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Am J Physiol Renal Physiol 293: F1751-F1757, 2007. First published August 8, 2007; doi:10.1152/ajprenal.00091.2007
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Downregulation of SGK1 by nucleotides in renal tubular epithelial cells

Liuzhe Li, Charles S. Wingo, and Shen-Ling Xia

North Florida/South Georgia Veterans Health System and Department of Medicine, University of Florida College of Medicine, Gainesville, Florida

Submitted 22 February 2007 ; accepted in final form 5 August 2007

ABSTRACT

This study determined whether nucleotides that bind to purinergic receptors (P2R) regulate the expression or function of serum- and glucocorticoid-inducible kinase-1 (SGK1) in mouse renal inner medullar collecting duct cells (mIMCD-3). The SGK1 protein was detected by Western blotting. A significant reduction of cytosolic SGK1 expression was observed in the cells pretreated with P2R agonist adenosine 5'-O-(3-thiotriphosphate) (ATP{gamma}S), and the reduction could be reversed by P2R antagonists. This reduction was also observed in cells that were pretreated with agonists for P2R subtypes. Using ELISA, we observed a reduced SGK1 kinase activity in ATP{gamma}S-pretreated cells. This effect was reversed by P2R antagonists. Furthermore, an increase of SGK1 kinase activity in aldosterone-pretreated cells was suppressed by ATP{gamma}S. These studies demonstrate for the first time that SGK1 can be downregulated by nucleotides in renal collecting duct epithelial cells, likely via the activation of P2R, and suggest that activation of renal purinergic signaling regulates a SGK1-dependent pathway that is known to modulate ion transport in the renal collecting duct.

purinergic signaling; phosphatidylinositol 4,5-bisphosphate; epithelial sodium channels; mineralocorticoid receptor; renal ion transport; serum- and glucocorticoid-inducible kinase-1

THE SERUM- AND GLUCOCORTICOID-INDUCIBLE KINASE-1 (SGK1) is an important serine-threonine kinase and plays a critical role in the regulation of renal function (10, 11, 26). It is a well-established early response gene to the action of aldosterone, which in turn increases apical membrane expression of epithelial Na+ channel (ENaC) and Na+ absorption in the renal collecting duct cells (4, 14, 27).

A recent in vivo study in the rat demonstrated that activation of purinergic receptors (P2R) by adenosine 5'-O-(3-thiotriphosphate) (ATP{gamma}S) inhibits Na+ reabsorption in the distal nephron and collecting duct (24). This observation is consistent with previous in vitro results showing that ATP inhibits amiloride-sensitive short-circuit current and amiloride-sensitive Na+ channel activity via P2R (12, 15, 17). A recent in vitro study in Xenopus oocyte also has demonstrated that ENaC current can be inhibited by activation of P2R subtype receptors (29). Although a growing body of evidence indicates that extracellular ATP can regulate renal epithelial Na+ transport, the cellular mechanisms involved are largely unknown.

The renal inner medullar collecting duct (IMCD) is the final regulatory segment for water and ion transport that plays a critical role in body salt and volume homeostasis. The mouse cell line mIMCD-3 has been widely used as a model to study renal transport physiology, because it retains many cellular characteristics of the intact mouse IMCD (21). Previous studies by our group (3, 30, 31) have shown that these cells express SGK1 message and have both P2R subtypes, P2X receptor (a ligand-gated ion channel) and P2Y receptor (a G protein-coupled receptor, GPCR). Therefore, the studies were performed to evaluate whether extracellular nucleotides regulate the expression or function of SGK1 in mIMCD-3 cells.

MATERIALS AND METHODS

Chemicals.

Polyclonal antibody for SGK1 was purchased from Cell Signaling Technology (Beverly, MA); polyclonal antibodies for phosphorylated SGK1 (p-SGK1) at serine residue (Ser422) and at threonine residue (Thr256) and GAPDH were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ATP{gamma}S, 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), {alpha},beta-methylene-ATP ({alpha},beta-MeATP), 2-methylthio-ATP (2-MeSATP), and uridine triphosphate (UTP), suramin, pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS), adenosine, and D-aldosterone were purchased from Sigma-Aldrich (St. Louis, MO). DMEM/F12 medium, phenol red-free DMEM-F-12, and charcoal/dextran-stripped fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Regular FBS was purchased from HyClone Laboratories (Logan, UT).

