Hormone-regulated transepithelial Na+ transport in mammalian CCD cells requires SGK1 expression

My N. Helms, Géza Fejes-Tóth, and Anikó Náray-Fejes-Tóth

Department of Physiology, Dartmouth Medical School, Lebanon, New Hampshire 03756-0001


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

To study the role of serum and glucocorticoid-inducible kinase-1 (SGK1) in mammalian cells, we compared Na+ transport rates in wild-type (WT) M1 cortical collecting duct cells with M1 populations stably expressing human full-length SGK1, NH2-terminal truncated (Delta N-60) SGK1, "kinase-dead" (K127M) SGK1, and cells that have downregulated levels of SGK1 mRNA (antisense SGK1). Basal rates of transepithelial Na+ transport were highest in full-length SGK1 populations, compared among the above populations. Dexamethasone treatment increased Na+ transport in WT and full-length SGK1 cells 2.7- and 2-fold, respectively. Modest stimulation of Na+ absorption was detected after dexamethasone treatment in Delta N-60 SGK1 populations. However, Delta N-60 SGK1 transport rates remained substantially lower than WT values. Importantly, a combination of high insulin, dexamethasone, and serum failed to significantly stimulate Na+ transport in antisense or K127M SGK1 cells. Additionally, expression of antisense SGK1 significantly decreased transepithelial resistance values. Overall, we concluded that SGK1 is a critical component in corticosteroid-regulated Na+ transport in mammalian cortical collecting duct cells. Furthermore, our data suggest that the NH2 terminus of SGK1 may contain a Phox homology-like domain that may be necessary for effective Na+ transport.

aldosterone; epithelial sodium channel; M1 cell line; serum and glucocorticoid-inducible kinase 1; cortical collecting duct


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SERUM AND GLUCOCORTICOID-INDUCIBLE KINASE-1 (SGK1) is a novel member of the serine/threonine family of kinases and is induced in response to a variety of extracellular stimuli (listed in Ref. 18). Implicit in its name, SGK1 was originally identified as a serum and glucocorticoid-induced gene in rat mammary tumor cells by differential display (37). Since then, posttranslational regulation of SGK1 has also been described. For example, components of the phosphoinositide 3-kinase signaling pathway are necessary for the phosphorylation of SGK1, which leads to its activation. Amino acid residues Thr256 (located in the activation loop of SGK1) and Ser422 (located in the COOH-terminal domain of SGK1) must both be phosphorylated by 3-phosphoinositide-dependent protein kinases-1 and -2 (PDK1 and PDK2) for complete activation of SGK1 (16, 26). Recently, our laboratory and others have demonstrated that the level of SGK1 transcript is rapidly upregulated in response to mineralocorticoids, independently of de novo protein synthesis, in target epithelial cells (2, 6, 23, 25, 30).

Mineralocorticoids are the key regulators of transepithelial Na+ transport. In the absence of 11beta -hydroxysteroid dehydrogenase-2 activity, glucocorticoids can also induce mineralocorticoid-like effects (11, 17, 19, 24). Mineralocorticoids and glucocorticoids are both effective in upregulating SGK1 transcript levels in epithelial cells, which implies a relationship between SGK1 and epithelial Na+ channel (ENaC) activity in collecting duct cells. This notion is strongly supported by several heterologous SGK1 expression studies. For instance, oocytes coexpressing SGK1 and ENaC subunits exhibited higher Na+ current and more channels localized to the plasma membrane compared with oocytes expressing ENaC alone (1, 6, 23, 30). Additionally, ectopic expression of SGK1 in Xenopus laevis A6 cells displayed exceptionally high levels of Na+ transport under basal conditions, which were sustained with aldosterone treatment (9). Moreover, inhibition of the phosphoinositide 3-kinase signaling pathway with LY-294002 was associated with a substantial decrease in the rate of Na+ transport in A6 cells (28, 36). Together, the above studies strongly implicate SGK1 as a mediator of aldosterone-stimulated transcellular Na+ reabsorption.

