HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: 290/2/C492    most recent 00556.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Alvarez de la Rosa, D. Articles by Canessa, C. M. Search for Related Content PubMed PubMed Citation Articles by Alvarez de la Rosa, D. Articles by Canessa, C. M.

MEMBRANE TRANSPORTERS, ION CHANNELS, AND PUMPS

SGK1 activates Na⁺-K⁺-ATPase in amphibian renal epithelial cells

¹Unidad de Farmacología, Facultad de Medicina, Universidad de La Laguna, La Laguna, Spain; ²Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut; and ³Departamento de Farmacología y Fisiología, Facultad de Medicina, Universidad de Zaragoza, Zaragoza, Spain

Submitted 7 November 2004 ; accepted in final form 22 September 2005

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Serum- and glucocorticoid-induced kinase 1 (SGK1) is thought to be an important regulator of Na⁺ reabsorption in the kidney. It has been proposed that SGK1 mediates the effects of aldosterone on transepithelial Na⁺ transport. Previous studies have shown that SGK1 increases Na⁺ transport and epithelial Na⁺ channel (ENaC) activity in the apical membrane of renal epithelial cells. SGK1 has also been implicated in the modulation of Na⁺-K⁺-ATPase activity, the transporter responsible for basolateral Na⁺ efflux, although this observation has not been confirmed in renal epithelial cells. We examined Na⁺-K⁺-ATPase function in an A6 renal epithelial cell line that expresses SGK1 under the control of a tetracycline-inducible promoter. The results showed that expression of a constitutively active mutant of SGK1 (SGK1^T_S425D) increased the transport activity of Na⁺-K⁺-ATPase 2.5-fold. The increase in activity was a direct consequence of activation of the pump itself. The onset of Na⁺-K⁺-ATPase activation was observed between 6 and 24 h after induction of SGK1 expression, a delay that is significantly longer than that required for activation of ENaC in the same cell line (1 h). SGK1 and aldosterone stimulated the Na⁺ pump synergistically, indicating that the pathways mediated by these molecules operate independently. This observation was confirmed by demonstrating that aldosterone, but not SGK1^T_S425D, induced an 2.5-fold increase in total protein and plasma membrane Na⁺-K⁺-ATPase ₁-subunit abundance. We conclude that aldosterone increases the abundance of Na⁺-K⁺-ATPase, whereas SGK1 may activate existing pumps in the membrane in response to chronic or slowly acting stimuli.

sodium transport; serum- and glucocorticoid-induced kinase; A6 cells; sodium pump

In the present study, we examined the effects of SGK1 and aldosterone on Na⁺-K⁺-ATPase activity in A6 cells derived from the Xenopus distal tubule. A6 cells endogenously express the three ENaC subunits (26) as well as the ₁- and ₁-subunits of Na⁺-K⁺-ATPase (32) and are capable of regulated vectorial Na⁺ transport when grown on permeable supports. Furthermore, A6 cells are able to express SGK1 when stimulated with serum or steroid hormones, although kinase levels are negligible in serum-depleted cells (1). We used a tetracycline-inducible system to control the expression of a constitutively active mutant of SGK1 (SGK1^T_S425D) independently of any other stimuli.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Cell culture. We previously described the generation and characterization of an A6 cell line expressing a constitutively active mutant of SGK1, SGK1^T_S425D, under the control of a tetracycline-inducible promoter (1). Briefly, A6 cells were transfected with two plasmids: one constitutively expresses the tetracycline repressor (tetR) under a cytomegalovirus (CMV) promoter; the other contains the full-length SGK1^T_S425D open-reading frame under the control of a CMV promoter coupled to a tandem repeat of the tetracycline operator (tetO). Under normal conditions, tetR binds tetO and prevents SGK1^T_S425D expression. Upon tetracycline stimulation, tetR is released from tetO and the CMV promoter drives SGK1^T_S425D expression.

Cells were grown at 27°C in amphibian 0.75x DMEM (GIBCO-BRL) buffered with NaHCO₃ and supplemented with 10% FBS and antibiotics for plasmid selection (500 µg/ml zeocin and 10 µg/ml blasticidin; Invitrogen) in a humidified incubator containing 1.5% CO₂. Cells were expanded in plastic dishes and subcultured onto 96-well microtiter plates 2 days before the flux experiments were begun. When indicated, cells were subcultured onto 4.7-cm² permeable supports (Transwell; Corning, Corning, NY) for at least 10 days to allow for differentiation and development of transepithelial transport properties (4). One day before the experiments were started, cells were washed twice with serum-free amphibian DMEM and kept in the same medium for 24 h. Cells were then treated with 1 µg/ml tetracycline (Invitrogen) or 100 nM aldosterone (Sigma) for different time periods for each experiment as indicated in the text.