Cell culture.

The mIMCD-3 cell line was obtained from American Type Culture Collection (Manassas, VA). Cells were incubated in DMEM/F12 medium containing 5% FBS at 37°C with 95% O2-5% CO2. Cells of 15–20 passages in a confluent monolayer were used. For the experiments with aldosterone, cells were first grown to confluent monolayers in normal culture medium and then switched to phenol red-free DMEM-F-12 plus 5% charcoal/dextran-stripped FBS that contained 1 µM aldosterone. After 20 h of incubation, the aldosterone-containing medium was replaced by the same fresh medium for another 30 min, with or without selected P2R agonist and antagonist.

Protein isolation.

For the isolation of total cell lysates, cells were washed twice with ice-cold PBS and then in the presence of a buffer solution containing 10 mM sodium orthovanadate, 1% SDS, and 10 mM Tris·HCl (pH 7.5). Afterward, cells were sonicated for 15 s and spun at ~15,000 g for 5 min. The supernatant was collected into a clean tube for Western blotting. For the isolation of the cytosolic fraction and membrane fraction, cells were washed twice with ice-cold PBS and scraped into 0.9 ml of cold PBS containing protease inhibitor cocktail. Afterward, cells were sonicated for 15 s and spun at ~500 g for 15 min at 4°C. The nuclei and unbroken cells were discarded, and the supernatant was further spun at ~148,000 g for 1 h at 4°C. The supernatant and pellet, for the respective cytosolic and membrane fractions, were transferred into clean tubes and prepared for Western blotting.

Western blotting.

The actual amount of protein sample from each testing group was measured via a fluorescence plate reader (FL 600; Bio-Tek Instruments, Winooski, VT), and the protein concentration was normalized. Equal amounts of protein were boiled in the presence of loading buffers and then separated on 10% SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was incubated in the blocking solution containing 5% nonfat milk and then in the Tris-buffered saline containing the primary antibody, SGK1 (1:1,000), p-SGK1 (Ser422) (1:500), p-SGK1 (Thr256) (1:500), or GAPDH (1:800), at room temperature for 2 h. After the membrane was washed three times in Tris-buffered saline containing 0.2% Tween 20, it was incubated in the antibody solution containing the horseradish peroxidase-conjugated secondary antibody. Enhanced chemiluminescent reagents were used to detect the antibody-bound proteins on the membrane according to the manufacturer's instructions (Amersham Biosciences).

SGK kinase activity assay.

A nonradioactive enzyme-linked immunosorbent assay (ELISA) kit for SGK1 kinase activity was purchased from Stressgen Bioreagents (EKS-430A; Ann Arbor, MI) and used to screen the activity of SGK1. The amount of protein sample from each testing group was measured, and the concentration was normalized. Equal amounts of cytosolic protein samples from control cells and from different pretreatment groups of cells were added to the appropriate wells of a microtiter plate that were precoated with the substrate of SGK1. The assay procedure was performed according to the manufacturer's instructions. The absorbance of the color from each well was measured with an absorbance detector (AD 340S; Beckman Coulter) at a wavelength of 450 nm. The assay was run at least in triplicate. The subtraction of the background optical density (OD) reading was applied to all the sample OD readings. The kinase activity is expressed as the ratio of OD reading from each sample and the OD reading from 16 ng of purified active recombinant SGK kinase (provided in the assay kit).

Statistical analysis.

Gel digitizing software UN-SCAN-IT 6.0.3 (Silk Scientific, Orem, UT) was used for the analysis of all immunoblots. The total pixel intensities with four-corner background correction were exported into Origin 6.0 (Microcal Software, Northampton, MA) for statistical analysis. Densitometric quantification of SGK1 bands was reported as a percentage of the density value versus its loading control of GAPDH density value for each treatment group and then normalized to that for the control group. The results are expressed as means ± SE. The data from each treatment group were compared with the data from the control group by using an unpaired Student's t-test. When data were compared among the treatment groups and the control group, a one-way ANOVA was performed with three groups or more before a t-test was performed. Differences were considered statistically significant if P < 0.05.