Despite these recent advances, however, the physiological role of SGK1 in mammalian kidney cells has not been definitively established. The goal of our study was to generate stable mouse cortical collecting duct (CCD) cells that either overexpress or downregulate SGK1 activity. Here, we report for the first time that SGK1 is a critical regulator of ENaC activity in mammalian CCD cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Generation of an antisense SGK1 construct. The antisense SGK1 construct was created by ligating a 708-bp fragment of cDNA located in the 3'-untranslated region of mouse SGK1 into the multiple cloning site of TRE vector (Clontech, Palo Alto, CA) in the reverse (antisense) orientation. Specifically, the DNA fragment is located in the mouse SGK1 between the BamHI and the HindIII restriction sites and includes the poly-A tail (Fig. 1A). The newly constructed antisense TRE construct carries both ampicillin and hygromycin resistance genes for selection in Escherichia coli and in mammalian cells, respectively. The subsequent transcription of antisense RNA downregulates the endogenous expression of the complement transcript (20, 27, 39).


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Fig. 1.   Effect of antisense serum and glucocorticoid-inducible kinase-1 (SGK1) construct expression on the level of SGK1 transcript in M1 cortical collecting duct cells. A: 3'-untranslated region fragment (UTR; 709 bp) from the mouse SGK1 genome cloned in the reverse (antisense) orientation into pTRE vector (Clontech, Palo Alto, CA). B: representative RT-PCR of SGK1 and beta -actin from control and antisense M1 populations grown in insulin- and dexamethasone-containing medium. Serial dilutions of cDNA (10-0.15 ng) were used as templates. Antisense SGK1 construct expression downregulated endogenous levels of SGK1 transcript. C: beta -actin-normalized SGK1 transcript levels were 4 times lower in the antisense cells (n = 4) vs. wild-type (WT; n = 5) M1 cells (P < 0.05); Student's paired t-test (1-tailed; 2-sample equal variance). Values are averages ± SE.

Retroviral cDNA constructs of human full-length SGK1, NH2-terminal truncated (Delta N-60) SGK1, and dominant-negative K127M SGK1 tagged with hemaglutinnin (HA) epitopes at the NH2 terminus were generously provided by Dr. Suzanne Conzen (University of Chicago). Retroviral vectors were produced by transient LipofectAMINE (Invitrogen, Carlsbad, CA) transfection of HA-tagged constructs into host amphotropic Pheonix cells (American Type Culture Collection, Rockville, MD) according to the manufacturer's protocol and as previously described (22).

Cell culture and construction of M1 CCD cell lines, which overexpress or downregulate SGK1 activity. M1 cells (33) were propagated in PC-1 growth medium (BioWhittaker, Walkersville, MD) containing an abundance of growth supplements. The supplementary growth factors include 15 µg/ml insulin, 5 µM dexamethasone, 5% FBS, 2 mM glutamine, and antibiotics (75 µg/ml penicillin, 100 µg/ml streptomycin, and 12.5 µg/ml tylosin). This medium is hereafter referred to as insulin- and dexamethasone-containing (IDC) medium.

Subconfluent M1 cells were cotransfected with antisense TRE vector concurrently with a Tet-On construct. The Tet-On construct carries neomycin resistance and was designed to facilitate TRE vector expression in the presence of tetracycline. However, it was our experience that Tet-On vector provided strong, constitutive cytomegalovirus promoter activity in trans, which did not require additional tetracycline stimuli. The cotransfections were carried out by using a modified LipofectAMINE protocol (15). M1 cells stably expressing antisense SGK1 cDNA were then selected by using 200 µg/ml hygromycin and 250 µg/ml neomycin.

Supernatant from amphotropic Phoenix cells producing retroviruses packaging truncated (Delta N-60), full-length, and dominant-negative (K127M) SGK1 were collected and passed through a 0.45-µm filter to remove cellular debris. Twenty-four hours before infection, 6 × 105 M1 cells were seeded onto a 60-mm plastic dish in 2 ml IDC medium. M1 cells were inoculated with 2 ml filtered amphotropic supernatant and allowed to incubate for an additional 48 h. Stable Delta N-60, full-length, and K127M SGK1 expression in M1 cells were selected with 700 ng/ml puromycin.

All clones surviving antibiotic selection were expanded without the process of clonal selection on the basis of SGK1 vector expression levels. Cell populations will be referred to by the expression vector transformed into parent M1 cells (full-length, Delta N-60, K127M, or antisense SGK1).