⁸⁶Rb⁺ uptake measurements. Na⁺-K⁺-ATPase activity was studied by measuring ouabain-sensitive ⁸⁶Rb⁺ influx into a confluent monolayer of A6 cells grown on 96-well microtiter plates. The flux experiments reported herein were performed in an automated 96-well plate flux machine (Fluxomatic; built by Dr. B. Forbush; Ref. 12). The basic flux solution contained (in mM) 96 NaCl, 2 RbCl, 1 CaCl₂, 1 MgCl₂, 1 Na₂HPO₄, 1 Na₂SO₄, and 5 Na⁺-HEPES, pH 7.4. After 20-min preincubation in basic flux medium, cells were transferred to basic medium with 2 µCi/ml ⁸⁶Rb⁺ for the time periods indicated for each experiment. Na⁺-K⁺-ATPase-mediated fluxes were calculated as the difference between ⁸⁶Rb⁺ uptake in the presence or absence of 100 µM ouabain. When indicated, 250 µM bumetanide was included in the flux medium. The flux was terminated by rinsing the cells three times with isotonic MgCl₂ (74 mM) supplemented with 100 µM ouabain and 250 µM bumetanide and then allowing the cells to dry. Cellular ⁸⁶Rb⁺ uptake was determined using PhosphorImager analysis (Molecular Dynamics) of the 96-well plates with ImageQuant software (12).

Membrane preparation and ouabain-sensitive ATPase activity assay. A6 cells grown to confluence on 10-cm-diameter dishes and incubated for 24 h in serum-free medium were treated overnight with 100 nM aldosterone or 1 µg/ml tetracycline. Membrane preparation and ouabain-sensitive ATPase activity assays were performed as previously described (23). All procedures were performed on ice unless otherwise indicated. Briefly, cells were disrupted by sonication in lysis buffer (250 mM sucrose, 20 mM Tris·HCl, 1 mM EDTA, protease inhibitors, pH 7.4) and spun at 3,000 g in a tabletop centrifuge. Supernatants were then combined with an equal volume of 1 M NaI, 5 mM MgCl₂, 20 mM EDTA, and 160 mM Tris·HCl, pH 8.3, and incubated for 10 min. Membranes were recovered by centrifugation at 100,000 g for 1 h, resuspended in 10 mM Tris·HCl and 1 mM EDTA, pH 7.4, and homogenized by sonication. The protein concentration in the membrane suspension was measured with the bicinchoninic acid (BCA) procedure (BCA kit; Pierce Biotechnology, Rockford, IL). ATPase assays were performed by measuring the release of P_i from ATP using a colorimetric assay as described previously (23). Before each assay, membranes were permeabilized by being incubated in 0.65 mg/ml deoxycholic acid, 2 mM EDTA, and 20 mM imidazole for 30 min. Assays were initiated by adding 100 µl of permeabilized membrane suspension to 500 µl of 1.2x assay buffer (final concentration, in mM: 110 NaCl, 20 KCl, 3 MgCl₂, 25 imidazole, and 3 ATP) in the absence or presence of 100 µM ouabain and transferring the tubes to a 37°C water bath. After the indicated incubation times, reactions were stopped with 1 ml of ice-cold stop solution (0.5 M HCl, 3% ascorbic acid, 0.5% ammonium molybdate, and 1% SDS). Tubes were incubated on ice for 10 min, and 1.5 ml of a solution containing 2% sodium meta-arsenite, 2% sodium citrate, and 2% acetic acid were added. After 10-min incubation at 37°C, the temperature of the tubes was adjusted to room temperature and absorbance was measured at 850 nm in a Varian Cary spectrophotometer. Each data point was calculated as the average value obtained from five replicate samples. Ouabain-sensitive ATPase activity is expressed as µmol P_i·mg of protein^–1·h^–1.

Cell surface protein biotinylation and Western blot analysis. Cell surface protein biotinylation was performed essentially as described previously (3). Briefly, after treatment, cells were washed with ice-cold PBS supplemented with 1 mM MgCl₂ and 100 µM CaCl₂ and incubated twice for 20 min with 1.5 mg/ml sulfosuccinimidyl-2-(biotinamido)-ethyl-1,3-dithiopropionate (sulfo-NHS-SS-biotin; Pierce Biotechnology) at 4°C. When cells were grown on filters, biotin was added only to the basolateral side. After biotinylation, free biotin was quenched with 150 mM glycine and cells were lysed in a 1% Triton X-100 buffer. The protein concentration in the cell lysates was measured using the BCA kit. Biotinylated proteins were recovered from 240 µg of total protein from each lysate using streptavidin beads (Pierce Biotechnology). After being washed, proteins were eluted with SDS-PAGE loading buffer. Samples were heated to 60°C for 10 min to avoid aggregation of Na⁺-K⁺-ATPase -subunits and then loaded onto SDS-PAGE gels. Samples containing 40 µg of protein from total cell lysates were analyzed on parallel gels. After performing electrophoresis and transferring the samples onto membranes, the Na⁺-K⁺-ATPase ₁-subunit was detected using ₅ MAb, which recognizes all -subunit isoforms in a broad range of species, including Xenopus laevis. The ₅ MAb was originally produced by Dr. D. Fambrough (20), obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the National Institute of Child Health and Human Development, and maintained by the Department of Biological Sciences, University of Iowa, Iowa City, IA. Anti-mouse IgG conjugated to peroxidase (Sigma) was used at a 1:10,000 dilution. Signals were developed using the ECL⁺ kit (Amersham) and detected using Biomax MR film (Kodak). After we detected the Na⁺-K⁺-ATPase -subunit, we stripped and probed the blots with anti-calnexin PAb (StressGen Biotechnologies), followed by anti-rabbit IgG conjugated to peroxidase (Sigma) at a 1:10,000 dilution. This control was performed to ensure that intracellular proteins were not biotinylated. The Western blot analysis results were quantified using a GS-800 scanning densitometer (Bio-Rad Laboratories, Hercules, CA) with QuantityOne software provided by the manufacturer. Data are expressed as arbitrary density values normalized to control conditions. Each experiment was performed in triplicate.

