mpkCCD_cL4 cells. Cells were incubated for 15 min at 37°C in a medium that contained 140 mM Na⁺ without or with 1 µg/ml of the Na⁺ ionophore amphotericin B, 10 ng/ml recombinant mouse TNF-, 10 ng/ml Escherichia coli LPS, or 10^–9 M vasopressin (AVP). As expected, AVP induced a large (approximately four-fold) increase in PKA activity, as compared with untreated cells. Amphotericin B, TNF-, and LPS all triggered PKA activation to comparable extents (Figure 1A). Because PKA activation classically relies on increased cellular cAMP content, we measured the effect of amphotericin B, TNF-, and LPS on cellular cAMP content. As expected, treatment of cells with 10^–9 M AVP for 10 min induced a large (approximately 25-fold) increase in cellular cAMP content. In contrast, incubation of cells for 10 min with 1 µg/ml amphotericin B, 10 ng/ml TNF-, or 10 ng/ml LPS did not alter cellular cAMP content (Figure 1B). Collectively, these results indicate that classical NF-B activators (TNF- and LPS) and increased cellular Na⁺ influx all activate PKA in a cAMP-independent manner.

View larger version (18K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 1. Effect of NF-B activators on protein kinase A (PKA) activity and cellular cAMP content in mpkCCDcL4 cells. Confluent mpkCCDcL4 cells that were grown on filters were preincubated in isotonic medium that contained 140 mM Na+ for 1 h at 37°C before 15 min of incubation at 37°C in the absence (CTL) or presence of 1 µg/ml amphotericin B, 10 ng/ml LPS, 10 ng/ml TNF-, or 10–9M vasopressin (AVP). (A) PKA activity was determined as described in the Materials and Methods section. Results, expressed as percentage of control, are means ± SEM from four independent experiments. (B) Cellular cAMP content was determined as described in the Materials and Methods section. Results, expressed as pmol cAMP/µg protein per h, are means ± SEM from three independent experiments. *P < 0.05 versus control values.

PKA-Dependent Stimulation of Transepithelial Na⁺ Transport by NF-B Activators

View larger version (19K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 2. LPS stimulates the amiloride-sensitive short-circuit current (ISC) in a PKA-dependent manner. Confluent mpkCCDcL4 cells were transferred to a Ussing chamber and Isc was measured under voltage-clamp (0 mV) as described in the Materials and Methods section. (A) Cells first were equilibrated for 30 min at 37°C in the presence of serum- and hormone-free culture medium and then exposed to 10 ng/ml LPS for 30 min before addition of 5 x 10–5 M H89. A representative recording is shown (n = 12). Arrows indicate addition of LPS, H89, or amiloride. (B) Cells were preincubated for 30 min in the absence or presence of 5 x 10–5 M H89 and then incubated for another 30 min without (CTL) or with 10 ng/ml LPS before addition of 10–5 M amiloride. The amiloride-sensitive ISC was calculated as the total Isc minus the amiloride-resistant Isc. Results, expressed as µA/cm2, are means ± SEM from four independent experiments. *P < 0.01 versus control values.

NF-B Activators Induce Na,K-ATPase Cell-Surface Recruitment

View larger version (30K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 3. LPS and TNF- induce Na,K-ATPase cell surface recruitment. Confluent mpkCCDcL4 cells that were grown on filters were incubated for 1 h at 37°C in the absence or presence of 10 ng/ml LPS or 10 ng/ml TNF- for 1 h at 37°C. The Na,K-ATPase -subunit was detected by Western blotting performed after biotinylation and streptavidin precipitation of cell-surface proteins. (Top) Representative immunoblots. Bars represent the quantification of labeled -subunit bands expressed as a percentage of the control optical density value. Results are means ± SEM from five to eight independent experiments *P < 0.05 versus control values.

