Sgk, a Putative Serine/Threonine Kinase, Is Differentially Expressed in the Kidney of Diabetic Mice and Humans
←
→
Page content transcription
If your browser does not render page correctly, please read the page content below
J Am Soc Nephrol 10: 2488 –2494, 1999
Sgk, a Putative Serine/Threonine Kinase, Is Differentially
Expressed in the Kidney of Diabetic Mice and Humans
JANET M. KUMAR, DAVID P. BROOKS, BARBARA A. OLSON, and
NICHOLAS J. LAPING
Department of Renal Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania.
Abstract. Differential display PCR was used to identify alter- other tissues from obese db/db mice. An increase in Sgk
nate expression of serum glucocorticoid-regulated kinase (Sgk) mRNA was also observed in the human diabetic kidney. In
mRNA in diabetes-induced renal disease. Differential expres- addition, thrombin, which may play a role in the progression of
sion of Sgk mRNA was identified in the kidneys of normal and renal disease, increased Sgk message in cell culture. Because
obese db/db mice, a model of select aspects of human diabetic the diabetes-induced increase in Sgk was only observed in the
nephropathy. Sgk mRNA was selectively increased in diabetic kidney, which is particularly susceptible to diabetes-induced
mouse kidneys. The Sgk mRNA levels remained constant in damage, Sgk may play a role in diabetic nephropathy.
Chronic renal failure is the progressive loss of functional renal tially expressed in obese db mouse kidneys. In addition, the
mass, accompanied by hypertrophy and remodeling of renal effect of thrombin, a factor implicated in chronic renal disease,
tissue. The molecular and cellular events that take place during on Sgk message expression in rat epithelial cells (REC) was
chronic renal failure include release of growth factors, mesan- examined.
gial and epithelial cell proliferation, and expansion of extra-
cellular matrix (1–3). Renal hypertrophy as a result of diabetes Materials and Methods
is due in part to high ambient glucose levels in the blood. RNA Isolation
Recent studies suggest that the effects of hyperglycemia on Total RNA was extracted from the entire kidneys, livers, brains,
mesangial matrix expansion and mesangial cell proliferation and hearts of 5-mo-old normal and diabetic db mice, strain C57KS/
may be mediated by serine/threonine protein kinases including J-m1/1Leprdb, six animals per group (Jackson Laboratories, Bar
protein kinase C and cyclin-dependent protein kinase-2 (4 – 6). Harbor, ME), and from a section of the cortex of normal and diabetic
Kinases play an essential role in the signal transduction path- patient kidneys (Department of Veterans Affairs, Case Western Re-
ways of many extracellular stimuli that ultimately affect gene serve University, Cleveland, OH). The creatinine levels for the normal
expression. Although kinase activity was thought to be largely and diabetic human kidneys were 0.1 to 1.1 mg/dl and 1.8 to 3.3
regulated by posttranslational phosphorylation, transcription- mg/dl, respectively. RNA was isolated from these samples as well as
ally regulated kinases that include the polo subfamily of serine/ the REC by guanidinium thiocyanate denaturation and acidified phe-
nol chloroform extraction (16).
threonine kinases have been described (7–12). These kinases
have been shown to be involved in cell proliferation and cell
cycle regulation. Differential Display PCR
This study was designed to identify additional transcription- Total RNA from normal and diabetic mouse kidneys was used as
ally regulated kinases that may be involved in diabetic ne- templates for reverse transcription with 200 U Superscript reverse
phropathy. Messenger RNA levels from lean and obese db transcriptase (Life Technologies/BRL, Gaithersburg, MD), 25 mM
mice, an animal model of select aspects of diabetic nephropa- dNTP, and 1 mM of a 39 primer (Table 1) at 42°C for 1 h. PCR
thy (13), were compared by differential display PCR amplification was carried out essentially as described previously
(17,18). The primers used for cDNA synthesis and PCR were de-
(DDPCR). This study found that serum glucocorticoid-regu-
signed from conserved regions of tyrosine kinase receptors using
lated kinase (Sgk), a putative serine/threonine protein kinase BLAST search algorithm. Approximately 1/5 volume of the single-
that is also transcriptionally regulated by serum, glucocorti- stranded cDNA mixture was used in the PCR reaction in combination
coids, and follicle-stimulating hormone (14,15), was differen- with 20 mM dNTP, 1 mM of each 59 and 39 primer (Table 1), 0.2 mCi
of [a-33P] dATP (2000 Ci/mmol), and 1.25 U Amplitaq polymerase
(Perkin-Elmer, Foster City, CA). Twenty-five different primer com-
Received March 30, 1998. Accepted June 2, 1999.
