1 Division of Nephrology, Department of Pediatrics, and 2 Division of Nephrology, Departments of Medicine and of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94143-0532; and 3 Department of Pediatrics, Oregon Health and Science University, Portland, Oregon 97201
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Serum and glucocorticoid-regulated
kinase-1 (SGK1) is a serine-threonine kinase that is regulated at the
transcriptional level by numerous regulatory inputs, including
mineralocorticoids, glucocorticoids, follicle-stimulating hormone, and
osmotic stress. In the distal nephron, SGK1 is induced by aldosterone
and regulates epithelial Na+ channel-mediated
transepithelial Na+ transport. In other tissues, including
liver and shark rectal gland, SGK1 is regulated by hypertonic stress
and is thought to modulate epithelial Na+ channel- and
Na+-K+-2Cl cotransporter-mediated
Na+ transport. In this report, we examined the regulation
of SGK1 mRNA and protein expression and Na+ currents in
response to osmotic stress in A6 cells, a cultured cell line derived
from Xenopus laevis distal nephron. We found that
in contrast to hepatocytes and rectal gland cells, hypotonic conditions
stimulated SGK1 expression and Na+ transport in A6 cells.
Moreover, a correlation was found between SGK1 induction and the later
phase of activation of Na+ transport in response to
hypotonic treatment. When A6 cells were pretreated with an inhibitor of
phosphatidylinositol 3-kinase (PI3K), Na+ transport was
blunted and only inactive forms of SGK1 were expressed. Surprisingly,
these results demonstrate that both hypertonic and hypotonic stimuli
can induce SGK1 gene expression in a cell type-dependent fashion.
Moreover, these data lend support to the view that SGK1 contributes to
the defense of extracellular fluid volume and tonicity in amphibia by
mediating a component of the hypotonic induction of distal nephron
Na+ transport.
serum and glucocorticoid-regulated kinase; osmoregulation; sodium transport; phosphatidylinositol-3-kinase; epithelial sodium channel
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
THE DISTAL NEPHRON (DN) of the kidney plays a central role in maintaining extracellular fluid (ECF) volume and tonicity in terrestrial and freshwater vertebrates. It performs this function by mediating transepithelial Na+ and water transport in response to regulatory factors such as aldosterone, insulin, atrial-natriuetic peptide, and osmotic stress (2, 17-19). The adaptation of the DN to changes in an organism's surrounding environment is thought to have played a critical role in vertebrate evolution and the survival of euryhaline species (e.g., salmon and tilapia) during migration between saltwater and brackish water or freshwater (3, 7). Moreover, renal mechanisms coordinating conservation of Na+ and water were critical to the evolution and survival of amphibia and higher vertebrates on land. In humans, disruption of the DN pathways promoting Na+ and water reabsorption has been associated with several genetic disorders, including Liddle's syndrome (20), apparent mineralocorticoid excess (55), pseudohypoaldosteronism (10), and nephrogenic diabetes insipidis (37).
Na+ transport also plays a critical role in maintaining the
volume of individual cells. In contrast to regulatory actions on the DN
from hormonal influences, Na+ transport in individual cells
is altered in response to changes in intracellular volume
(9). When intracellular volume is decreased in cells
exposed to hypertonic conditions, cells restore appropriate volume by
initiating a cascade of events that includes Na+ influx
from the extracellular into the intracellular space (32). An example of this phenomenon is illustrated by hepatocytes, which respond to osmotic loads within the portal circulation by increasing Na+ influx to preserve cellular volume (21,
51). The intracellular mechanisms involved in these responses
are complex, utilizing several Na+, K+,
Cl channels and/or transporters in conjunction with water
channels (6).
The cellular mechanisms that control the volume of individual cells are potentially in conflict with mechanisms that modulate ECF volume and tonicity. For example, Na+ influx serves as a component of the regulatory volume increase in cells exposed to hypertonic conditions (6, 51); however, Na+ entry into DN epithelial cells is an essential step in the defense of ECF volume and tonicity in response to hypotonic conditions (29, 53). Recently, serum and glucocorticoid-regulated kinase-1 (SGK1) (50), a serine-threonine kinase, has been independently isolated by several laboratories as an early-response gene that is induced by nonhormonal and hormonal factors involved in stress responses. Hypertonic stress has been shown to increase SGK1 mRNA or protein levels in nonrenal epithelia such as human hepatocytes (47), acinar cells of the pancreas (25), mouse mammary tumor cells (4), and the shark rectal gland (48). SGK1 mRNA and/or protein expression also is induced by aldosterone in the DN of several species (11, 31) as well as in the mammalian colon (39).
SGK1 affects ion transport in pathways mediated by the
amiloride-sensitive epithelial Na+ channel (ENaC) and
furosemide-sensitive Na+-K+-2Cl
cotransporter (11, 25). Several recent reports have begun to elucidate the mechanisms by which SGK1 mediates the effects of
mineralocorticoids on ENaC-mediated Na+ transport.