Increased Intracellular [Na⁺] Induces Dissociation of IB from p65–NF-B via IKK Activation

View larger version (27K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 4. Increasing intracellular [Na+] induces dissociation of IB from p65–NF-B. (A) Determination of the amount of p65–NF-B associated with IB and isoforms. mpkCCDcL4 cell lysates were incubated overnight at 4°C with polyclonal anti-IB and/or anti-IB antibodies. An equal volume of cell lysate taken before (–) and after (+) immunoprecipitation was submitted to 7% SDS-PAGE, and p65–NF-B (p65) was detected by Western blotting. (B) Effects of TNF- and high Na+ influx on IB dissociation from p65–NF-B. Confluent mpkCCDcL4 cells that were grown on filters were preincubated for 1 h at 37°C in isotonic medium that contained low (30 mM) or high (140 mM) Na+ concentration. Cells then were incubated in the absence or presence of 10 ng/ml TNF- (left) or with 1 µg/ml amphotericin B (middle and right) for 30 min at 37°C. Cell lysates were incubated overnight at 4°C with an anti-IB antibody (middle) or an anti-IB antibody (right), and co-precipitated p65–NF-B (p65) was detected by Western blotting. (A and B, top) Representative immunoblots. (Bottom) Densitometric quantification of labeled p65–NF-B bands expressed as a percentage of the control optical density value. Results are means ± SEM from four independent experiments.

Our next step was to characterize the kinetics of Na⁺-induced dissociation of IB from p65–NF-B in mpkCCD_cL4 cells. The amount of p65–NF-B that co-precipitated with IB after immunoprecipitation with an anti-IB antibody was first significantly reduced in amphotericin B–treated cells after 15 to 30 min of incubation with high concentrations of Na⁺ (140 mM) and then returned to baseline levels after 60 min of incubation (Figure 5). Conversely, no change in the amount of p65–NF-B that co-precipitated with the anti-IB antibody was observed throughout the 60-min incubation period in amphotericin B–treated cells that were incubated with isotonic medium that contained low concentrations of Na⁺ (30 mM; Figure 5). These results indicate that an increase of intracellular [Na⁺] caused by a Na⁺ ionophore induces a transient dissociation of IB from p65–NF-B in cultured mpkCCD_cL4 cells.

View larger version (37K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 5. Time course of p65–NF-B dissociation from IB induced by high cellular Na+ influx. (A) Confluent mpkCCDcL4 cells that were grown on filters were preincubated for 1 h at 37°C in isotonic medium that contained low (30 mM) or high (140 mM) Na+ concentrations before addition of 1 µg/ml amphotericin B for various periods (0 to 60 min). Cell lysates then were incubated overnight at 4°C with an anti-IB antibody, and co-precipitated p65–NF-B (p65) was detected by Western blotting. (A) Representative immunoblot showing the time course of dissociation of IB from p65-NF-B. (B) Bars represent the densitometric quantification of labeled p65–NF-B bands expressed as a percentage of the control optical density value (time 0). Results are means ± SEM from three independent experiments *P < 0.05 versus control values.

View larger version (22K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 6. TNF- and increased intracellular [Na+] stimulates IB kinase (IKK) activity. Confluent mpkCCDcL4 cells that were grown on filters were preincubated for 1 h at 37°C in isotonic medium that contained low (30 mM) or high (140 mM) Na+ concentration. Cells then were incubated in the absence or presence of 10 ng/ml TNF- (left) or with 1 µg/ml amphotericin B (middle and right) for 30 min at 37°C, and IKK activity was measured as described in Materials and Methods. The results, expressed as pmol ATP/min per µg protein, are means ± SEM from three independent experiments. *P < 0.05 versus control.

Increased Intracellular [Na⁺] Induces Dissociation of an Endogenous PKAc/IB/p65–NF-B Complex

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 7. Increased intracellular [Na+] induces dissociation of an endogenous PKAc/p65–NF-B complex. Confluent mpkCCDcL4 cells that were grown on filters were preincubated in isotonic medium that contained low (30 mM) or high (140 mM) Na+ concentrations for 1 h at 37°C in the presence or absence of 10–5 M MG-132 before addition of 1 µg/ml amphotericin B (30 min at 37°C). Equal amounts of protein were subjected to immunoprecipitation using an anti-PKAc antibody, and co-precipitated p65–NF-B (p65) was detected by Western blotting. Representative immunoblot is shown (top). Bars represent the densitometric quantification of labeled p65–NF-B bands expressed as the percentage of the control optical density value (bottom). Results are means ± SEM from three independent experiments.