RESULTS

SGK1 protein is expressed in mIMCD-3 cells and is downregulated in cells treated with ATP{gamma}S. SGK1 gene expression was previously demonstrated in the mIMCD-3 cell line by Northern analysis (3). We first investigated the protein expression of SGK1 with Western blotting. Using an SGK1 polyclonal antibody, we detected a single immunoband at ~55 kDa from the total cell lysates, total membrane, and cytosolic fraction, respectively (Fig. 1, A–C). The expression of SGK1 immunoband in cells pretreated with ATP{gamma}S (a more stable analog of ATP, at 100 µM for 30 min; same treatment throughout the study) was slightly reduced in total cell lysates (–4%; P = 0.03; n = 3), but there was a large reduction of SGK1 immunoreactivity in the total membrane (–40%; P = 0.004; n = 3) and in the cytosol (–60%; P = 0.007; n = 3) (Fig. 1E).


Figure 1
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  		Fig. 1. Reduction of serum- and glucocorticoid-inducible kinase-1 (SGK1) protein in adenosine 5'-O-(3-thiotriphosphate) (ATP{gamma}S)-treated mouse renal inner medullar collecting duct (mIMCD-3) cells. Expression of SGK1 was examined in cells with and without ATP{gamma}S pretreatment by Western blot analysis (C, control; T, treatment). Antibody against SGK1 was used in total lysate (A), total membrane (B), and cytosol (C). Antibody against phosphorylated SGK1 (p-SGK1) was also used in cytosol (D). An equal amount of protein (20 µg) was loaded per well in triplicate. GAPDH was used as a control to show that equal amounts of protein were loaded per well. E: semiquantitative analysis of the immunoblots by densitometry. Asterisk indicates p-SGK1 in the cytosol.

 
We used p-SGK1 polyclonal antibodies to further examine the protein expression, because previous studies indicated that Thr256 and Ser422 of SGK1 are involved in the regulation of the kinase activity (5, 11, 19). Figure 1D shows that in cells pretreated with ATP{gamma}S, cytosolic p-SGK1 (Ser422) was decreased significantly compared with the control cells (–49%; P = 0.02; n = 4). A similar percentage of reduction was obtained with p-SGK1 at Ser422 compared with the reduction of total cytosolic SGK1 (see Fig. 1E), indicating that both the inactive and active forms of the kinase can be affected by ATP{gamma}S. However, we were unable to detect an appreciable immunoband with p-SGK1 (Thr256) in the cytosolic fractions of either control cells or cells pretreated by ATP{gamma}S (data not shown).

ATP{gamma}S-induced downregulation of cytosolic SGK1 protein is reversed by P2R antagonists. To verify that the downregulation of SGK1 by ATP{gamma}S was mediated by the activation of P2R, we applied suramin and PPNDS, antagonists of P2R, to cells 10 min before ATP{gamma}S treatment (all at 100 µM). The reduction of the cytosolic SGK1 protein by ATP{gamma}S in those cells was partially reversed (Fig. 2; ATP{gamma}S + suramin and ATP{gamma}S + PPNDS compared with ATP{gamma}S only, P < 0.002 and P < 0.004, respectively; compared with control, P = 0.05 and P = 0.04, respectively; n = 3 in each group). Suramin and PPNDS themselves had no significant effect on the expression of cytosolic SGK1 protein (P = 0.3 and P = 0.7, respectively; n = 3 in each group). Together, these data suggest that the ATP{gamma}S-induced decrease in cytosolic SGK1 protein was mediated by P2R.


Figure 2
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  		Fig. 2. ATP{gamma}S-induced reduction of cytosolic SGK1 protein is not observed in the presence of purinergic receptor (P2R) antagonists. A: triplicate samples of the cell cytosol in equal amounts (25 µg) were loaded from cells without treatment (control) and from cells pretreated (100 µM for 30 min) with ATP{gamma}S, ATP{gamma}S + suramin, ATP{gamma}S + pyridoxal-5'-phosphate-6-(2'-naphthylazo-6'-nitro-4',8'-disulfonate) (PPNDS), suramin only, and PPNDS only. B: semiquantitative analysis of the immunoblots by densitometry. NS, no significant difference from the control.