Aldosterone increases DN SGK1 mRNA and protein expression substantially
within 15-30 min (5, 35, 43). This precedes and
correlates with the so-called "early phase" of aldosterone action
(30 min-3 h), during which the augmentation of transepithelial Na+ transport in the DN occurs almost exclusively by
increasing the activity of apical ENaC and does not occur via other
effectors of Na+ transport, such as
Na+-K+-ATPase (22, 33). SGK1, in
turn, has been shown to increase ENaC-mediated Na+
transport as well as ENaC localization to the plasma membrane in an
oocyte coexpression system (1, 27). Moreover, recent evidence establishing that SGK1 knockout mice are unable to conserve Na+ further substantiates the importance of SGK1 in
mediating mineralocorticoid effects (56).
These same time frames have been observed with renal tubular A6 cells, a well-established cell line for studying ENaC-mediated Na+ transport of Xenopus laevis DN origin (38). In A6 cells, the regulation of SGK1 and its correlation with aldosterone-induced Na+ transport depend on two features: 1) abundance of the enzyme is transcriptionally regulated by corticosteroids and 2) activation of the kinase requires phosphorylation of specific threonine and serine residues by the phosphoinositide-dependent kinases (23, 49). These same regulatory principles of SGK1 appear to be essential in other cell types, such as Hep G2, HEK-239, and mammary tumor cells, but with hypertonic stress as the condition inducing SGK1 gene expression (34, 46, 51).
We were interested in studying the effect of osmotic stress on SGK1 expression in A6 cells. However, because hypotonic stress induces amiloride-sensitive Na+ transport in these cells, we postulated that if SGK1 were to play a role in mediating effects on Na+ transport, its expression would correspondingly be induced by this treatment, in contrast to other cell types. We further sought to characterize potential second- messenger systems involved in osmotic control of SGK1 expression and Na+ transport in this cell type.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture. A6 cells were obtained from the American Tissue Culture Collection (Manassas, VA). Cells were grown as previously described in DMEM with 5% fetal bovine serum (FBS) at ~250 mosM, an osmolarity suitable for growth of these amphibian cells. Maintenance growth conditions included humidified air at 30°C with 1% CO2.
Na+ transport measurements. A6 cells were plated onto 24-mm-diameter Transwell plates (Costar, Corning, NY) with 0.4-µm permeabilized membranes coated with collagen type VI (Sigma, St. Louis, MO). Cells were plated at a concentration of 1.0 × 106 cells/cm2, grown for 7-10 days until formation of tight epithelia with an additional 2-3 days of growth in A6 media with 5% FBS stripped of endogenous steroids, and prepared by a previously described charcoal treatment (12). By using a voltage meter (Millicell-ERS, Millipore, Bedford, MA) to measure potential difference and resistance across the layer of A6 epithelia, equivalent current [potential difference/resistance (PD/R)] was calculated to estimate Na+ transport. Amiloride was used to verify that currents were ENaC dependent.
Osmotic studies. A6 cells were treated with hypotonic, isotonic, and hypertonic media. All media contained 5% FBS stripped of endogenous steroids. Hypotonic media were set at ~150 or 200 mosM as follows: 150 mosM media included (in mM) 56.7 NaCl, 7.7 Na HCO3, and 5.3 KCl as the major osmolar components, in addition to other components previously described (12); and 200 mosM media included 86 mM NaCl with no other differences compared with 150 mosM media. Isotonic media were prepared in two ways, standard A6 media at ~250 mosM and 150 mosM media with a supplement of 100 mM sorbitol. Solutions of 300 and 350 mosM were prepared as standard A6 media supplemented with 50 and 100 mM sorbitol, respectively.
Pharmacological agents. Reagents used to study Na+ transport and SGK1 expression are referenced here according to supplier, preparative concentration, and experimental concentration, respectively: dexamethasone, Sigma, 100 µM in ethanol, 100 nM; LY-294002, Calbiochem (La Jolla, CA), 50 mM in DMSO, 50 µM; amiloride, Sigma, 50 mM in ethanol, 50 µM; cycloheximide, Sigma, 20 mM in ethanol, 20 µM; and actinomycin D, Sigma, 5 mg/ml in water, 5 µg/ml.
Detection and quantitation of SGK1 mRNA.
Total RNA was isolated from A6 cells by using RNA Stat-60 (Tel-Test,
Friendswood, TX) according to the manufacturer's instruction. RNA was
electrophoretically separated on a 0.95% agarose gel containing 2.2 M
formaldehyde, transferred to a nylon membrane (Hybond-NX, Amersham-Pharmacia, Piscataway, NJ), and cross-linked to the membrane by ultraviolet cross-linking (Strategene UV Crosslinker, Stratagene, La
Jolla, CA). 32P-radiolabeled probes of X. laevis
SGK1 and -actin were produced by using a random primer method of
incorporating [
-32P]dCTP into complementary strands of
the respective cDNA (Prime-a-Gene, Promega, Madison, WI). After
3-6 h of incubation at 42°C in a standard prehybridization
solution supplemented with 100 µg/ml denatured salmon testes DNA,
membranes were exposed overnight to 2 × 106
counts · min
1 · ml
1
radiolabeled probe in a 50% formamide standard hybridization solution
at 42°C. Membranes were rinsed and washed twice in 2× SSC-0.1% SDS
at 50°C before undergoing autoradiography.