Statistical analysis. Data are expressed as means ± SE. Statistical analysis of ⁸⁶Rb⁺ uptake experiment results was performed using one-way ANOVA, followed by Dunnett's multiple-comparison test using Prism software (GraphPad, San Diego, CA). Analysis of cell surface biotinylation and ATPase activity data was performed using a nonpaired t-test.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
SGK1 effects on Na⁺-K⁺-ATPase transport activity in A6 cells. To investigate the effects of SGK1 on Na⁺-K⁺-ATPase activity, we used an inducible A6 cell line that expresses a constitutively active mutant of SGK1, SGK1^T_S425D, under the control of tetracycline (1). A6 cells express endogenous SGK1, but the levels of the kinase are negligible when cells are grown in serum-free medium (1). Na⁺-K⁺-ATPase transport activity in A6-SGK1^T_S425D cells was assayed as ouabain-sensitive ⁸⁶Rb⁺ uptake by the cells. Rb⁺ ions behave in the same manner as K⁺ ions and are transported from the extracellular medium into the cytosol by Na⁺-K⁺-ATPase. ⁸⁶Rb⁺ included in the flux medium initially accumulated in the cell until it reached a steady-state concentration. To ensure that the flux assay was conducted under linear uptake conditions, the time course of ⁸⁶Rb⁺ uptake was measured. ⁸⁶Rb⁺ accumulation was measured in the absence of inhibitors, in the presence of 100 µM ouabain, and with a combination of ouabain and 250 µM bumetanide (Fig. 1A). Ouabain-sensitive ⁸⁶Rb⁺ uptake accounted for 38% of total uptake, consistent with previously reported values in A6 cells (16). The remaining ⁸⁶Rb⁺ uptake was a result of the activity of the bumetanide-sensitive Na⁺-K⁺-2Cl^– cotransporter (NKCC) (16), as well as from K⁺ channels, which are abundantly expressed in the basolateral membrane of A6 cells (7, 13). Because ouabain-sensitive ⁸⁶Rb⁺ uptake was linear for at least 40 min (Fig. 1A), all subsequent experiments were performed with 20-min flux periods.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Ouabain-sensitive 86Rb+ uptake in A6 cells. A: A6-tetracycline-inducible promoter of constitutively active mutant of SGK1 (A6-SGK1TS425D) cells grown on 96-well plates were incubated with flux medium containing 2 µCi/ml 86Rb+ for the indicated time periods in the absence of inhibitors, in the presence of 100 µM ouabain, or in the presence of 100 µM ouabain and 250 µM bumetanide. After being washed 3 times, cells were air dried, exposed to a PhosphorImager screen, and quantified using ImageQuant software. Results are presented as the averages of 4 wells ± SE. 86Rb+ uptake was linear between time 0 and 40-min incubation in the absence or presence of inhibitors (R > 0.990). B: parental (nontransfected) A6 cells were treated with 1 µg/ml tetracycline (Tet+) for the indicated time periods. At the end of treatment, ouabain-sensitive 86Rb+ uptake was measured during a 20-min flux period. In parallel, ouabain-sensitive 86Rb+ uptake was measured in untreated cells (Tet–). Bars represent average values ± SE (n = 4 wells) normalized to control conditions (time 0). *P < 0.01 vs. control.

The effects of SGK1^T_S425D expression on Na⁺-K⁺-ATPase transport activity were studied at different times after induction with tetracycline. At the end of tetracycline treatment, cells were washed and then incubated for an additional 20 min in ⁸⁶Rb⁺ flux medium, air dried, and exposed to a PhosphorImager screen. Signals were then quantified, and average values were normalized to control (time 0). Expression of SGK1^T_S425D for 24 h increased Na⁺-K⁺-ATPase activity 2.5-fold compared with the time 0 control (Fig. 2). When normalized to the control time course shown in Fig. 1B, the increase was 3.3-fold. Shorter expression time of SGK1^T_S425D (6 h or less) did not significantly alter Na⁺-K⁺-ATPase activity.