 
Cytosolic SGK1 protein is downregulated by other nucleotides. To further test the involvement of activation of the P2R subtypes P2X and P2Y in the downregulation of SGK1 expression, we incubated cells with the nucleotides BzATP, {alpha},beta-MeATP, 2-MeSATP, and UTP individually (100 µM; 30 min). BzATP, {alpha},beta-MeATP, and UTP caused a decrease in the expression of cytosolic SGK1 (Fig. 3A), indicating that both P2X and P2Y receptor subtypes were involved in the regulation of SGK1. The functional potencies of nucleotides in reducing cytosolic SGK1 are in the order of ATP{gamma}S (–55%; P < 0.02) > UTP (–50%; P < 0.02) > BzATP (–32%; P < 0.05) > {alpha},beta-MeATP (–19%; P = 0.07) (Fig. 3B). However, 2-MeSATP did not decrease the expression of SGK1. It is possible that either the cells lacked specific 2-MeSATP-sensitive P2R subtypes or 2-MeSATP-activated P2R subtypes were not involved in the downregulation of SGK1.


Figure 3
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  		Fig. 3. Cytosolic SGK1 expression can be altered by other nucleotides. A: a typical Western blot shows that samples in equal amounts (25 µg) were loaded from cells without treatment (control) and from cells pretreated (100 µM for 30 min) with ATP{gamma}S, 3'-O-(4-benzoyl)benzoyl-ATP (BzATP), {alpha},beta-methylene-ATP ({alpha},beta-MeATP), 2-methylthio-ATP (2-MeSATP), and uridine triphosphate (UTP). B: the band intensities of SGK1 were examined by densitometry. The average SGK1 data were taken from 3 individual experiments. C: a typical Western blot with equal amounts (25 µg) of cell cytosol loaded from cells treated without (control) or with ATP{gamma}S for 5, 10, or 30 min and with adenosine for 30 min. Ade, adenosine. D: the band intensities of SGK1 were examined by densitometry. SGK1 data averages were taken from 3 individual experiments.

 
In a previous study by our group (30) using this cell line, the P1 receptor (P1R) agonist adenosine could induce a small increase in the intracellular Ca2+ concentration. Since activation of P1R has been shown to inhibit ENaC (28), we examined whether activation of P1R could affect cytosolic SGK. In cells that were pretreated with the P1R agonist adenosine (100 µM; 30 min), a small reduction of cytosolic SGK was observed via Western blotting analysis (–10%; P = 0.04; n = 3) (Fig. 3, C and D). Since the reduction level of cytosolic SGK was greater in the activation of P2R than in the activation of P1R (45–60 vs. 10%), we postulate that the downregulation of SGK1 by P2R agonists in our study was mainly mediated by P2R.

We also looked at the effects of ATP{gamma}S-treated cells after 5- and 10-min treatment periods. The reduction of cytosolic SGK protein expression was seen only in the 5-min treatment group (–12%; P = 0.002; n = 3) (Fig. 3, C and D). We were unable to detect changes of SGK1 with 10-min ATP{gamma}S treatment under similar experimental settings.

SGK1 kinase activity in the cell cytosol is reduced by ATP{gamma}S. The Western blotting data indicate that activation of purinergic receptors affects the cytosolic SGK1 protein. To evaluate whether the function of SGK1 was also affected by activation of P2R in the cytosol, we next examined SGK1 kinase activity via ELISA analysis. Figure 4A shows that the basal level of SGK1 kinase activity was decreased significantly in cells pretreated with ATP{gamma}S (100 µM; 30 min) (ATP{gamma}S, 1.00 ± 0.04 vs. control, 1.20 ± 0.03; P < 0.03; n = 3).