Dr. Kumar’s present address: Biology Department, Cabrini College, 610 King
binations were used for PCR (Table 1). The cycling parameters were
of Prussia Road, Radnor, PA 19087-3698. as follows: 40 cycles at 94°C for 30 s, 45°C for 2 min and 72°C for
Correspondence to Dr. Nicholas J. Laping, Department of Renal Pharmacology, 30 s with a final extension time of 5 min at 72°C. Amplification
UW2521, 709 Swedeland Road, Box 1539, King of Prussia, PA 19406-0939. Phone: products were resolved on a 6% denaturing polyacrylamide gel.
610-270-5310; Fax: 610-270-5381; E-mail: Nicholas_J_Laping@sbphrd.com Fragments were eluted from the gel by incubation in boiling water for
1046-6673/1012-2488 15 min, reamplified with the same primer pair used to generate them,
Journal of the American Society of Nephrology and cloned into PCRII vector (Invitrogen, Carlsbad, CA). The frag-
Copyright © 1999 by the American Society of Nephrology ments were then sequenced using the fmol® DNA sequencing systemJ Am Soc Nephrol 10: 2488 –2494, 1999 Expression of Sgk in Kidney 2489
Table 1. Sequence of primers used in differential display PCR analysis
59 Specific Primers 39 Specific Primers
#1 59-CAC TAA AGA GCG TGC #6 59-CAG TAT ACA GGT CTC
#2 59-CCT TAA GAA AAC CAC #7 59-TTA GCT TGT ACA GCT
#3 59-TGC TAG GAA CGT CCT #8 59-CCA TCA GTA TAC AGG
#4 59-GGA AGC AGC TTG CAT #9 59-CAG TAT ACA GGT TGT
#5 59-AAC CCG TAC ATC GTG #10 59-TAG GAA ACA AGT CTC
(Promega, Madison, WI). BLAST-N and FASTA programs (Smith- used for cDNA synthesis and PCR were designed from con-
Waterman algorithm) were used to compare the generated sequences served regions of tyrosine kinase receptors (using BLAST
with those in the databases. The 500-bp mouse cDNA fragment, search algorithm) (Table 1). These primers were designed to
referred to as clone RK15 and found to be homologous to the human increase the likelihood of identifying kinases that are differen-
and rat Sgk, was amplified using primer pair #3 and #10 (Table 1).
tially regulated with renal disease. After the PCR, gel electro-
phoresis, and autoradiography, 15 different gel bands showed
Cell Culture a difference in intensity between the normal and diabetic
REC were obtained from the kidney cortex glomeruli of 55- to 70-g
kidney samples. One of these fragments was a 500-bp cDNA
Sprague Dawley rats (Charles River, Wilmington, MA) as described
(19). Lyophilized human plasma thrombin (average activity 1000
whose levels increased in all three diabetic kidneys sampled
U/mg), actinomycin D, and cycloheximide were purchased from (Figure 1A). Cloning and sequencing this cDNA revealed it to
Sigma Chemical Co. (St. Louis, MO). The protein kinase inhibitor have 91% homology with the rat Sgk gene and 92% homology
RO-32-0432 (20) and peptides corresponding to the tethered ligand of with human Sgk. To confirm that expression levels increased at
the activated thrombin receptor SFLLRN, FLLRN were synthesized at the mRNA level, the 500-bp cDNA, designated RK15, was
SmithKline Beecham (King of Prussia, PA). REC were routinely used as a probe for a Northern blot containing the total RNA
cultured in RPMI 1640 containing 10% fetal bovine serum. Before from the normal and diabetic mouse kidneys. The message size
treatment, subconfluent cells were placed in serum-depleted media for was 2.4 kb, which is similar to the 2.4- to 2.6-kb message
24 h. Experiments using thrombin, actinomycin D, and cycloheximide observed in rat and human cells (14,23,24) (Figure 1B). The
were carried out in serum-free RPMI 1640. For concentrations and Northern blot results indicate that in all of the samples, Sgk
duration of thrombin treatments, see figure legends. Actinomycin D
message levels were sixfold more abundant in the diabetic
and cycloheximide were used at 5 and 10 mg/ml, respectively, for the
times indicated. After incubation in serum-free RMPI 1640, the cells
kidney compared with the normal kidney.