View larger version (14K): [in this window] [in a new window]   Fig. 2. Effect of SGK1TS425D expression on ouabain-sensitive 86Rb+ uptake in A6 cells. Cells grown on 96-well plates were left untreated (time 0) or were treated with 1 µg/ml tetracycline for the indicated time periods before we performed the uptake assay. At the end of the experiment, cells were incubated with 86Rb+ in the presence or absence of 100 µM ouabain for 20 min. After being washed 3 times, cells were air dried and exposed to a PhosphorImager screen. Signals were quantified using ImageQuant software. Bars represent average values ± SE (n = 10 experiments) normalized to control conditions (time 0). *P < 0.01 vs. control.

View larger version (21K): [in this window] [in a new window]   Fig. 3. The effects of aldosterone and SGK1TS425D on ouabain-sensitive 86Rb+ uptake are synergistic. Cells grown on 96-well plates were left untreated (time 0) or were treated with 100 nM aldosterone, 1 µg/ml tetracycline, or a combination of aldosterone and tetracycline for the indicated time periods. A 20-min flux assay was performed to determine ouabain-sensitive 86Rb+ uptake. Bars represent average values ± SE (n = 10 experiments) normalized to control conditions (time 0). Black bars represent aldosterone treatment, dark gray bars represent tetracycline treatment, and light gray bars represent combined aldosterone and tetracycline treatment. *P < 0.01 vs. control (time 0).

View larger version (18K): [in this window] [in a new window]   Fig. 4. SGK1TS425D effects on Na+-K+-ATPase activity are direct and independent of intracellular Na+ concentration. A: cells grown on 96-well plates were treated with 1 µg/ml tetracycline for the indicated time periods. Cells were then washed and incubated in basic flux medium in the absence or presence of 2.5 µM monensin for 20 min. After incubation, cells were washed and ouabain-sensitive 86Rb+ uptake was assayed for a 20-min flux time period. Black bars represent average values (n = 6 experiments) ± SE of cells not treated with monensin. Gray bars represent average values (n = 2 experiments) ± SE of cells preincubated with 2.5 µM monensin before 86Rb+ uptake assay. *P < 0.05 vs. control. B: cells grown on 10-cm-diameter plastic dishes were treated overnight with 1 µg/ml tetracycline or 100 nM aldosterone. After treatment, a crude membrane preparation was obtained. Ouabain-sensitive Vmax ATPase activity was determined using 10 µg of proteins from a permeabilized membrane suspension incubated at 37°C for 30 min in reaction buffer containing 3 mM ATP. Pi released from ATP was quantified using a colorimetric assay. Bars represent average values (n = 5 replicate samples) ± SE of ouabain-sensitive ATPase activity. *P < 0.05 vs. control.

Aldosterone, but not SGK1, increases Na⁺-K⁺-ATPase expression and plasma membrane abundance. To further study the molecular mechanisms involved in Na⁺-K⁺-ATPase activation by SGK1, we examined the total and plasma membrane abundance of Na⁺-K⁺-ATPase -subunit in the absence or presence of SGK1^T_S425D. We compared SGK1 effects on protein abundance with the effects of aldosterone, which was previously shown to increase Na⁺-K⁺-ATPase ₁-subunit mRNA and protein abundance (21, 33). A6 cells grown on filters for at least 10 days were serum starved and treated overnight with 100 nM aldosterone or 1 µg/ml tetracycline. After treatments, plasma membrane proteins were biotinylated and recovered by streptavidin pull-down. Samples containing 40 µg of total protein from cell lysates and the products of streptavidin pull-down were analyzed in parallel using SDS-PAGE followed by Western blot analysis with anti-Na⁺-K⁺-ATPase -subunit antibody. Western blot analysis showed a single band migrating at 100 kDa, consistent with the predicted size of the ₁-subunit of Na⁺-K⁺-ATPase (Fig. 5A). A control sample containing proteins from nonbiotinylated A6 cells was included to check for nonspecific protein binding to the agarose-streptavidin beads. The ₁-subunit of Na⁺-K⁺-ATPase was detected in the total lysate but not in the streptavidin pull-down from nonbiotinylated cells (Fig. 5A), indicating that the signal obtained in the biotinylation experiments was specific for biotinylated proteins. A second control experiment was performed to check the possibility of intracellular biotinylation due to permeation of sulfo-NHS-SS-biotin. Blots were stripped and reprobed with anti-calnexin PAb (Fig. 5B). Calnexin is an abundant endoplasmic reticulum-resident membrane protein and should not be biotinylated by impermeant derivatives of biotin. The results show that although a strong calnexin signal was detected in the total protein lysate, only a faint signal was detected in the biotinylated samples (Fig. 5B), indicating that contamination caused by intracellular proteins was low in our experiments and did not interfere with data interpretation.