Detection of phosphorylated and unphosphorylated forms of SGK1. Protein from A6 cells grown on Transwell filters was isolated by using a lysis buffer and protocol described previously (12). By using 70 µg of protein from a given sample, protein was separated by 7.5% SDS-PAGE and electroblotted to nitrocellulose membranes (Micron Separations, Westborough, MA). After nonspecific binding was reduced by 3 h preincubation in 5% dry milk dissolved in a PBS-0.1% Tween 20 (PBS-T) solution, the membranes were exposed overnight to a 1:500 rabbit polyclonal anti-rat SGK1 antibody (courtesy of Dr. Gary Firestone, University of California, Berkeley, CA). The membranes were then washed with PBS-T solution and exposed to rabbit Ig-horseradish peroxidase-linked whole antibody (Amersham-Pharmacia) at 1:5,000 in 7.5% dry milk in PBS-T solution for 1 h at room temperature. After another wash with PBS-T solution, bound antibody was detected by a chemiluminescence protocol (ECL; Amersham-Pharmacia). Phosphorylated and unphosphorylated forms of SGK1 were distinguished by size because of their different migrations during electrophoresis (12, 49).
Statistical analysis. Significance was determined by using a two-tailed Student's t-test to compare test samples with control samples. A P value of <0.01 was considered significant.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of hypotonic and hypertonic stress on Na+ transport in A6 cells. When grown on permeable supports, A6 cells form a high-resistance monolayer that is useful in the study of transepithelial Na+ transport. Previous work demonstrated that a variety of physiologically relevant factors, including corticosteroids, vasopressin, insulin, and osmotic stress, stimulate amiloride-sensitive transepithelial Na+ transport (15, 38). However, unlike other cell types, hypotonic conditions, rather than hypertonic conditions, stimulated A6 Na+ transport (52).
We initially compared the effects of osmotic stress and corticosteroids on the electrical properties of A6 monolayers by using PD/R as a surrogate for short-circuit current (SCC). To establish optimal osmotic conditions to stimulate Na+ current, we first performed a dose-response experiment, varying the medium osmolarity from 150 (hypotonic) to 350 mosM (hypertonic) by altering the NaCl concentration; an osmolarity of 250 mosM was considered isotonic and equal to the tonicity of the growth medium. As shown in Fig. 1A, exposing the basolateral membrane to hypotonic conditions stimulated PD/R, whereas hypertonic conditions decreased it. PD/R was significantly increased by osmolarity of 200 mosM and further increased by osmolarity of 150 mosM. In contrast, osmotic changes to the medium bathing the apical surface of the monolayers had no apparent effect on current, as previously demonstrated (14, 36, 52). Moreover, PD/R was not appreciably induced over basal when medium NaCl concentration was reduced while osmolarity was maintained at 250 mosM by addition of sorbitol (data not shown). This latter result confirms that Na+ current was induced by changes in osmolarity, not by changes in Na+ concentration per se (52).
|
Effect of hypotonic stress on SGK1 mRNA and protein expression.
SGK1 is strongly stimulated by hypertonic stress in a variety of cell
types, as well as by aldosterone and dexamethasone in A6 cells
(4, 11, 45, 46). In all of the cases that have been
examined, the induction of SGK1 paralleled the induction of
Na+ transport. With this in mind, we next examined the
effect of osmotic stress on SGK1 expression in A6 cells. A6 cells were
grown to high resistance on permeable supports, exposed to hypotonic (150 mosM), isotonic (250 mosM), or hypertonic (350 mosM) conditions, and RNA and protein were harvested at the time points shown in Fig.
2. In contrast to other cell types, but
consistent with its effect on Na+ current, hypotonic stress
induced SGK1 expression in A6 cells. Hypertonic stress, on the other
hand, appeared to reduce SGK1 mRNA, although the background was too
high to conclude this with certainty (Fig. 2 and data not shown). As
with induction of the Na+ current, no effect was seen when
the osmotic gradient was applied to the apical membrane. As shown in
Fig. 2B, SGK1 mRNA was induced within 15 min and peaked
around 45 min after hypotonic treatment, a time course similar to that
observed for dexamethasone induction of SGK1 mRNA in these same cells
(11).
|
Effect of inhibition of transcription or protein synthesis on hypotonic induction of Na+ transport and SGK1 expression. The initial rise in PD/R in response to hypotonic treatment occurred within 5 min and preceded the induction of either SGK1 mRNA or protein (Figs. 1B and 2, B and D). PD/R during the later phase after osmotic treatment (>30 min) paralleled the response to dexamethasone treatment, a treatment in which induction of PD/R is known to be dependent on changes in gene transcription. In light of the strong induction of SGK1 protein by osmotic stress and the evidence supporting a role for SGK1 in stimulating ENaC-mediated Na+ transport (11, 27, 31, 39), we wanted to examine the possibility that transcriptional induction of SGK1 might play a role in the second phase of hypotonic induction of PD/R (between 30 and 120 min).
