Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520-8026
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ABSTRACT |
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The purpose of this study was to examine
the role of the serum- and glucocorticoid-induced kinase (SGK) in the
activation of the epithelial sodium channel (ENaC) by aldosterone,
arginine vasopressin (AVP), and insulin. We used a
tetracycline-inducible system to control the expression of wild-type
(SGK
sodium reabsorption; aldosterone; insulin; vasopressin; serum- and glucose-induced kinase
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INTRODUCTION |
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THE ACTIVITY OF the epithelial sodium channel (ENaC) is essential for sodium reabsorption in the kidney. In the distal segment of the renal tubule ENaC is regulated mainly by aldosterone. However, the mechanisms underlying the effects of aldosterone are only partially understood. A number of models for aldosterone action have been proposed, including an increase in the number of active channels in the plasma membrane, in the channel's open probability, or a combination of both (for recent reviews see Refs. 24, 29).
Different lines of evidence suggest that the serum- and glucocorticoid-induced kinase (SGK) mediates the aldosterone upregulation of ENaC. SGK is an aldosterone-induced protein in mammalian and amphibian renal cells (5, 17), and, most significantly, SGK activates ENaC when the two proteins are expressed together in Xenopus oocytes (5, 17) or in renal epithelial cells (8). Activation of SGK is controlled by the phosphoinositide 3-kinase (PI3-kinase) pathway, which promotes the phosphorylation of SGK at serine 422 (human SGK), which in turn increases the phosphorylation of threonine 256 by phosphatidylinositol-dependent kinase (PDK) 1. Phosphorylation of these two residues renders the kinase active (13, 19). There is indirect evidence linking aldosterone to the activation of SGK by PI3-kinase. First, inhibition of PI3-kinase by the specific and reversible blocker LY-294002 decreases the basal levels of ENaC activity and abolishes the aldosterone effects (4, 20). Second, aldosterone increases one of the products of PI3-kinase, phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P3], in A6 cells (4).
Hormones other than aldosterone, such as insulin and arginine vasopressin (AVP), also modulate the activity of ENaC in the kidney. It has been suggested that the actions of these hormones may also require SGK. In muscle and adipose tissues PI3-kinase plays a central role in insulin signaling, making possible the participation of SGK in the insulin-mediated regulation of ENaC (21). Recently, Faletti et al. (8) found that expression of one kinase-inactive SGK mutant (SGKD222A), but not expression of a different inactive mutant (SGKS422A), abrogated the insulin response in A6 cells.
Involvement of SGK in the AVP response has been proposed in light of the finding that cAMP analogs can activate SGK (22), although this result was not confirmed by another group (26). AVP binding to V2 receptors in principal cells of the rat cortical collecting duct increases the intracellular levels of cAMP, which in turn activates amiloride-sensitive sodium permeability (25). Faletti et al. (8) found that transfection of wild-type SGK in A6 cells increased the AVP effect, whereas SGKD222A abolished the response and another kinase-inactive mutant, SGKS422A, did not show any effect. However, a caveat in this latter study is that the authors could not detect expression of the transfected SGK proteins.
In this study, we investigated the role of SGK in the modulation of ENaC activity by hormones in renal epithelial cells. We used as a model the A6 cell line derived from frog kidney, which expresses endogenous ENaC and SGK (5) and responds to aldosterone, insulin, and AVP by increasing amiloride-sensitive transepithelial sodium transport. The experiments indicate that SGK targets a mechanism that is common to the insulin and AVP signaling pathways but does not seem to be shared by the aldosterone response.
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MATERIALS AND METHODS |
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Plasmid constructs. Full-length Xenopus SGK cDNA was amplified by RT-PCR from aldosterone-treated A6 cells. Specific primers were designed according to the published sequence (5), and restriction sites were added to the 5'- and 3'-ends to facilitate cloning. The amplified fragment was cloned in pcDNA4/TO (Invitrogen, Carlsbad, CA). Point mutations were introduced by PCR with the QuickChange mutagenesis kit (Stratagene, La Jolla, CA). All constructs were sequenced at the Keck Facility at Yale University. Plasmid pcDNA6/TR, containing the coding sequence for the tetracycline repressor, was obtained from Invitrogen.