View larger version (51K): [in this window] [in a new window]   Fig. 5. Effect of aldosterone stimulation or SGK1TS425D expression on steady-state abundance of total and plasma membrane Na+-K+-ATPase 1-subunit. A: A6 cells were treated with 100 nM aldosterone or 1 µg/ml tetracycline for 24 h, followed by basolateral plasma membrane protein biotinylation. Aliquots of cell lysates containing 240 µg of protein were used for streptavidin pull-down to recover biotinylated proteins. Samples containing 40 µg of cell lysates (total protein) and the products of streptavidin-pull downs (plasma membrane) were run in parallel using SDS-PAGE and analyzed using Western blot analysis with an antibody against the Na+-K+-ATPase 1-subunit. A Western blot representative of an experiment performed in triplicate is shown. The experiment included a sample of cells without biotin to control for nonspecific binding of the Na+-K+-ATPase 1-subunit to the streptavidin-agarose beads. Migration of molecular mass markers (numbers at left indicate mass in kDa) is indicated by arrows. B: to control for specific biotinylation of plasma membrane proteins, Western blots of total protein and plasma membrane protein samples were stripped and reprobed using a PAb against calnexin, an abundant membrane protein resident in the endoplasmic reticulum. Only total protein samples showed significant signals. Migration of molecular mass markers (numbers at left indicate mass in kDa) is indicated by arrows. C: Na+-K+-ATPase 1-subunit signals were quantified using scanning densitometry and normalized to control conditions. Bars represent average ± SE of 3 independent experiments, each of which was performed in triplicate. Black bars indicate total protein, and gray bars indicate plasma membrane protein. Student's t-test was used to compare each condition with control values. *P < 0.05.

We also performed the same set of experiments in A6 cells grown on plastic culture dishes (data not shown). Aldosterone increased total and plasma membrane ₁-subunit abundance 2.3- and 2.2-fold, respectively, changes that are comparable to those detected in cells grown on filters. On the other hand, SGK1^T_S425D expression did not induce any changes in total or plasma membrane ₁-subunit abundance, confirming the results obtained in cells grown on filters.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The results presented herein demonstrate that SGK1 increases the activity of endogenously expressed Na⁺-K⁺-ATPase 2.5-fold in a Na⁺-transporting epithelial cell line (Fig. 2). The effect of SGK1 persisted after [Na⁺]_i was increased by monensin (Fig. 4A). Most important, ouabain-sensitive ATPase activity was higher in membranes isolated from cells expressing SGK1^T_S425D than in membranes from control cells (Fig. 4B). Together, these results indicate that increased [Na⁺]_i is not the means by which SGK1 affects Na⁺-K⁺-ATPase activity. Our results are in agreement with the observations of Setiawan et al. (27), who showed that SGK1 cRNA injected into Xenopus oocytes increased the activity of endogenous Na⁺-K⁺-ATPase. In contrast, Zecevic et al. (36) found that SGK1 activated only exogenous Na⁺ pumps formed by coinjection of rat ₁-subunits and Xenopus ₁-subunits, whereas endogenous pumps remained unaffected. Although the latter observation is difficult to explain, most of the results support the role of SGK1 in the regulation of endogenous Na⁺-K⁺-ATPase.

SGK1 and Na⁺-K⁺-ATPase are coexpressed in epithelial cell types other than the principal cells of the distal tubule (2). The thick ascending limb of Henle expresses both proteins together with the kidney-specific isoform of the Na⁺-K⁺-2Cl^– cotransporter (NKCC2), which localizes to the apical membrane. The activity of NKCC2 is also stimulated by SGK1 when the two proteins are coinjected into Xenopus oocytes (19). Unfortunately, we could not examine the effects of SGK1 on NKCC2 directly, because the A6 cell line does not express apical NKCC2 (Alvarez de la Rosa D, Gimenez I, Forbush B, and Canessa CM, unpublished observations). The presence of SGK1 in various types of Na⁺-transporting epithelial cells suggests that one important function of this kinase may be to coordinate the activity of the transporters that mediate entrance of Na⁺ in the apical membrane, such as ENaC and NKCC2, and the exit of Na⁺ in the basolateral membrane by the Na⁺-K⁺-ATPase.