We therefore examined the effect of inhibiting gene transcription on hypotonic induction of PD/R and SGK1 expression. Thirty minutes before the addition of hypotonic media, A6 monolayers were treated with actinomycin D (5 µg/ml) or vehicle, and the time course of PD/R was followed. As shown in Fig. 3, during the first 30 min after hypotonic treatment, the rise in PD/R appeared similar in monolayers treated without or with actinomycin D, suggesting that the rise in current during this time period does not require transcriptional events. However, after 30 min, the two curves began to diverge, with the actinomycin D-treated monolayers demonstrating a more flattened response compared with cells not treated with the inhibitor. Figure 3 also shows that inhibition of transcription with actinomycin D did not affect PD/R under isotonic conditions, supporting the conclusion that the cells remained healthy with intact Na+ translocation mechanisms during the time course of the experiment. Figure 3 also reveals the strong dependence of steroid induction of PD/R on changes in gene transcription.
|
|
Phosphatidylinositol 3-kinase inhibition blunts and delays
hypotonic induction of Na+ transport.
Because dexamethasone induction of SGK1 activity and Na+
transport has been shown to be phosphatidylinositol 3-kinase (PI3K) dependent, we investigated whether the correlation between SGK1 and
Na+ transport persisted under hypotonic conditions. A6
monolayers were pretreated with the PI3K inhibitor LY-294002
(44) or vehicle 30 min before altering of medium tonicity.
As shown in Fig. 5, PI3K inhibition
reduced baseline SCC by ~60% and completely prevented the early
increase (<30 min) of PD/R in response to hypotonic media (Fig.
5A). Moreover, the later sustained rise in PD/R (after 30 min) in response to hypotonic conditions was also markedly blunted by
LY-294002, to an extent equal to or greater than the inhibition of the
dexamethasone-induced current (Fig. 5, A and B).
Cells harvested for protein 1 h after addition of stimulus in the
presence or absence of LY-294002 were then probed in immunoblots with
SGK1-specific antibody (Fig. 5C). Although hypotonic media and dexamethasone each increased the abundance of SGK1, it is notable
that in the presence of PI3K inhibitor, only the unphosphorylated form
of the SGK1 was expressed.
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Hypotonic induction of SGK1 in A6 cells correlates with increased Na+ transport. Similar to previous investigations, the present study found that SGK1 gene expression is osmotically regulated. However, in marked contrast to previous reports in which hypertonic conditions in the shark rectal gland and hepatocytes induced SGK1 mRNA and protein levels, our results demonstrate that SGK1 expression is increased under hypotonic conditions in renal tubular cells of amphibian origin. The study further provided evidence that augmented SGK1 expression correlates with activation of Na+ transport, a finding that may have evolutionary significance.
The osmotic induction of SGK1 mRNA and protein in A6 cells occurred rapidly and paralleled the induction seen by corticosteroids (Fig. 2). SGK1 mRNA began to increase within 15 min and protein within 30 min of exposure to hypotonic conditions. The data (Fig. 1) also confirm previous reports that hypotonicity induced a rapid and marked rise in amiloride-sensitive Na+ current. The current increased within 5 min, and its initial rise within the first 30 min was not blocked by inhibitors of mRNA or protein synthesis. The later phase of hypotonic-induced Na+ current (after 30 min and up to 120 min), by contrast, was blunted by inhibition of gene transcription or protein synthesis (Figs. 3 and 4A). Indeed, as revealed in Fig. 4B, the component of PD/R induced by hypotonicity that is dependent on new protein synthesis is similar in its magnitude and time course to the increase observed after exposure to corticosteroids. The data therefore lend support to the hypothesis that transcriptional induction of SGK1 plays a role in the sustained increase in Na+ current across A6 monolayers 30 min after exposure to corticosteroids or hypotonic conditions. Although it is important to emphasize that the present observations between increased SGK1 expression and increased Na+ transport are not causal but rather only correlative, the parallel between these two observations has remained consistent across cell types despite the contrasting stimuli such as hypotonicity and hypertonicity. Moreover, this consistency remains functionally appropriate for the homeostatic needs of the organism. In amphibia and other freshwater or euryhaline vertebrates, the ability of the DN to reabsorb Na+ and generate dilute urine is essential to the adaptation of decreases in environmental tonicity. This is particularly true for the euryhalines, which migrate between saltwater and freshwater; however, amphibia, such as X. laevis, are exposed to less extreme transitions between brackish water and freshwater. Thus it is physiologically appropriate for Na+ transport to increase in the DN in response to reduced ECF tonicity, a response that has been described for isolated frog kidney tubule, as well as for other natriferic amphibian tissues including frog skin, toad bladder, and salamander kidney (26, 28, 30, 41, 42). In contrast, in elasmobranchs (sharks, skates, and rays), the transition to saltwater is buffered by the action of the rectal gland, a NaCl-excreting organ that in many regards functions oppositely to the DN (40). Finally, hepatocytes must respond to large feeding-dependent changes in osmotic load via the portal circulation and rely on Na+ influx, mediated by the ENaC and the Na+-K+-2ClMechanism of SGK1 induction by hypotonic stress. SGK1 activation by hypotonic stress in A6 cells appears to have little effect on the degradation of mRNA, suggesting that the increase in mRNA abundance under these conditions is mediated transcriptionally. The mechanism underlying hypotonic induction of SGK1 transcription remains unknown. More is known about its hypertonic induction, which appears to involve p38 mitogen-activated protein kinase (MAPK) acting through an Sp1-like site in the SGK1 5'-flanking region (4, 48). Initial inhibitor experiments (not shown) suggest that hypotonic stress does not require p38 MAPK, because it (unlike the effect of hypertonic stress on SGK1 expression in mammary epithelial cells) is not inhibited by MAPK antagonists SB-202190 or SB-203580. Regardless of the physiological role of SGK1 in mediating osmotic effects on Na+ transport, it appears that SGK1 gene transcription can be oppositely regulated by osmotic stress in a cell type-dependent fashion. This is, to our knowledge, the first description of such paradoxical regulation by a single stimulus of an endogenous gene's transcription. It will be of mechanistic interest to determine how these opposing signals are channeled into a common effect on the SGK1 gene.