Quantitative RT-PCR. To compare the relative abundance of SGK1 mRNA between wild-type (WT) M1 cells and populations of M1 cells stably expressing antisense SGK1 construct, we used quantitative RT-PCR methods, previously described in (23). Briefly, total RNA was extracted from WT and antisense SGK1 M1 cells by using TRI Reagent per the manufacturer's protocol (Molecular Research Center, Cincinnati, OH). Then, 2 µg of isolated total RNA was reverse transcribed by extension of random primers. SGK1 upper (5'-CTC AGT CTC TTT TGG GCT CTTT-3') and lower (5'-TTT CTT CTT CAG GAT GGC TTTC -3') PCR primers generated a 450-bp product. Reactions were performed with AmpliTaq DNA polymerase (Roche, Indianapolis, IN) under standard conditions with varying amounts of template (10, 2.5, 0.62, and 0.15 ng of cDNA) originating from WT or antisense cells cultured in IDC medium. Thermal cycling conditions included a 2-min denaturing step at 96°C, followed by 25 cycles of 95°C for 1 min for denaturing, 57°C for 1 min to reanneal, and 72°C for 1 min for elongation of template, and then a final extension of 72°C for 8 min. The abundance of beta -actin mRNA for each sample was also determined by using primers and PCR conditions optimized for beta -actin, as previously described (34). Amplified SGK1 PCR products were separated on a 5% polyacrylamide gel, stained with ethidium bromide, quantified by using a FluoroImager and ImageQUANT software (Molecular Dynamics, Sunnyvale, CA), and then normalized for beta -actin mRNA levels.

Electrophysiological measurements. Cells were seeded onto Millicell permeable membranes (Millipore, Bedford, MA) at a density of 4 × 105 cells/chamber. After seeding, cells were bathed in IDC medium on the basolateral and apical sides until they formed confluent monolayers (~3-7 days). After the cells reached confluency, the bathing medium was changed to an insulin and steroid hormone-free (I-SterF) medium composed of DMEM/F-12 (Mediatech, Herndon, VA) supplemented with 5% FBS stripped twice with charcoal, 15 mM HEPES, 2 mM glutamine, and antibiotics. Cells were cultured in I-SterF medium for 48 h to establish basal levels of Na+ transport. Cells were then treated with 1 µM dexamethasone (in I-SterF medium) for a 24-h period to study the effects of corticosteroids. The transepithelial voltage and transepithelial resistance (RTE) values of each population of cells were regularly determined by using an Epithelial Voltohmmeter (World Precision Instruments, Sarasota, FL), and the equivalent short-circuit current (Isc) was calculated. Our laboratory has previously demonstrated that Isc mainly represents transepithelial Na+ current in this model system (7). Cells were regarded as confluent monolayers when voltage values remained steady for two independent readings over a 48-h time period and RTE values reached threshold levels of 900 Omega  · cm2. All membrane resistance values reported represent the difference between measured RTE and porous membrane RTE (~125 Omega  · cm2) in which the cells were grown.

Western blot analysis. Confluent Delta N-60 SGK1, full-length SGK1, K127M SGK1, and WT M1 cells were rinsed twice with ice-cold PBS before lysing with 500 µl of SDS solubilization buffer [48.2 mM 2-(N-hexylamino) ethanesulfonic acid, 1% SDS, 10% glycerol, and 1% protease and phosphatase inhibitor cocktail (Sigma, Palo Alto, CA)]. The protein concentration of cellular lysate was determined by using BCA Protein Assay Reagent (Pierce Chemical, Rockford, IL). Then, 7 µg of total protein lysate or immunoprecipitated product concentrated from a 60-mm dish was electrophoresed on a 10% acrylamide gel under denaturing conditions. The proteins were then transferred to Immobilon-P membrane (Millipore) and blocked in buffer consisting of 5% dry milk, 10 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween, 124 µM thimerosal, and 1% Mega Block (PGC Scientifics, Frederick, MD) for 1 h. Next, the membrane was incubated in 4 µg of rabbit polyclonal anti-HA antibody (Upstate Biotechnology, Cleveland, OH) diluted in 4 ml of blocking buffer at room temperature for 1 h. After extensive washes, anti-rabbit IgG-horseradish peroxidase (HRP)-labeled antibody (Cell Signaling Technology, Beverly, MA) was added at a concentration of 1 µg/ml and incubated for another hour at room temperature. After thorough washes, HRP signal was detected by using the ECL substrate (Amersham Pharmacia Biotech, Piscataway, NJ) or Super Signal West Dura Substrate (Pierce Chemical). The membrane was finally exposed to Kodak X-OMAT AR scientific imaging film (Kodak, Rochester, NY).