Antibody generation. A polyclonal anti-SGK antibody was generated in rabbits. Animals received subcutaneous injections of a glutathione S-transferase (GST) protein fused to a segment of the mouse SGK comprised of amino acids 301 to 405. The GST-SGK fusion protein was expressed in Escherichia coli and affinity purified with a glutathione-agarose column.
Cell culture, transfection, and cell line generation. Experiments were performed on A6-S2 cells (11), a clone obtained by limiting dilution of A6 cells derived from the kidney of Xenopus laevis and selected for high transepithelial resistance (RT) and responsiveness to hormones. These cells were kindly provided by Dr. John Hayslett, Yale University. Cells were maintained in amphibian medium (0.75× DMEM, 10% FBS, buffered with sodium bicarbonate) in an incubator set at 27°C and 1.5% CO2. Cells expanded in plastic dishes were seeded on Transwell permeable supports (Corning, Corning, NY) for biochemical experiments (4.7-cm2 filters) or for electrical measurements (0.33-cm2 filters). After 10-14 days in culture, cells were washed twice in serum-free medium and maintained without serum for two more days before experiments were performed. Aldosterone (Sigma, St. Louis, MO) was added to a final concentration of 100 nM. Tetracycline (Invitrogen) was added to a final concentration of 1 µg/ml. LY-294002 was obtained from Calbiochem (La Jolla, CA) and was used at a final concentration of 50 µM. Insulin and AVP were obtained from Sigma and used at final concentrations of 100 nM and 1 µM, respectively. When ethanol or DMSO was the solvent of stock solutions, the final dilution was 1:500 or 1:1,000, depending on the experiment. Control experiments included ethanol or DMSO at the same dilution and showed no effect.
Transfection of A6 cells grown on plastic was performed with Lipofectamine 2000 (Invitrogen) following the manufacturer's instructions. A 5:1 mixture of pcDNA6/TR and pcDNA4/TO-SGK plasmids was used in every transfection. Stable cell lines expressing the tetracycline repressor and pcDNA4/TO-SGK constructs were obtained by growth on selective media containing 500 µg/ml zeocin and 10 µg/ml blasticidin. Clones were tested for SGK expression on induction with 1 µg/ml tetracycline and development of high RT when grown on filters. CHO cells were obtained from the American Type Culture Collection and transiently transfected with Lipofectamine 2000 (Invitrogen).RT-PCR.
Total RNA was extracted from A6 cell lines grown on filters with the
RNeasy kit from Qiagen (Valencia, CA) following the manufacturer's instructions. RNA concentration was measured by absorption
spectroscopy. One microgram of RNA was used for first-strand cDNA
synthesis with the SuperScript system (Invitrogen). Five percent of
each RT reaction was used as a template for amplification of
full-length heterologous SGK by PCR with a forward primer specific for
Xenopus SGK and a reverse primer specific for the pcDNA4/TO
poly-linker region. Full-length Xenopus -actin was
amplified as a control for each sample with specific primers designed
by using the published sequence (GenBank accession number AF079161).
Western blotting. A6 cells grown on plastic or filters were washed twice with ice-cold PBS, scraped in the same buffer, recovered by centrifugation, and lysed with SDS-PAGE loading buffer. Samples were separated by electrophoresis in 10% SDS-PAGE and transferred to Immobilon-P (Millipore, Bedford, MA). After blocking with 5% dry milk, the membranes were probed with anti-SGK antibody at 1:5,000 dilution. Secondary anti-rabbit IgG labeled with peroxidase (Sigma) was used at 1:10,000. The signal was developed with ECL+ (Amersham), and blots were exposed to BioMax MR film (Eastman Kodak, New Haven, CT). In some experiments the intensity of the SGK signal was quantified by densitometric analysis with a GS-800 densitometer (Bio-Rad Laboratories, Hercules, CA).
Equivalent short-circuit current measurement. Transepithelial voltage (VT) and equivalent short-circuit current (Isc) across A6 monolayers were measured as previously described (1). RT was calculated from VT and Isc by using Ohm's law.
Statistical analysis.
Data points represent the means ± SE of n independent
experiments. Differences between groups were evaluated with nonpaired t-test. P and n values are given in
the text or Figs. 1-10 for each experiment.