View larger version (21K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 8. Pull-down of p65–NF-B and IB by recombinant PKAc. Confluent mpkCCDcL4 cells that were grown on filters were preincubated in isotonic medium that contained low (30 mM) or high (140 mM) Na+ concentrations for 1 h at 37°C before addition of 1 µg/ml amphotericin B for 30 min at 37°C before lysis. Lysates were subjected to a pull-down assay using biotinylated recombinant PKAc (biotin-PKAc) bound to streptavidin-agarose beads used as bait. For control experiments, biotinylation of purified PKAc was omitted (unbound PKAc). Precipitated p65–NF-B and IB were detected by Western blotting. Representative immunoblots are shown in the top panel. Bars represent the densitometric quantification of labeled bands expressed as the percentage of the control optical density value (30 mM Na+). Results are means ± SEM from three independent experiments.

Inhibition of NF-B Activity Prevents PKA Activation and Na,K-ATPase Cell-Surface Recruitment Induced by Increased Intracellular [Na⁺]

View larger version (20K): [in this window] [in a new window] [as a PowerPoint slide]   Figure 9. Effect of NF-B inhibition on PKA activation and Na,K-ATPase cell-surface recruitment induced by high cellular Na+ influx. Confluent mpkCCDcL4 cells that were grown on filters were preincubated with isotonic medium that contained either low (30 mM) or high (140 mM) Na+ concentrations for 1 h at 37°C without or with 10 µM pyrrolidine dithiocarbamate (PDTC) or 1 µM BAY11-70859 before addition of 1 µg/ml amphotericin B for an additional 15 min (for PKA activity) or 60 min (for Na,K-ATPase measurement) at 37°C. (A) effect of NF-B inhibition on PKA activation induced by high cellular Na+ influx. As a positive control, mpkCCDcL4 cells were treated for 10 min at 37°C with 10–3 M db-cAMP. PKA activity was determined as described in Materials and Methods. Results, expressed as percentage of control, are means ± SEM from three independent experiments. *P < 0.05 versus control values. (B) Effect of NF-B inhibition on increased Na,K-ATPase cell-surface expression induced by high cellular Na+ influx. The Na,K-ATPase -subunit was detected by Western blotting performed after biotinylation and streptavidin precipitation of cell-surface proteins. (Top) Representative immunoblots. Bars represent the quantification of labeled -subunit bands expressed as a percentage of the control optical density value (30 mM Na+). Results are means ± SEM from five independent experiments *P < 0.05 versus control values.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

We showed previously that increasing intracellular [Na⁺] activates PKAc independent of cAMP and induces Na,K-ATPase cell-surface recruitment in mammalian renal CD principal cells (5). Therefore, Na⁺-dependent PKA activation does not depend on the classical cAMP-induced dissociation of PKAc from PKA regulatory subunits (7–9). Several lines of experimental evidence from our study indicate that Na⁺-induced PKA activation results from the release of free PKAc from an endogenous PKAc/IB/p65–NF-B complex present in CD principal cells: (1) A PKAc/IB/p65–NF-B complex dissociates in response to increased Na⁺ influx, (2) inhibitors of the proteasome prevent the Na⁺-induced dissociation of the PKAc/IB/p65–NF-B complex, (3) compounds that inhibit NF-B activation prevent Na⁺-induced PKA activation, and (4) classical activators of NF-B (LPS and TNF-) stimulate PKA in a cAMP-independent manner. Our results are in agreement with a previous observation demonstrating PKA activation through dissociation of PKAc from IB and p65–NF-B in response to cytokines (10). As previously demonstrated for dissociation of the IB/p65–NF-B complex (14,15), dissociation of the endogenous PKAc/IB/p65–NF-B complex requires proteasomal degradation of IB (see Figure 7). However, it is still a matter of debate whether activated PKA directly induces Na,K-ATPase redistribution, e.g., through phosphorylation of the Na,K-ATPase -subunit (23,29–32), and/or whether other signaling intermediates are involved.