Role of PI3K in hypotonic induction of Na+ transport. In contrast to gene transcription, which appears to be implicated only in the later phase of the hypotonic induction of Na+ current in A6 cells, PI3K activity is required for both the initial and the later phases of the response. Because both the kinase activity and the phosphorylation state of SGK1 protein appear to be PI3K dependent (Fig. 5) (23, 34), it is tempting to speculate that SGK1 is implicated in the early, transcription-independent phase of osmotic induction. However, initial levels of SGK1 are low and might not be adequate to mediate the early effect. Moreover, there are several other PI3K-dependent kinases, most notably SGK2, SGK3, and protein kinase B/Akt, that could be implicated (24). Indeed, recent evidence suggests that SGK2 and SGK3, but not protein kinase B/Akt, stimulate ENaC-mediated Na+ transport (Lang F, personal communication). Additional studies will be needed to examine the roles of these and other kinases in mediating the effects under hypotonic conditions. Whichever the mechanism, our data suggest that two distinct, convergent pathways are required for the full range of Na+ transport regulation: 1) a PI3K-dependent pathway that promotes the active form of SGK1 and/or other related kinases and 2) a transcriptional pathway that increases the abundance of SGK1 (and possibly other transport regulators).
Role of differential osmotic regulation of SGK1 in evolution. Although A6 cells are the first cell line in which hypotonic treatment has been shown to induce SGK1, the role of hypotonic activation of Na+ transport has been known for years in amphibia and other freshwater vertebrates as they adapt to osmotic changes in their environments. Indeed, Ussing originally proposed a link between increased Na+ transport and hypotonic stress in frog skin nearly 40 years ago (28, 42), with subsequent reports of this linkage made in the frog kidney tubule (30), toad bladder (26), and larval salamander kidney (41). It is thus not surprising that A6 cells, derived from the DN of the freshwater African claw-toed frog (52), show similar behavior. We have examined whether osmotic stress stimulates Na+ transport or SGK1 expression in renal tubular cells of mammals. Interestingly, neither hypotonic nor hypertonic conditions had much effect on Na+ transport and, importantly, on SGK1 expression (Rozansky DJ, unpublished observations).
SGK homologues have been identified in all eukaryotic species examined, including Saccharomyces cerravisiae (8), Caenorhabditis elegans (54), and a variety of vertebrates (11, 31, 46, 47, 50). Although SGK1 regulation in invertebrates and yeast has not been extensively studied, it is known that yeast has two SGK homologues, YPK1 and YKR2, which appear to be at least partially redundant. Yeast deficient in both these genes is nonviable, although the cause of the lethal phenotype remains unknown. Interestingly, one study found that the YPK1/YKR2-deficient yeast can be rescued by expression of rat SGK1 (8). Taken together with our present data, these observations suggest the possibility that SGK1 has served throughout eukaryotic evolution as a facilitator of volume homeostasis, initially of individual cells and subsequently of ECF. Perhaps in amphibia, tonicity is the primary regulator of DN SGK1 expression through its direct effects on DN cells and through the renin-angiotensin-aldosterone system, which appears to be regulated by tonicity in amphibia (53). In mammals, the regulation of tonicity and ECF volume is independent, and tonicity per se appears to have little effect on either DN Na+ transport or SGK1 expression. Rather, tonicity is regulated primarily by vasopressin (which affects mainly water transport), whereas ECF volume is regulated primarily by the renin-angiotensin-aldosterone system through its tonicity-independent effects on DN ion transport. ![]() |
ACKNOWLEDGEMENTS |
---|
This work was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (1K08-DK-02723 to D. J. Rozansky and R01-DK-51151 and R01-DK-56695 to D. Pearce), National Kidney Foundation (D. J. Rozansky) and American Heart Association Western States Affiliate (9950699Y to D. Pearce).