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RESULTS |
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Tetracycline-inducible expression of SGK in A6 cells. By using a tetracycline-regulated expression system we could control the expression of SGK independently from aldosterone and other extracellular stimuli. Thus we generated stable A6 cell lines coexpressing the tetracycline repressor protein (TetR) together with various forms of SGK (SGKT). Transcription of both cDNAs was controlled by a cytomegalovirus (CMV) promoter, but SGKT had in addition two copies of the tetracycline operator (TetO) inserted in tandem downstream of the CMV promoter (Fig. 1A). Constitutively expressed TetR binds to the TetO, repressing the transcription of SGKT. Addition of tetracycline releases the TetR from the TetO, and the CMV promoter drives expression of the kinase (Fig. 1A).
Tetracycline-induced SGKT expression was examined by RT-PCR with a pair of primers specific for the transfected SGK to differentiate it from the endogenous transcript. The tetracycline effect was rapid because a transcript could be detected by RT-PCR in as short as 30 min after the addition of the drug (Fig. 1B). The repression system was remarkably tight, because no transcript could be detected in the absence of tetracycline even with RT-PCR. Actin was amplified in every condition as a control for the integrity and amount of the RNA added to the reaction (Fig. 1B). Control reactions without RT or without template were always negative. To detect SGKT protein, we raised an anti-SGK polyclonal antibody by immunizing rabbits with the carboxy terminus of mouse SGK fused to GST. The immune serum specifically detected Xenopus SGK in transiently transfected CHO cells examined by Western blotting (Fig. 1C). No signal was detected in nontransfected cells or when the cognate peptide was added to the reaction. The preimmune serum also did not produce a signal (Fig. 1C). It is worth noting that we consistently observed slower than predicted SGK migration on SDS-PAGE. SGK molecular mass deduced from the sequence is 49.1 kDa, and it migrates at around 56 kDa. Another group, using an unrelated antibody, also noted abnormal electrophoretic migration of SGK (5). The newly developed antibody detected the endogenous SGK protein induced by 100 nM aldosterone added to serum-starved A6 cells (Fig. 1D). The tetracycline-inducible system was used to generate A6 cell lines conditionally expressing wild-type SGK (SGKEffects of SGK expression on transepithelial sodium transport and
aldosterone action.
To examine the effects of SGK expression on ENaC function we measured
transepithelial sodium transport in the parental A6 cell line and in A6
cells transfected with SGK · cm2. Cells
were then maintained in serum-free medium for 2 days before experiments
were performed. Control experiments showed that tetracycline does not
affect Isc, VT, or
RT in the A6 parental cell line or in A6 cells
transfected with an empty vector and grown in the presence of the
selective antibiotics (not shown).
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PI3-kinase role in SGK-stimulated sodium transport.
We took advantage of the A6 cell line expressing the
constitutively active mutant SGK
SGK effects on insulin response. We also studied the effects of SGK on insulin-mediated ENaC upregulation in A6 cells. Cells grown on filters and placed in serum-free medium for 2 days were treated with aldosterone, tetracycline, or a combination of both for 24 h. Insulin was then added to the basolateral side to a final concentration of 100 nM. Isc was measured after 30 min of incubation, 50 µM amiloride was added to the apical chamber, and Isc was measured again.
The addition of insulin to A6-SGK
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SGK effects on AVP response. The effects of SGK on AVP-mediated upregulation of ENaC activity were examined in the transfected A6 cell lines (Fig. 10). We followed the experimental protocol described above for the insulin response experiments. Briefly, control cells or cells previously stimulated with aldosterone, tetracycline, or both were exposed to 1 µM AVP. After 30 min, total and amiloride-sensitive Isc were measured. The treatment with AVP induced a significant amiloride-insensitive Isc because of the cAMP-dependent activation of an apical chloride conductance, as previously described (30).