Immunoprecipitation. Confluent cells grown on a 60-mm plate were washed with ice-cold PBS and then lysed with buffer consisting of 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton, and 1% protease and phosphatase inhibitor cocktail, further complemented with 1 mM beta -glycerol phosphate, 2.5 mM sodium pyrophosphate, 1 mM Na3VO4, and 1 µg/ml leupeptin. Lysates were then sonicated for 5-10 s. To immunoprecipitate SGK1 protein, 500 µl of supernatant from hybridomas-producing SGK1 antibody or 7 µg anti-HA antibody diluted in lysis buffer were added to the cell lysate. SGK1 monoclonal antibodies were generated in mice immunized with mouse SGK1 amino acids 147-437, by using standard techniques previously described (10). Then, the antibody containing lysates was incubated overnight at 4°C with gentle rocking. The following morning, 20 µl of protein A-Sepharose beads (Sigma) were added to the sample and incubated for an additional 2 h at 4°C with gentle rocking. The sample was then briefly centrifuged at 14,000 g. The precipitated pellet was washed in lysis buffer and then resuspended in 25 µl SDS solubilization buffer. The immunoprecipitated complex was analyzed by Western blot technique, as described above.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Stable integration of SGK1 constructs in the M1 CCD cell line. To study the role of SGK1 on transepithelial Na+ transport in mammalian cells, we generated M1 CCD cells stably expressing the human full-length SGK1, Delta N-60 SGK1, and dominant-negative K127M SGK, as well as antisense SGK1, which downregulates the endogenous level of SGK1 transcript.

The abundance of SGK1 transcript in WT and antisense M1 cells grown in IDC medium was determined by using quantitative PCR techniques. The SGK1 primer pair selected amplifies a region located within the endogenous mouse SGK1 transcript. Expression of antisense SGK1 effectively downregulated the endogenous level of SGK1 transcript and did not affect the expression levels of beta -actin (Fig. 1, B and C). Normalized mouse SGK1 transcript levels were ~4 times lower in the antisense cell populations compared with WT M1 cells (P < 0.05).

To demonstrate that HA-tagged full-length, Delta N-60, and K127M SGK1 were constructs incorporated into the M1 genome and were expressed, we determined HA-protein expression by using Western blot analysis. Delta N-60 SGK1 signal was robust from 7 µg total protein lysate (Fig. 2, lane 4). Similarly, as previously reported for human embryonic kidney 293 and mammary epithelial MCF10A cells (16, 22), we found that full-length SGK1 proteins are expressed at levels much lower than truncated SGK1. Therefore, it was necessary to immunoprecipitate full-length and K127M SGK1 protein by using an SGK1 antibody to enrich for the signal in Western blot analysis. Western blot analysis of immunoprecipitated protein using SGK1 antibody from M1 cells expressing full-length or K127M SGK1 displayed the appropriate size bands when probed with the HA antibody (Fig. 2, lanes 2 and 5). As a positive control, we used protein from COS-7 cells transiently transfected with full-length HA-tagged SGK1 (Fig. 2, lane 3). We are confident of the specificity of the HA antibody, because immunoprecipitated protein from WT M1 cells subjected to Western blot analysis was negative for the HRP signal (Fig. 2, lane 6). Furthermore, the immunoprecipitation was performed with a mouse antibody, which does not cross-react with the HRP-labeled anti-rabbit antibody used in Western blot analysis. This is important because immunoprecipitation of SGK1 using HA antibody raised in rabbit gave an immensely strong signal in Western blot (Fig. 2, lane 1). The saturated signal in lane 1 most likely corresponds to the secondary antibody bound to heavy-chain rabbit immunoglobulins carried over from the immunoprecipitation. These data demonstrate that the stable M1 cells we generated effectively express the truncated, full-length, and dominant-negative SGK1 proteins.


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Fig. 2.   Western blot analysis confirms stable expression of hemaglutinnin (HA) SGK1 proteins in M1 cortical collecting duct cells. Protein was separated on a polyacrylamide gel and with an anti-HA antibody as described in MATERIALS AND METHODS. Lane 1: protein immunoprecipitated (IP) with HA-antibody from M1 cells expressing K127M SGK1. Lane 2: protein immunoprecipitated with SGK1 antibody from M1 cells expressing K127M SGK1. Lane 3: positive control (+ctr); protein immunoprecipitated with SGK1 antibody from COS-7 cells transfected with 1 µg full-length HA SGK1 construct. Lane 4: 7 µg total protein from M1 cells expressing NH2-terminal truncated (Delta N-60) SGK1. Lane 5: protein immunoprecipitated with SGK1 antibody from M1 cells expressing full-length SGK1. Lane 6: negative control; protein immunoprecipitated with SGK1 antibody from WT cells. Western blot analysis and immunoprecipitation procedures (see MATERIALS AND METHODS) were performed twice independently (n = 2). Signal was detected by using ECL substrate in lanes 1-4 and West Dura substrate in lanes 5 and 6.