![]() |
FOOTNOTES |
---|
Address for reprint requests and other correspondence: D. Pearce, Division of Nephrology, Dept. of Medicine and Dept. of Cellular and Molecular Pharmacology, Box 0532, Univ. of California, San Francisco, CA 94143-0532 (E-mail: pearced{at}medicine.ucsf.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 February 19, 2002;10.1152/ajprenal.00176.2001
Received 6 June 2001; accepted in final form 7 February 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Alvarez de la Rosa, D,
Zhang P,
Naray-Fejes-Toth A,
Fejes-Toth G,
and
Canessa CM.
The serum and glucocorticoid kinase sgk increases the abundance of epithelial sodium channels in the plasma membrane of Xenopus oocytes.
J Biol Chem
274:
37834-37839,
1999
2.
Ausiello, DA,
Skorecki KL,
Verkman AS,
and
Bonventre JV.
Vasopressin signaling in kidney cells.
Kidney Int
31:
521-529,
1987[ISI][Medline].
3.
Barron, MG.
Endocrine control of smoltification in anadromous salmonids.
J Endocrinol
108:
313-319,
1986[Abstract].
4.
Bell, LM,
Leong ML,
Kim B,
Wang E,
Park J,
Hemmings BA,
and
Firestone GL.
Hyperosmotic stress stimulates promoter activity and regulates cellular utilization of the serum- and glucocorticoid-inducible protein kinase (Sgk) by a p38 MAPK-dependent pathway.
J Biol Chem
275:
25262-25272,
2000
5.
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
6.
Bohmer, C,
Wagner CA,
Beck S,
Moschen I,
Melzig J,
Werner A,
Lin JT,
Lang F,
and
Wehner F.
The shrinkage-activated Na(+) conductance of rat hepatocytes and its possible correlation to rENaC.
Cell Physiol Biochem
10:
187-194,
2000[ISI][Medline].
7.
Bray, AA.
The evolution of the terrestrial vertebrates: environmental and physiological considerations.
Philos Trans R Soc Lond B Biol Sci
309:
289-322,
1985[ISI][Medline].
8.
Casamayor, A,
Torrance PD,
Kobayashi T,
Thorner J,
and
Alessi DR.
Functional counterparts of mammalian protein kinases PDK1 and SGK in budding yeast.
Curr Biol
9:
186-197,
1999[ISI][Medline].
9.
Chamberlin, ME,
and
Strange K.
Anisosmotic cell volume regulation: a comparative view.
Am J Physiol Cell Physiol
257:
C159-C173,
1989
10.
Chang, SS,
Grunder S,
Hanukoglu A,
Rosler A,
Mathew PM,
Hanukoglu I,
Schild L,
Lu Y,
Shimkets RA,
Nelson-Williams C,
Rossier BC,
and
Lifton RP.
Mutations in subunits of the epithelial sodium channel cause salt wasting with hyperkalaemic acidosis, pseudohypoaldosteronism type 1.
Nat Genet
12:
248-253,
1996[ISI][Medline].
11.
Chen, SY,
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
12.
Chen, SY,
Wang J,
Liu W,
and
Pearce D.
Aldosterone responsiveness of A6 cells is restored by cloned rat mineralocorticoid receptor.
Am J Physiol Cell Physiol
274:
C39-C46,
1998
13.
Crowe, WE,
Ehrenfeld J,
Brochiero E,
and
Wills NK.
Apical membrane sodium and chloride entry during osmotic swelling of renal (A6) epithelial cells.
J Membr Biol
144:
81-91,
1995[ISI][Medline].
14.
De Smet, P,
Simaels J,
and
Van Driessche W.
Regulatory volume decrease in a renal distal tubular cell line (A6). II Effect of Na+ transport rate.
Pflügers Arch
430:
945-953,
1995[ISI][Medline].
15.
Edinger, RS,
Rokaw MD,
and
Johnson JP.
Vasopressin stimulates sodium transport in A6 cells via a phosphatidylinositide 3-kinase-dependent pathway.
Am J Physiol Renal Physiol
277:
F575-F579,
1999
16.
Ehrenfeld, J,
Raschi C,
and
Brochiero E.
Basolateral potassium membrane permeability of A6 cells and cell volume regulation.
J Membr Biol
138:
181-195,
1994[ISI][Medline].
17.
Fidelman, ML,
May JM,
Biber TU,
and
Watlington CO.
Insulin stimulation of Na+ transport and glucose metabolism in cultured kidney cells.
Am J Physiol Cell Physiol
242:
C121-C123,
1982
18.
Funder, JW.
Aldosterone action: new answers, new questions.
Mol Cell Endocrinol
151:
1-3,
1999[ISI][Medline].
19.
Garty, H,
and
Palmer LG.
Epithelial sodium channels: function, structure, and regulation.
Physiol Rev
77:
359-396,
1997
20.
Hansson, JH,
Nelson-Williams C,
Suzuki H,
Schild L,
Shimkets R,
Lu Y,
Canessa C,
Iwasaki T,
Rossier B,
and
Lifton RP.