Our finding that increased Na⁺ influx activates a p65–NF-B–dependent signaling pathway is strengthened by results obtained with classical activators of NF-B. Indeed, cAMP-independent PKA activation that was observed in response to increasing intracellular [Na⁺] (5) was also observed in response to LPS and TNF- (see Figure 1). Moreover, measurement of the amiloride-sensitive Isc revealed that both LPS and TNF- stimulated transepithelial Na⁺ transport in a PKA-dependent manner in cultured CD principal cells (see Figure 2). It should be mentioned that previous studies reported an inhibitory effect of cytokines (IL-1 and TNF-) and LPS on renal transepithelial Na⁺ transport (33–35). However, these reports focused on long-term effects (up to 24 h) that led to an increased expression of nitric oxide (NO) synthase, which in turn increases production of NO, a potent inhibitor of tubular Na⁺ reabsorption (36,37). Altogether, these results suggest that proinflammatory cytokines and LPS may first transiently stimulate transepithelial Na⁺ transport and then produce a sustained inhibitory effect via induction of NO production in renal tubule epithelial cells.

Our results strongly suggest that increased intracellular [Na⁺] induces dissociation of the PKAc/IB/p65–NF-B complex via the classical IKK-dependent pathway (11). Indeed, we showed that increased intracellular [Na⁺] stimulated IKK activity (see Figure 6) and the observed time course of dissociation and re-association between IB and p65–NF-B (see Figure 5) in response to increased intracellular [Na⁺] or to cytokines is similar in murine epithelial cells and macrophages (38), respectively. Re-association of IB with p65–NF-B observed after 60 min of exposure of CD principal cells to increased intracellular [Na⁺] may be explained by the following feedback mechanism: NF-B stimulates IB transcription, and neosynthesized IB translocates into the nucleus, where it enhances the dissociation of NF-B from DNA (39). The newly formed IB/NF-B complex then is translocated back to the cytoplasm, thereby restoring the inducible cytoplasmic pool of NF-B (40). We also show that p65–NF-B does not dissociate from IB in response to a short-term increase of cellular Na⁺ influx (see Figure 4). This result is consistent with the fact that IB might be involved mostly in rapid and transient NF-B activation, whereas IB responds more slowly to IKK and might be responsible for the sustained late phase of NF-B activation in response to persistent stimuli (41,42). Our findings suggesting that the IB/p65–NF-B signaling pathway can be triggered by increased intracellular [Na⁺] in mpkCCD_cL4 cells are in line with the recent report of Lebowitz et al. (16) showing that IKK associates with the ENaC -subunit and that overexpression of IKK stimulates ENaC activity in Xenopus oocytes (16). These authors also showed that aldosterone, which stimulates Na⁺ transport in CD principal cells, increased IKK expression and activated NF-B in cultured mpkCCD_cL4 cells.

In summary, this study provides evidence that increased intracellular [Na⁺] triggers the dissociation of an endogenous PKAc/IB/p65–NF-B complex, leading to PKA activation and Na,K-ATPase cell-surface recruitment in mammalian CD principal cells. This novel signaling pathway may play an important role in the intracellular [Na⁺] sensing mechanism. In addition, our results suggest that proinflammatory cytokines and bacterial products may locally alter Na⁺ reabsorption mechanisms in renal epithelial cells.

	Acknowledgments

 
This work was supported by the Swiss National Foundation for Science Grant 3100-067878.02 and by a grant from the Carlos and Elsie De Reuter Foundation to E.F.

We warmly thank Dr. Florence Margottin-Goguet for critical reading of the manuscript.

	Footnotes

 
Published online ahead of print. Publication date available at www.jasn.org.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

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