Effect of changes in SGK1 expression on transepithelial Na+ transport in M1 cells. To observe the effect of expressing full-length, Delta N-60, antisense, and K127M SGK1 on transepithelial Na+ transport, each cell line was cultured on permeable filters concurrently with WT M1 cells. Fig. 3A, illustrates the steady-state Isc of each cell line grown in IDC medium. Noticeably, Isc in cells expressing full-length SGK1 was significantly higher than that in WT M1 cells (P < 0.005). In IDC medium, Isc values of Delta N60 SGK1 populations were comparable to WT values. Importantly, the expression of antisense and K127M SGK1 in M1 cells decreased Isc values markedly, to ~20% of Isc in WT cells (P < 0.0005) in the presence of "complete" medium that includes serum and growth factors.


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Fig. 3.   Transepithelial Na+ transport levels [short-circuit current (Isc)] in WT M1 cells and cells stably expressing full-length , Delta N-60, antisense, and K127M SGK1. A: cells were maintained in complete insulin- and dexamethasone-containing growth medium until confluent monolayers developed. Isc values were determined 48 h after development of confluency. B: growth medium was then replaced with insulin and steroid hormone-free (I-SterF) medium for 48 h to establish basal levels of Na+ transport. In I-SterF medium, basal rates of Na+ absorption were significantly higher in cells expressing full-length SGK1 compared with other cell populations. C: 1 µM dexamethasone was added to the I-SterF medium and incubation continued for 24 h. Dexamethasone significantly increased Na+ current in WT and full-length SGK1 cells but failed to substantially stimulate it in antisense and K127M SGK1 cells. The Isc values represent average values ± SE from independent Millicell cultures. Each experiment was repeated from 2 created progenitor populations. Statistical comparison between each respective cell population and WT within each experimental parameter was made by using Student's paired t-test (1-tailed, 2-sample equal variance); ***P < 0.0005, **P < 0.005, *P < 0.05. Bars left to right: WT, full-length, Delta N-60, antisense, and K127M.

In I-SterF, Isc values typically declined to levels below 10 µA/cm2 (see Fig. 3B). However, a noticeable difference was observed in the cell lines overexpressing full-length SGK1, for which Isc remained high (16.18 µA/cm2) even in the absence of serum and hormone stimulation. To demonstrate that these changes were not simply due to a "time effect," we kept separate cultures in complete growth medium for an additional 11 days. Within this time frame, cells maintained steady-state levels of Isc and RTE (data not shown).

After basal levels of transepithelial Isc were established, cells were treated with 1 µM dexamethasone in I-SterF medium for 24 h (Fig. 3C). Dexamethasone, as expected, increased Isc values (2.7-fold) in WT M1 cells. Similarly, Isc values in cells expressing full-length SGK1 increased twofold after dexamethasone treatment. These cells reached rates of Na+ transport that were significantly higher than those observed in WT cells (P < 0.0005). Although Isc values in the Delta N-60 populations increased approximately twofold with corticosteroid stimulation, the resulting Isc was still significantly lower than that in WT levels (P < 0.05).

Most importantly, dexamethasone failed to stimulate Na+ absorption in cells expressing antisense SGK1 and did not substantially increase Na+ transport above basal levels in cells expressing the dominant-negative K127M SGK1. Isc values for antisense and K127M SGK1 cell populations remained significantly lower than WT M1 cells in the presence of 1 µM dexamethasone (P < 0.0005 and P < 0.005, respectively).