The use of a tetracycline-inducible SGK1 expression system in an epithelial cell line that responds to aldosterone allowed us to test whether SGK1 mediates the early actions of aldosterone on transepithelial Na⁺ transport (25, 27). The response to aldosterone has been divided into two phases. During the early phase (i.e., the first 2 h), the increase in Na⁺ transport is thought to be mediated by the activation of preexisting ENaCs and Na⁺ pumps, whereas the late phase is mediated by the synthesis of new channels and transporters (30). Time course experiments (Fig. 2) indicated a delay of at least 6 h between SGK1^T_S425D expression and the increase in Na⁺-K⁺-ATPase activity. This late effect of SGK1 on Na⁺-K⁺-ATPase activity is in contrast to our previous observation of SGK1^T_S425D stimulating ENaC activity by 50% 1 h after SGK1^T_S425D induction in the same cell line (1). In light of these data, it is unlikely that SGK1 participates in rapid stimulation of Na⁺-K⁺-ATPase, although it could have an influence on the late phase of the aldosterone response. The latter possibility implies that SGK1 should reflect, at least partially, the effects of aldosterone. In contrast, the results indicate that the actions of aldosterone and SGK1 on the two key components of the transcellular pathway of Na⁺ transport, ENaC (1) and Na⁺-K⁺-ATPase, are additive, implying that they operate through independent mechanisms. Furthermore, cell surface biotinylation and Western blot analysis demonstrated that aldosterone increased expression and cell surface abundance of Na⁺-K⁺-ATPase, whereas SGK1 had no effect on protein abundance. Again, these results differ from those reported in Xenopus oocytes (36), in which SGK1 increased total and plasma membrane abundance of the injected Na⁺-K⁺-ATPase ₁-subunit but did not increase the activity or abundance of endogenous Na⁺-K⁺-ATPase (36). When oocytes were Na⁺ loaded via expression of ENaC, SGK1 still increased Na⁺-K⁺-ATPase abundance in the plasma membrane but did not have any effect on total protein expression (36). These results suggest that in oocytes, SGK1 could potentially enhance Na⁺-K⁺-ATPase ₁-subunit translation as well as trafficking to the plasma membrane. The different results obtained in A6 cells and Xenopus oocytes may be attributed to differences in the expression of proteins required for the regulation of the Na⁺-K⁺-ATPase by SGK1 and underscore the importance of using renal epithelial cells to study transport regulation.

Regarding the mechanism underlying the activation of Na⁺-K⁺-ATPase by SGK1, cell surface biotinylation and Western blot analysis demonstrated that SGK1 does not increase the synthesis or incorporation of new pumps into the plasma membrane. Instead, SGK1 increases the activity of pumps already present in the plasma membrane. Na⁺-K⁺-ATPase is the target of multiple regulatory mechanisms, including changes in substrate affinities, interaction with other proteins, and phosphorylation of specific residues (18, 29). The delay in the onset of SGK1 action suggests that the effect of the kinase is not direct but most likely occurs through the induction of new proteins. It has been demonstrated previously that SGK1 regulates transcriptional factors (8, 35), raising the possibility that SGK1 could increase the expression of proteins that modulate Na⁺-K⁺-ATPase activity. Our system, however, did not allow us to test this hypothesis, because the addition of actinomycin D or cycloheximide to block the synthesis of new proteins would have interfered with the expression of SGK1^T_S425D.

In summary, our results demonstrate that SGK1 expression increases Na⁺-K⁺-ATPase activity in renal epithelial cells independently of changes in protein expression or abundance in the plasma membrane. At least in A6 cells, SGK1 and aldosterone modulated Na⁺ pump activity through independent processes. The precise nature of these mechanisms is yet to be identified.

	GRANTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
This work was supported by National Heart, Lung, and Blood Institute Grant P05 HL-55007-01 (to C. M. Canessa), National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-17433 (to B. Forbush), and Fundación Canaria de Investigación y Salud Grant 88/04 (to D. Alvarez de la Rosa). D. Alvarez de la Rosa and I. Gimenez are fellows of the "Ramón y Cajal" program, Ministerio de Educación y Ciencia, Spain.

	ACKNOWLEDGMENTS

 
We are grateful to Dr. Joseph F. Hoffman and Dr. Ricardo Borges for critical reading of the manuscript and to Dr. Alessandra Zatti for helpful advice regarding ATPase assays.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. Alvarez de la Rosa, Unidad de Farmacología, Facultad de Medicina, Universidad de La Laguna, 38071 La Laguna, Tenerife, Spain (e-mail: diego.alvarez{at}ull.es)

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

	REFERENCES

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
1. Alvarez de la Rosa D and Canessa CM. Role of SGK in hormonal regulation of epithelial sodium channel in A6 cells. Am J Physiol Cell Physiol 284: C404–C414, 2003.[Abstract/Free Full Text]

2. Alvarez de la Rosa D, Coric T, Todorovic N, Shao D, Wang T, and Canessa CM. Distribution and regulation of expression of serum- and glucocorticoid-induced kinase-1 in the rat kidney. J Physiol 551: 455–466, 2003.[Abstract/Free Full Text]

3. Alvarez de la Rosa D, Li H, and Canessa CM. Effects of aldosterone on biosynthesis, traffic, and functional expression of epithelial sodium channels in A6 cells. J Gen Physiol 119: 427–442, 2002.[Abstract/Free Full Text]