Under control conditions, addition of AVP to A6-SGK ![]() |
DISCUSSION |
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In this study we sought to further examine the contribution
of SGK to ENaC regulation on renal epithelial cells. With the tetracycline-inducible system in the A6 cell line we were able to
control the expression of SGK independently of hormonal stimulation. This experimental maneuver allowed us to study the interactions between
SGK and the regulatory pathways involved in activation of ENaC by
aldosterone, insulin, and AVP. The importance of a system in which each
clone serves as its own control is illustrated by the large variability
in basal Isc among clones transfected with the
same DNA construct. For instance, basal Isc
values in four different clones of A6 cells transfected with
SGKwt measured 3 days after removal of serum were 11.2 ± 2.9, 4.0 ± 0.2, 5.0 ± 1.8, and 21.1 ± 0.7 µA/cm2. The response to hormones also varied among
clones, as illustrated by one clone transfected with
SGK
It has been proposed that induction and activation of SGK are responsible for the early increase in ENaC activity induced by aldosterone. The mechanism of induction has been well documented, and it represents enhanced transcription of the SGK gene by steroids (5, 17). Activation depends on increased levels of PtdIns(3,4,5)P3; however, the mechanism(s) by which aldosterone may increase PtdIns(3,4,5)P3 levels is not known.
Our results showed that expression of SGKT
induced by tetracycline increased Isc in a
time-dependent manner. There was a significant delay between the onset
of expression of
SGK
Another issue is whether or not aldosterone promotes activation of SGK.
As indicated by the results shown in Fig. 2, addition of aldosterone to
cells expressing high levels of exogenous
SGK
How then can our results be reconciled with the effect of blockers of PI3-kinase that suppress basal and aldosterone-induced Isc (4, 20)? Here we provide evidence (Fig. 8) that the effects of LY-294002 on ENaC are independent of SGK activity because Isc was significantly decreased despite a fully active SGK. Considering the diverse cellular processes regulated by PtdIns(3,4,5)P3, including differentiation, cytoskeletal rearrangements, and membrane trafficking, it is possible that blockage of any of those processes may disrupt the maintenance and/or regulation of the Isc.
In our experiments, SGK
Another important finding in our study is that
SGK
An increase in channel density at the plasma membrane could result from increased delivery, slower retrieval, or a combination of both processes. Our results cannot be used to distinguish which of those processes is mediated by SGK. It has been proposed that ENaC abundance in the plasma membrane is controlled by the ubiquitin-ligase Nedd4, possibly by regulating endocytosis through interaction with the PY motifs of the carboxy terminal of ENaC subunits (12, 28). Recent publications have described in vitro phosphorylation of Nedd4-2 by SGK and subsequent downregulation of the ubiquitin-ligase activity (6, 27), although the requirement of ENaC PY motifs for SGK action is controversial (reviewed in Ref. 12). On the other hand, both insulin and AVP induce mobilization of intracellular pools of vesicles containing GLUT-4 in fat and muscle cells (9) or aquaporin (AQP)-2 in principal cells of the distal tubule (18). The fast time course of insulin and AVP action, which increases Isc by twofold in 10-15 min (3, 11), argues in favor of the insertion hypothesis. It is difficult to explain how Isc can increase so rapidly with a decrease or block of ENaC endocytosis, even if channels have a short half-life in the membrane (1 h in MDCK cells, ~15 min in A6 cells) (1, 10).
It is important to emphasize that insulin and AVP are able to activate ENaC in the absence of any detectable SGK. We cannot exclude that a small amount of SGK, not detectable by Western blotting, is sufficient to mediate the response to these hormones; however, the dose-dependent nature of ENaC stimulation by active SGK does not support this interpretation.
In conclusion, we show that the effects of SGK in the aldosterone response are modest, indicating that most of the stimulation of ENaC induced by aldosterone is mediated by a mechanism(s) distinct from SGK. In contrast, the responses to AVP and insulin share a common step, which is saturated by the activity of SGK.
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ACKNOWLEDGEMENTS |
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We thank Dr. Joseph F. Hoffman for reading the manuscript and giving helpful suggestions.
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FOOTNOTES |
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This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-54062 (to C. Canessa) and by a research fellowship from the National Kidney Foundation (to D. Alvarez de la Rosa).
Address for reprint requests and other correspondence: C. M. Canessa, Dept. of Cellular and Molecular Physiology, Yale Univ. School of Medicine, 333 Cedar St., SHM, B-121, New Haven, CT 06510 (E-mail: cecilia.canessa{at}yale.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 October 16, 2002;10.1152/ajpcell.00398.2002
Received 29 August 2002; accepted in final form 7 October 2002.
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