Effect of changes in SGK1 expression on RTE. Generally, high-RTE values characterize cell types with very tight junctions, whereas low RTE values are associated with leaky epithelia or multiple open ion channels. In the present study, as well as in previously published reports (33), M1 cells predominantly develop RTE values >900 Omega  · cm2. However, antisense populations incubated in I-SterF medium and 1 µM dexamethasone-containing medium displayed exceptionally low-RTE values of 421.2 ± 71.8 and 569.8 ± 87.4 Omega  · cm2, respectively (Fig. 4, B and C). The apical to basolateral permeability of Millicell cultures were inspected to determine the integrity, or "leakiness," of the monolayer. Four hundred microliters of tissue culture medium were applied to the apical compartment of the Millicell chamber, and 600 µl of the same medium were applied to the basolateral compartment. During regular changes of the culture medium, potential leakage through the monolayer was determined by measuring fluid volume from each compartment. Under each experimental growth parameter, there was no significant change in fluid volumes from each compartment. Additionally, [3H]mannitol paracellular permeability assays (described in Ref. 41) were performed to further verify that the monolayer was not leaky in antisense cell populations cultured on permeable supports. The percent leakage of radioactively labeled mannitol measured from WT cells cultured in IDC medium did not differ significantly compared with antisense cells incubated in I-SterF- and dexamethasone-containing medium (data not shown). Together, these observations are highly suggestive that downregulation of SGK1 alters RTE values. The resulting epithelial monolayer is not leaky and therefore is suitable for the determination of transepithelial Na+ transport.


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Fig. 4.   Transepithelial resistance (RTE) in WT M1 cells and cells stably expressing full-length, Delta N-60, antisense, or K127M SGK1. The cell populations were maintained as described for Fig. 3. RTE values were measured with an epithelial voltohmmeter. On the whole, RTE values in cells expressing different SGK1 constructs did not substantially deviate from WT M1 values. However, cells expressing antisense SGK1 displayed significantly lower RTE values. Values are averages ± SE. Statistical comparison among WT and each cell population is made by using Student's paired t-test (1-tailed; 2-sample equal variance); ***P < 0.0005, **P < 0.005, *P < 0.05. Bars left to right: WT, full-length, Delta N-60, antisense, and K127M.

Figure 4A also demonstrates that cells overexpressing full-length SGK1, grown in IDC medium, exhibited lower RTE values compared with control (P < 0.0005). In this instance, presumably, the depressed RTE values are a result of very high-ENaC activity, maximally stimulated by high concentrations of hormone and serum.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Since the recent identification of SGK1 as an early aldosterone-induced gene, it has been a priority to characterize the physiological role of SGK1 in epithelial Na+ transport. Prior studies utilizing X. laevis oocytes and A6 cell lines are demonstrative of the ability of SGK1 to mediate ENaC activity. However, the role of SGK1 in effecting mineralocorticoid- and corticosteroid-induced Na+ transport in mammalian CCD cells had not been established before this study.

This study contributes to establishing SGK1 as a corticosteroid-regulated mediator of Na+ reabsorption in mammalian collecting duct cells. The overexpression of SGK1 in M1 cell lines increased transepithelial Isc significantly above control levels under basal growth conditions and after dexamethasone treatment. Furthermore, the downregulation of SGK1 transcript using an antisense SGK1 construct and the expression of a dominant-negative SGK1 decreased transepithelial Isc. Because we performed these studies by using a mixed population of cells stably expressing SGK1 constructs, the results cannot be attributed to mere clonal variations. Furthermore, studies published by Faletti et al. (9) similarly ascertain that SGK1 is a necessary component of ENaC-mediated Na+ transport in X. laevis A6 cells. Faletti et al. included overexpression of full-length human and dominant-negative D222A mutant SGK1, which is unresponsive to phosphorylation by PDK2. Basal rates of Na+ transport in A6 cells overexpressing human SGK1 were 3.5 times higher than untransfected control. Conversely, D222A SGK1 clones did not respond to hormone stimulation, which is characteristic of A6 model epithelial cells.

However, our studies also examined populations of cells that overexpressed truncated SGK1. These cells did not respond to corticosteroid treatment to the extent observed in WT and full-length SGK1 populations. Because Delta N-60 SGK1 is expressed at very high levels in both M1 cells and other cell types (16, 22) and maintains its phosphorylation (activation) sites, the reason for this observed effect on ENaC function is not obvious. We speculate that the first 60 amino acids in SGK1 are necessary for stimulation of ENaC activity. It is reasonable to propose this, because research within the past decade has identified several proteins that contain NH2-terminal Phox homology domains, which specifically bind to phosphoinositides to target to their subcellular site of activation (5, 8, 14, 32, 40). Interestingly, the mouse homolog of human SGK3, cytokine-independent survival kinase (CISK), contains a PX domain in the NH2-terminal region (21). Mutation of this region inhibited CISK localization to endosomal compartments and therefore inhibited CISK function (38). The highly conserved PX sequence motif identified in CISK [R(R/K)xxLxx(Y/F)] is also conserved within the NH2-terminal region of SGK1 that was truncated in our Delta N-60 cell lines. Perhaps, in a manner similar to the Pleckstrin homology domain of PKB and the PX domain of CISK, SGK1 possesses a targeting motif necessary for PI-3 kinase-dependent activation at the membrane. We speculate that this may in part explain the lack of effect on Na+ transport in Delta N-60 SGK1 cells when they are treated with corticosteroid.