4. Alvarez de la Rosa D, Punescu TG, Els WJ, Helman SI, and Canessa CM. Mechanisms of regulation of epithelial sodium channel by SGK1 in A6 cells. J Gen Physiol 124: 395–407, 2004.[Abstract/Free Full Text]

5. Alzamora R, Marusic ET, Gonzalez M, and Michea L. Nongenomic effect of aldosterone on Na⁺,K⁺-adenosine triphosphatase in arterial vessels. Endocrinology 144: 1266–1272, 2003.[Abstract/Free Full Text]

6. Bhargava A, Fullerton MJ, Myles K, Purdy TM, Funder JW, Pearce D, and Cole TJ. The serum- and glucocorticoid-induced kinase is a physiological mediator of aldosterone action. Endocrinology 142: 1587–1594, 2001.[Abstract/Free Full Text]

7. Broillet MC and Horisberger JD. Basolateral membrane potassium conductance of A6 cells. J Membr Biol 124: 1–12, 1991.[CrossRef][ISI][Medline]

8. Brunet A, Park J, Tran H, Hu LS, Hemmings BA, and Greenberg ME. Protein kinase SGK mediates survival signals by phosphorylating the Forkhead transcription factor FKHRL1 (FOXO3a). Mol Cell Biol 21: 952–965, 2001.[Abstract/Free Full Text]

9. Chen S, Bhargava A, Mastroberardino L, Meijer OC, Wang J, Buse P, Firestone GL, Verrey F, and Pearce D. Epithelial sodium channel regulated by aldosterone-induced protein sgk. Proc Natl Acad Sci USA 96: 2514–2519, 1999.[Abstract/Free Full Text]

10. Chigaev A, Lu G, Shi H, Asher C, Xu R, Latter H, Seger R, Garty H, and Reuveny E. In vitro phosphorylation of COOH termini of the epithelial Na⁺ channel and its effects on channel activity in Xenopus oocytes. Am J Physiol Renal Physiol 280: F1030–F1036, 2001.[Abstract/Free Full Text]

11. Coric T, Hernandez N, Alvarez de la Rosa D, Shao D, Wang T, and Canessa CM. Expression of ENaC and serum- and glucocorticoid-induced kinase 1 in the rat intestinal epithelium. Am J Physiol Gastrointest Liver Physiol 286: G663–G670, 2004.[Abstract/Free Full Text]

12. Darman RB and Forbush B. A regulatory locus of phosphorylation in the N terminus of the Na-K-Cl cotransporter, NKCC1. J Biol Chem 277: 37542–37550, 2002.[Abstract/Free Full Text]

13. De Smet P, Li J, and Van Driessche W. Hypotonicity activates a lanthanide-sensitive pathway for K⁺ release in A6 epithelia. Am J Physiol Cell Physiol 275: C189–C199, 1998.[Abstract/Free Full Text]

14. Efendiev R, Bertorello AM, Zandomeni R, Cinelli AR, and Pedemonte CH. Agonist-dependent regulation of renal Na⁺,K⁺-ATPase activity is modulated by intracellular sodium concentration. J Biol Chem 277: 11489–11496, 2002.[Abstract/Free Full Text]

15. Faletti CJ, Perrotti N, Taylor SI, and Blazer-Yost BL. sgk: an essential convergence point for peptide and steroid hormone regulation of ENaC-mediated Na⁺ transport. Am J Physiol Cell Physiol 282: C494–C500, 2002.[Abstract/Free Full Text]

16. Fan PY, Haas M, and Middleton JP. Identification of a regulated Na/K/Cl cotransport system in a distal nephron cell line. Biochim Biophys Acta 1111: 75–80, 1992.[Medline]

17. Farjah M, Roxas BP, Geenen DL, and Danziger RS. Dietary salt regulates renal SGK1 abundance: relevance to salt sensitivity in the Dahl rat. Hypertension 41: 874–878, 2003.[Abstract/Free Full Text]

18. Féraille E and Doucet A. Sodium-potassium-adenosinetriphosphatase-dependent sodium transport in the kidney: hormonal control. Physiol Rev 81: 345–418, 2001.[Abstract/Free Full Text]

19. Lang F, Klingel K, Wagner CA, Stegen C, Wärntges S, Friedrich B, Lanzendörfer M, Melzig J, Moschen I, Steuer S, Waldegger S, Sauter M, Paulmichl M, Gerke V, Risler T, Gamba G, Capasso G, Kandolf R, Hebert SC, Massry SG, and Bröer S. Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy. Proc Natl Acad Sci USA 97: 8157–8162, 2000.[Abstract/Free Full Text]

20. Lebovitz RM, Takeyasu K, and Fambrough DM. Molecular characterization and expression of the (Na⁺ + K⁺)-ATPase -subunit in Drosophila melanogaster. EMBO J 8: 193–202, 1989.[ISI][Medline]