Hypertension caused by a truncated epithelial sodium channel gamma subunit: genetic heterogeneity of Liddle syndrome.
Nat Genet
11:
76-82,
1995[ISI][Medline].
21.
Haussinger, D,
Lang F,
and
Gerok W.
Regulation of cell function by the cellular hydration state.
Am J Physiol Endocrinol Metab
267:
E343-E355,
1994
22.
Helman, SI,
Liu X,
Baldwin K,
Blazer-Yost BL,
and
Els WJ.
Time-dependent stimulation by aldosterone of blocker-sensitive ENaCs in A6 epithelia.
Am J Physiol Cell Physiol
274:
C947-C957,
1998
23.
Kobayashi, T,
and
Cohen P.
Activation of serum- and glucocorticoid-regulated protein kinase by agonists that activate phosphatidylinositide 3-kinase is mediated by 3- phosphoinositide-dependent protein kinase-1 (PDK1) and PDK2.
Biochem J
339:
319-328,
1999[ISI][Medline].
24.
Kobayashi, T,
Deak M,
Morrice N,
and
Cohen P.
Characterization of the structure and regulation of two novel isoforms of serum- and glucocorticoid-induced protein kinase.
Biochem J
344:
189-197,
1999[ISI][Medline].
25.
Lang, F,
Klingel K,
Wagner CA,
Stegen C,
Warntges S,
Friedrich B,
Lanzendorfer 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
Broer S.
Deranged transcriptional regulation of cell-volume-sensitive kinase hSGK in diabetic nephropathy.
Proc Natl Acad Sci USA
97:
8157-8162,
2000
26.
Lipton, P.
Effect of changes in osmolarity on sodium transport across isolated toad bladder.
Am J Physiol
222:
821-828,
1972
27.
Loffing, J,
Zecevic M,
Feraille E,
Kaissling B,
Asher C,
Rossier BC,
Firestone GL,
Pearce D,
and
Verrey F.
Aldosterone induces rapid apical translocation of ENaC in early portion of renal collecting system: possible role of SGK.
Am J Physiol Renal Physiol
280:
F675-F682,
2001
28.
MacRobbie, E,
and
Ussing H.
Osmotic behavior of the epithelial cells of frog skin.
Acta Physiol Scand
53:
348-365,
1961[ISI].
29.
Mayer, N.
Adaptation de Rana escuelta a des milieux varies. Etude speciale de L'excretion renale del'eau et deselectrolytes au cours des chngements de milieux.
Comp Biochem Physiol
29:
27-50,
1969[ISI][Medline].
30.
McBean, RL,
and
Goldstein L.
Renal function during osmotic stress in the aquatic toad Xenopus laevis.
Am J Physiol
219:
1115-1123,
1970
31.
Naray-Fejes-Toth, A,
Canessa C,
Cleaveland ES,
Aldrich G,
and
Fejes-Toth G.
sgk is an aldosterone-induced kinase in the renal collecting duct effects on epithelial Na+ channels.
J Biol Chem
274:
16973-16978,
1999
32.
O'Neill, WC.
Physiological significance of volume-regulatory transporters.
Am J Physiol Cell Physiol
276:
C995-C1011,
1999
33.
Paccolat, MP,
Geering K,
Gaeggeler HP,
and
Rossier BC.
Aldosterone regulation of Na+ transport and Na+-K+-ATPase in A6 cells: role of growth conditions.
Am J Physiol Cell Physiol
252:
C468-C476,
1987
34.
Park, J,
Leong ML,
Buse P,
Maiyar AC,
Firestone GL,
and
Hemmings BA.
Serum and glucocorticoid-inducible kinase (SGK) is a target of the PI 3-kinase-stimulated signaling pathway.
EMBO J
18:
3024-3033,
1999
35.
Pearce, D,
Verrey F,
Chen SY,
Mastroberardino L,
Meijer OC,
Wang J,
and
Bhargava A.
Role of SGK in mineralocorticoid-regulated sodium transport.
Kidney Int
57:
1283-1289,
2000[ISI][Medline].
36.
Perkins, FM,
and
Handler JS.
Transport properties of toad kidney epithelia in culture.
Am J Physiol Cell Physiol
241:
C154-C159,
1981[Abstract].
37.
Rosenthal, W,
Seibold A,
Antaramian A,
Lonergan M,
Arthus MF,
Hendy GN,
Birnbaumer M,
and
Bichet DG.
Molecular identification of the gene responsible for congenital nephrogenic diabetes insipidus.
Nature
359:
233-235,
1992[ISI][Medline].
38.
Schmidt, TJ,
Husted RF,
and
Stokes JB.
Steroid hormone stimulation of Na+ transport in A6 cells is mediated via glucocorticoid receptors.
Am J Physiol Cell Physiol
264:
C875-C884,
1993
39.
Shigaev, A,
Asher C,
Latter H,
Garty H,
and
Reuveny E.
Regulation of sgk by aldosterone and its effects on the epithelial Na(+) channel.
Am J Physiol Renal Physiol
278:
F613-F619,
2000
40.