Additionally, the NH2 terminus of SGK1 may be responsible for targeting it to the appropriate ENaC-activating substrate. In this instance, the overexpression of truncated SGK1 in our studies could possibly be acting as a dominant-negative and thus inhibits full ENaC activity in M1 cells. Recent evidence from Brickley et al. (3) supports our contention that the NH2-terminal region targets SGK1 to the site of Na+ transport action. Their studies demonstrated a difference between cellular localization of Delta N-60 SGK1 green fluorescent protein (GFP) and SGK1 GFP (3). Although full-length SGK1 GFP is predominantly located in the cytoplasm and plasma membrane of HEK293T, Cos, SK-BR-3, and Madin-Darby canine kidney cells, truncated SGK1 GFP is homogenously distributed and fails to localize to the membrane (3, 29). This group has previously demonstrated that expression of truncated and WT SGK1 similarly inhibit apoptosis in MCF10A-Myc cell lines. For this reason, Delta N-60 SGK1 does not behave as a dominant-negative kinase in relation to apoptosis (22). We therefore suggest that the putative SGK1-targeting motif may specifically regulate SGK1 activity in ENaC-mediated Na+ transport.

Analysis of the RTE values obtained in this study implicates SGK1 as a key component in the maintenance of epithelial tight junctions, in addition to its role in mediating corticosteroid-induced Na+ reabsorption. Populations of M1 cells stably expressing antisense SGK1 exhibited significantly lower RTE values compared with WT cells in each experimental medium. We demonstrated that the substantial decrease in RTE values observed did not result in a leaky epithelium by using apical-to-basolateral permeability assays. Additionally, the cells maintained viability, as suggested by a modest rise in RTE values when treated with corticosteroid.

Of particular interest and relevance to the present study, several investigators have reported that steroid hormones positively modulate RTE in other epithelial cells. In these studies, the RTE of rabbit distal colon (12, 13, 35) and mammary epithelial cells (4, 31, 41) revealed a significant increase in RTE values after aldosterone and dexamethasone treatment, respectively. Although the precise molecular mechanism of steroid-regulated tight junction formation is not clearly established, Singer et al. (31) demonstrated that specific serine/threonine kinase inhibitors, such as H7, moderately reduced RTE values in mouse mammary epithelial cells after dexamethasone treatment. Accordingly, the effect of downregulation of SGK1 on RTE values in M1 cells in the present study suggests that SGK1 may be an important regulator of tight junction formation. Given that CCD cells, in vivo, necessarily develop very tight junctions to minimize back-flux of ions, SGK might have dual physiological roles in renal cells. Perhaps aldosterone concurrently modulates RTE and ENaC activity, by way of SGK1, to efficiently reabsorb Na+.

In summary, our data affirms that SGK1 is a critical player in the molecular pathway of ENaC activation. In our mammalian collecting duct cell lines, SGK1 activated transepithelial Na+ transport and ostensibly affected tight junction formation. Our study also suggests that the NH2 terminus of SGK1 is necessary for modulating ENaC activity, possibly through a putative PX domain.


    ACKNOWLEDGEMENTS

We thank Dr. Suzanne D. Conzen for providing the HA-tagged full-length, Delta N-60, and K127M SGK1 constructs.


    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants T32-DK-7508-17, DK-41841, DK-58898, and DK-55845.

Address for reprint requests and other correspondence: A. Náray-Fejes-Tóth, Dartmouth Medical School, Dept. of Physiology, Borwell Bldg., 1 Medical Center Dr., Lebanon, NH 03756-0001 (E-mail: Aniko.Fejes-Toth{at}Dartmouth.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.

First published November 12, 2002;10.1152/ajprenal.00299.2002

Received 9 August 2002; accepted in final form 1 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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