Figure 4
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  		Fig. 4. ELISA analysis of downregulation of cytosolic SGK1 kinase activity by ATP{gamma}S in mIMCD-3 cells. A: equal amounts of cytosolic protein (25 µg) were loaded per well in triplicate, from control cells and from cells pretreated with ATP{gamma}S and/or P2R antagonist (100 µM for 30 min). B: in a separate experiment, equal amounts of cytosolic protein (50 µg) were loaded per well in quadruplicate, from control cells and from cells pretreated with ATP{gamma}S (100 µM), vehicle (ethanol only; control for aldosterone), aldosterone (1 µM), and aldosterone + ATP{gamma}S. The relative SGK activity is expressed as the ratio of the optical density (OD) reading of a sample to the OD reading of a given amount (16 ng) of purified active SGK kinase.

 
The specificity of the inhibition via activation of P2R was further confirmed by experiments in which cells were treated with P2R antagonists before ATP{gamma}S application. ATP{gamma}S- inhibited SGK1 kinase activity was reversed by the presence of either suramin or PPNDS (all at 100 µM) (Fig. 4A, ATP{gamma}S + suramin, 1.19 ± 0.12 and ATP{gamma}S + PPNDS, 1.19 ± 0.06 vs. control, 1.20± 0.03; not significantly different; n = 3 in each group).

Since it is well established that aldosterone upregulates SGK1 protein in the renal cells (3, 4, 10, 12, 14), we used aldosterone as a positive control in the ELISA analysis. In cells pretreated with aldosterone (1 µM; 20 h), a significant increase of SGK1 kinase activity was detected (Fig. 4B, aldosterone, 1.63 ± 0.07 vs. control, 1.29 ± 0.06; P < 0.02; n = 4). However, this increase was suppressed completely in cells that were also pretreated with ATP{gamma}S (Fig. 4B, aldosterone + ATP{gamma}S, 1.20 ± 0.04 vs. control, 1.29± 0.06; not significantly different; n = 4). Together, these data suggest that functional SGK kinase can be downregulated by activation of P2R.

DISCUSSION

In the current study, we demonstrated for the first time to our knowledge that SGK1 could be downregulated by nucleotides in renal collecting duct epithelial cells (mIMCD-3), likely via the activation of P2R. Our findings show that 1) in ATP{gamma}S-pretreated cells, the cytosolic fraction of SGK1 protein was reduced between 45 and 60% (Figs. 1–3); 2) the reduction of cytosolic SGK1 protein by ATP{gamma}S was not observed in the presence of P2R antagonists (Fig. 2); 3) activation of P2X and P2Y receptors by other nucleotides could reduce the expression of cytosolic SGK1 ~20–50% (Fig. 3A); activation of P1R by adenosine could reduce the expression of cytosolic SGK1 ~10% (Fig. 3C); 4) the basal level of cytosolic SGK1 kinase activity was decreased significantly in ATP{gamma}S-pretreated cells but not in those treated with P2R antagonists (Fig. 4A); and 5) aldosterone-stimulated increases in cytosolic SGK1 kinase activity could be suppressed by ATP{gamma}S (Fig. 4B).

Mechanism of downregulation of SGK1 by nucleotides. SGK1 is activated by phosphorylation at both the transcriptional and posttranslational levels by many extracellular signals (11). SGK1 engages in the regulation of renal function, including Na+ retention and K+ elimination of the kidney, thereby contributing to body salt homeostasis and blood pressure (10, 26). Although the exact signaling cascade of activation of P2R-mediated downregulation of SGK1 remains to be explored, GPCR (i.e., the P2Y receptor)-mediated downstream components of the phosphatidylinositol pathway may be involved, and this conceivably permits modulation of ion transport activity.

Activation of GPCR stimulates a phospholipase C (PLC)-mediated signaling cascade that hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 is a precursor not only of inositol 1,4,5-trisphosphate (IP3; which triggers the release of Ca2+ from the endoplasmic reticulum) and diacylglycerol (which activates protein kinase C, PKC), but also of phosphatidylinositol 3,4,5-trisphosphate (PIP3), which is produced by the action of phosphatidylinositol (PI) 3-kinase (22). Phosphorylation of SGK1 occurs through a cascade by PIP2- and PIP3-dependent protein kinases (PDK) (5, 10, 19). Therefore, activation of P2Y receptors by ATP{gamma}S in our cell model could stimulate PLC and enhance degradation of PIP2 by increasing the hydrolysis of membrane PIP2, which could downregulate the activity of SGK1. In addition, reduced conversion of PIP2 to PIP3 might negatively affect PDK and downstream SGK1, as well.