Silva, P,
Solomon RJ,
and
Epstein FH.
Transport mechanisms that mediate the secretion of chloride by the rectal gland of Squalus acanthias.
J Exp Zool
279:
504-508,
1997[ISI][Medline].
41.
Stiffler, DF,
and
Alvarado RH.
Renal function in response to reduced osmotic load in larval salamanders.
Am J Physiol
226:
1243-1249,
1974
42.
Ussing, H.
Relationship between osmotic reactions and active sodium transport in the frog skin epithelium.
Acta Physiol Scand
63:
141-155,
1964[ISI].
43.
Verrey, F.
Early aldosterone action: toward filling the gap between transcription and transport.
Am J Physiol Renal Physiol
277:
F319-F327,
1999
44.
Vlahos, CJ,
Matter WF,
Hui KY,
and
Brown RF.
A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002).
J Biol Chem
269:
5241-5248,
1994
45.
Waldegger, S,
Barth P,
Forrest JN, Jr,
Greger R,
and
Lang F.
Cloning of sgk serine-threonine protein kinase from shark rectal glanda gene induced by hypertonicity and secretagogues.
Pflügers Arch
436:
575-580,
1998[ISI][Medline].
46.
Waldegger, S,
Barth P,
Raber G,
and
Lang F.
Cloning and characterization of a putative human serine/threonine protein kinase transcriptionally modified during anisotonic and isotonic alterations of cell volume.
Proc Natl Acad Sci USA
94:
4440-4445,
1997
47.
Waldegger, S,
Erdel M,
Nagl UO,
Barth P,
Raber G,
Steuer S,
Utermann G,
Paulmichl M,
and
Lang F.
Genomic organization and chromosomal localization of the human SGK protein kinase gene.
Genomics
51:
299-302,
1998[ISI][Medline].
48.
Waldegger, S,
Gabrysch S,
Barth P,
Fillon S,
and
Lang F.
h-sgk serine-threonine protein kinase as transcriptional target of p38/MAP kinase pathway in HepG2 human hepatoma cells.
Cell Physiol Biochem
10:
203-208,
2000[ISI][Medline].
49.
Wang, J,
Barbry P,
Maiyar AC,
Rozansky DJ,
Bhargava A,
Leong ML,
Firestone GL,
and
Pearce D.
SGK regulates insulin and mineralocorticoid regulation of epithelial sodium transport.
Am J Physiol Renal Physiol
280:
F303-F313,
2001
50.
Webster, MK,
Goya L,
Ge Y,
Maiyar AC,
and
Firestone GL.
Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum.
Mol Cell Biol
13:
2031-2040,
1993[Abstract].
51.
Wehner, F,
Sauer H,
and
Kinne RK.
Hypertonic stress increases the Na+ conductance of rat hepatocytes in primary culture.
J Gen Physiol
105:
507-535,
1995[Abstract].
52.
Wills, NK,
Millinoff LP,
and
Crowe WE.
Na+ channel activity in cultured renal (A6) epithelium: regulation by solution osmolarity.
J Membr Biol
121:
79-90,
1991[ISI][Medline].
53.
Wilson, JX.
The renin-angiotensin system in nonmammalian vertebrates.
Endocr Rev
5:
45-61,
1984[Abstract].
54.
Wilson, R,
Ainscough R,
Anderson K,
Baynes C,
Berks M,
Bonfield J,
Burton J,
Connell M,
Copsey T,
Cooper J,
Coulson A,
Craxton M,
Dear S,
Du Z,
Durbin R,
Favello A,
Fraser A,
Fulton L,
Gardner A,
Green P,
Hawkins T,
Hillier L,
Jier M,
Johnston L,
Jones M,
Kershaw J,
Kirsten J,
Laisster N,
Latreille P,
Lightning J,
Lloyd C,
Mortimore B,
O'Callaghan M,
Parsons J,
Percy C,
Rifken L,
Roopra A,
Saunders D,
Shownkeen R,
Sims M,
Smaldon N,
Smith A,
Smith M,
Sonnhammer E,
Staden R,
Sulston J,
Thierry-Mieg J,
Thomas K,
Vaudin M,
Vaughan K,
Waterson R,
Watson A,
Weinstock L,
Wilkinson-Sproat J,
and
Wohldman P.
22 Mb of contiguous nucleotide sequence from chromosome III of C elegans.
Nature
368:
32-38,
1994[ISI][Medline].
55.
Wilson, RC,
Krozowski ZS,
Li K,
Obeyesekere VR,
Razzaghy-Azar M,
Harbison MD,
Wei JQ,
Shackleton CH,
Funder JW,
and
New MI.
A mutation in the HSD11B2 gene in a family with apparent mineralocorticoid excess.
J Clin Endocrinol Metab
80:
2263-2266,
1995[Abstract].
56.
Wulff, P,
Vallon V,
Huang D,
Pfaff I,
Klinel K,
Kauselmann VH,
Lang F,
and
Kuhl D.
Deficient salt retention in the SGK1 knockout mouse (Abstract).
J Am Soc Nephrology
12:
44A,
2001.