21. Lebowitz J, An B, Edinger RS, Zeidel ML, and Johnson JP. Effect of altered Na⁺ entry on expression of apical and basolateral transport proteins in A6 epithelia. Am J Physiol Renal Physiol 285: F524–F531, 2003.[Abstract/Free Full Text]

22. May A, Puoti A, Gaeggeler HP, Horisberger JD, and Rossier BC. Early effect of aldosterone on the rate of synthesis of the epithelial sodium channel subunit in A6 renal cells. J Am Soc Nephrol 8: 1813–1822, 1997.[Abstract]

23. Mense M, Dunbar LA, Blostein R, and Caplan MJ. Residues of the fourth transmembrane segments of the Na,K-ATPase and the gastric H,K-ATPase contribute to cation selectivity. J Biol Chem 275: 1749–1756, 2000.[Abstract/Free Full Text]

24. Náray-Fejes-Tóth A, Canessa C, Cleaveland ES, Aldrich G, and Fejes-Tóth G. sgk is an aldosterone-induced kinase in the renal collecting duct: effects on epithelial Na⁺ channels. J Biol Chem 274: 16973–16978, 1999.[Abstract/Free Full Text]

25. Pearce D. The role of SGK1 in hormone-regulated sodium transport. Trends Endocrinol Metab 12: 341–347, 2001.[CrossRef][ISI][Medline]

26. Puoti A, May A, Canessa CM, Horisberger JD, Schild L, and Rossier BC. The highly selective low-conductance epithelial Na channel of Xenopus laevis A6 kidney cells. Am J Physiol Cell Physiol 269: C188–C197, 1995.[Abstract/Free Full Text]

27. Setiawan I, Henke G, Feng Y, Böhmer C, Vasilets LA, Schwarz W, and Lang F. Stimulation of Xenopus oocyte Na⁺,K⁺ATPase by the serum and glucocorticoid-dependent kinase sgk1. Pflügers Arch 444: 426–431, 2002.[CrossRef][ISI][Medline]

28. Stockand JD. New ideas about aldosterone signaling in epithelia. Am J Physiol Renal Physiol 282: F559–F576, 2002.[Abstract/Free Full Text]

29. Therien AG and Blostein R. Mechanisms of sodium pump regulation. Am J Physiol Cell Physiol 279: C541–C566, 2000.[Abstract/Free Full Text]

30. Verrey F. Early aldosterone action: toward filling the gap between transcription and transport. Am J Physiol Renal Physiol 277: F319–F327, 1999.[Abstract/Free Full Text]

31. Verrey F. Sodium reabsorption in aldosterone-sensitive distal nephron: news and contributions from genetically engineered animals. Curr Opin Nephrol Hypertens 10: 39–47, 2001.[ISI][Medline]

32. Verrey F, Kairouz P, Schaerer E, Fuentes P, Geering K, Rossier BC, and Kraehenbuhl JP. Primary sequence of Xenopus laevis Na⁺-K⁺-ATPase and its localization in A6 kidney cells. Am J Physiol Renal Fluid Electrolyte Physiol 256: F1034–F1043, 1989.[Abstract/Free Full Text]

33. Verrey F, Schaerer E, Zoerkler P, Paccolat MP, Geering K, Kraehenbuhl JP, and Rossier BC. Regulation by aldosterone of Na⁺,K⁺-ATPase mRNAs, protein synthesis, and sodium transport in cultured kidney cells. J Cell Biol 104: 1231–1237, 1987.[Abstract/Free Full Text]

34. Wulff P, Vallon V, Huang DY, Völkl H, Yu F, Richter K, Jansen M, Schlünz M, Klingel K, Loffing J, Kauselmann G, Bösl MR, Lang F, and Kuhl D. Impaired renal Na⁺ retention in the sgk1-knockout mouse. J Clin Invest 110: 1263–1268, 2002.[CrossRef][ISI][Medline]

35. You H, Jang YJ, You-Ten AI, Okada H, Liepa J, Wakeham A, Zaugg K, and Mak TW. p53-dependent inhibition of FKHRL1 in response to DNA damage through protein kinase SGK1. Proc Natl Acad Sci USA 101: 14057–14062, 2004.[Abstract/Free Full Text]

36. Zecevic M, Heitzmann D, Camargo SM, and Verrey F. SGK1 increases Na,K-ATP cell-surface expression and function in Xenopus laevis oocytes. Pflügers Arch 448: 29–35, 2004.[CrossRef][ISI][Medline]

This Article Abstract Full Text (PDF) All Versions of this Article: 290/2/C492    most recent 00556.2004v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Alvarez de la Rosa, D. Articles by Canessa, C. M. Search for Related Content PubMed PubMed Citation Articles by Alvarez de la Rosa, D. Articles by Canessa, C. M.