Functional role of P2X receptors in downregulation of SGK1. In the present study, ATP{gamma}S-induced downregulation of SGK1 was partially reversed by PPNDS (a potent antagonist for P2X1) and suramin (a potent antagonist for P2X1–3, P2X5, P2Y2, P2Y6, and P2Y11) (9, 18). In addition, BzATP (a potent agonist for P2X1, P2X7, and P2Y11), {alpha},beta-MeATP (a potent agonist for P2X1 and P2X3), and UTP (a potent agonist for P2Y2 and P2Y4) reduced the level of cytosolic SGK1 protein to a lesser extent. Although the specific P2R subtypes remain to be determined, the pharmacological profile of P2R agonists and antagonists suggests that both P2X and P2Y receptor subtypes are involved in the SGK1 regulation.

If the role of P2Y receptors is to interact with intracellular G protein-mediated PLC and to increase plasma membrane PIP2 hydrolysis (i.e., upstream control of SGK1 and ENaC), then it may be that the role of P2X receptors is to facilitate the action of P2Y receptors. This conjecture is supported by the following: 1) plasma membrane PLC is Ca2+ dependent (actually all 4 subtypes of PLC require Ca2+ for catalytic function) (22), and activation of P2X receptors could provide a local influx of Ca2+ near the plasma membrane to enhance the activity of PLC; 2) activation of P2Y receptors may require an activation of P2X receptors concurrently, since a previous study by our group (30) demonstrated that ATP, an agonist for both P2X and P2Y receptors, failed to induce a significant increase in intracellular Ca2+ signal in the presence of P2X receptor antagonists, indicating that P2X receptor-mediated signaling pathways could interact with P2Y receptor-mediated and PLC-dependent signaling pathways.

Physiological significance of downregulation of SGK1 by nucleotides. Previous studies in A6 epithelial cells by Ma et al. (15) showed that activation of P2R by ATP{gamma}S in the isolated membrane patch via patch-clamp pipette blocks ENaC activity in the cell-attached patch, and the authors proposed the involvement of PLC-mediated phosphatidylinositol pathway. In their follow-up work using the excised inside-out patch technique, Ma et al. (16) further demonstrated that increased PIP2 hydrolysis accelerated ENaC activity rundown. This mechanism has been confirmed by other investigators (for review, see Ref. 20) and is consistent with our findings, since our data showed that activation of both P2Y and P2X receptors by nucleotides reduced the expression of cytosolic SGK1 significantly. Although we did not perform a direct study to access ENaC activity by ATP{gamma}S, it is conceivable that as a consequence of reduction of SGK1 in the cell, a downregulation of ENaC activity might be expected (4, 10, 14, 27).

Although inhibition of amiloride-sensitive Na+ transport by nucleotides has been demonstrated in many epithelial cells, including those in the kidney and lung (13), the various mechanisms remain controversial. For example, an earlier study (6) showed that inhibition of benzamil-sensitive Na+ current by activation of P2Y receptors in rabbit collecting duct cells requires PKC activity but does not require changes in intracellular Ca2+. Another study with mouse cortical collecting duct (12) seems to support this mechanism, since the inhibition of amiloride-sensitive current was mediated by activation of P2Y receptors and the inhibition occurred independently of an increase of intracellular Ca2+. In contrast, experiments in mouse IMCD cells (17) demonstrated that inhibition of amiloride-sensitive Na+ current can occur through activation of both P2X and P2Y receptors. A more recent report (7) suggested that neither an increase in intracellular Ca2+ nor an activation of PKC was responsible for the inhibition of ENaC by activation of P2Y receptors in mouse trachea. Thus other mechanisms different from changes in intracellular Ca2+ or the activation of PKC appear to modulate ENaC activity (8, 15, 16). Our data showing downregulation of SGK1 by nucleotides provide one plausible explanation for the observed inhibition of amiloride-sensitive Na+ transport in the above studies.

Physiological significance of downregulation of aldosterone-stimulated SGK1 by ATP{gamma}S. The action of aldosterone has been shown to control Na+ reabsorption in the distal nephron through regulation of ENaC activity, and this regulation is associated with SGK genes (3, 4, 11, 27). Our data demonstrate that aldosterone-stimulated SGK1 kinase activity could be suppressed by the activation of P2R. There is a growing appreciation for nontraditional regulatory mechanisms that regulate Na+ transport in the collecting duct. The renal tubular purinergic signaling is emerging as one such potential mechanism (13, 23, 25). Although the interaction between nucleotides and aldosterone on the regulation of SGK1 function requires further investigation, the current study supports the hypothesis that renal tubular purinergic signaling provides a "yin" mechanism to potentially inhibit Na+ transport, at least in part, via downregulation of SGK1 expression, in contrast to other "yang" mechanisms to upregulate Na+ transport in the collecting duct. Therefore, the current results suggest that P2R agonists have the therapeutic potential to treat hypertension especially stimulated by mineralocorticoid.

Redistribution of cellular SGK1 by ATP{gamma}S. SGK1 is found in the cell membrane and cytosol and has a rapid turnover rate with a half-life of ~30 min (1, 10). SGK1 protein in the total cell lysates was reduced only 4% compared with the reduced levels in the membrane (40%) and in the cytosol (up to 65%) in ATP{gamma}S-treated cells (Fig. 1, A vs. B and C), suggesting that translocation of SGK1 occurs. It is possible that the downregulation of SGK1 by nucleotides involves the redistribution of SGK1 from the membrane and cytosol to the nucleus. This speculation is supported by previous studies (2, 10, 19). For instance, the subcellular translocalization of SGK1 in mammary epithelial cells has been observed from cytoplasmic and perinuclear regions to the nucleus after stimulation with serum (2).

Downregulation of both active and nonactive forms of SGK1 by ATP{gamma}S. By using p-SGK1 antibodies against specific phosphorylation sites, we observed the reduction of cytosolic phosphorylated form of SGK1 at Ser422 in ATP{gamma}S-pretreated cells. Furthermore, the proportion of the reduction of p-SGK1 at Ser422 is nearly the same as the reduction in total SGK (~50 vs. 45–60%; Fig. 1E vs. Figs. 2B and 3B). It is plausible that activation of P2R by ATP{gamma}S downregulates both phosphorylated and nonphosphorylated forms of SGK1. Both Ser422 and Thr256 of SGK1 are considered as phosphorylation sites involved in the regulation of functional SGK kinase activity (10), because mutations of these two sites cause significant decreases in the SGK1 kinase activity (19). Therefore, the observed reduction of p-SGK1 at Ser422 by ATP{gamma}S should decrease functional cytosolic SGK1 activity and consequently contribute to reduction of ENaC activity. We could not detect an appreciable immunoband with p-SGK1 at Thr256 in the cytosolic fractions from control cells and from cells pretreated by ATP{gamma}S, and thus we were unable to determine a difference between treated and untreated cells. It is possible that either p-SGK1 (Thr256) antibody is not working well or SGK1 is not phosphorylated at the Thr256 position in our experimental conditions.

In summary, the present study has shown that extracellular nucleotides can downregulate the expression of cytosolic SGK1 protein and its activity in the renal epithelial cells. Given aldosterone's known effect to stimulate ENaC activity, our data suggest that activation of purinergic signaling provides an additional mechanism for control of Na+ transport through downregulation of SGK1 in the renal collecting duct.

GRANTS

This work was supported in part by funds from the Department of Veterans Affairs, National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-49750, and the Division of Nephrology, Hypertension and Renal Transplantation, University of Florida.

ACKNOWLEDGMENTS

We thank Drs. Jill Verlander and Brian Cain for helpful discussions and Dr. Ruth Schwalbe for constructive suggestions during the writing of this article. We also thank Alicia Rudin, Armin Shivazad, Jeanette Lynch, and Melanie Cash for providing technical assistance.

FOOTNOTES


Address for reprint requests and other correspondence: S.-L. Xia, PO Box 100224, Univ. of Florida, Gainesville, FL 32610-0224 (e-mail: xiasl{at}ufl.edu)

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.

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