received 10 nM thrombin pretreatment for 2 h. The cells were treated To determine whether the diabetes-induced increase in Sgk
with the tethered ligands at 10 mM for 2 h. REC were pretreated with message was specific to the kidney, RNA from other organs
RO-32-0432 at 1 mM for 4 h. from normal and diabetic mice were examined. We observed
that the Sgk message levels were similar in the liver, brain, and
Northern Analysis heart of these animals. Of the four tissues examined in this
Approximately 10 mg of total RNA was fractionated on a 1.2% study, Sgk mRNA was increased only in the kidney of diabetic
agarose/formaldehyde gel by electrophoresis, transferred to nylon animals compared with the normal littermates (Figure 2).
membrane, prehybridized, hybridized, washed, and stripped according Sgk message in diabetic human kidneys was examined to
to an established protocol (21). The mouse Sgk fragment, used as a determine whether Sgk expression in diabetic mouse kidneys
probe in these experiments, was generated by digesting RK15 (de- resembles expression in human diabetic kidneys. Total RNA
scribed above) with EcoRI. The glyceraldehyde 3-phosphate dehy- was obtained from the kidney cortex of patients with no overt
drogenase (GAPDH) cDNA was generated by PCR according to the
renal disease and from diabetic patients. In addition, RNA
GAPDH Control Amplimer Set protocol (Clontech, Palo Alto, CA).
samples from a renal tumor and polycystic kidney were ana-
The probe for ribosomal protein L32 (rpl32) was generated by PCR
(22). The cDNA were labeled with Prime It® II (Stratagene, La Jolla, lyzed. These samples were probed with the 500-bp mouse Sgk
CA), using [32P] dATP. Membranes were exposed to phosphorimag- cDNA RK15. Sgk mRNA levels in both diabetic kidneys were
ing plates, and bands were visualized and quantified with ImageQuant increased more than twofold compared with the control when
software (Molecular Dynamics, Sunnyvale, CA). corrected for loading and transfer efficiency with GAPDH
(Figure 3). In the tumor sample, there was a slight decrease in
Statistical Analyses Sgk expression. In the polycystic kidney, there was no change
Data were analyzed with SuperANOVA software to determine in Sgk mRNA compared with the control. Thus, it appears that
statistical significance (Abacus Concepts, Berkeley, CA). increased levels of Sgk mRNA are associated with diabetes but
not cancer or polycystic kidney disease.
Results Because thrombin activity has been demonstrated in patients
To identify genes involved in chronic renal failure, DDPCR with renal disease (25,26) and has been implicated in mediat-
was performed with mRNA from the kidneys of 5-mo-old ing renal remodeling during chronic renal failure (19,27–29),
obese diabetic db/db mice and normal littermates. The primers the effect of thrombin on Sgk mRNA levels was examined.2490 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2488 –2494, 1999
Figure 2. A comparison of Sgk message expression in the liver, brain,
heart, and kidney of normal (N) and diabetic (D) db mice. Ten
micrograms of total RNA was loaded per lane. The Sgk and GAPDH
mRNA are indicated by arrows.
Figure 1. Differential display PCR analysis of normal and diabetic
mouse kidney cDNA. (A) A 6% denaturing polyacrylamide gel con-
taining normal and diabetic db mouse kidney cDNA after differential
display PCR (DDPCR) using primers #3 and #10 (Table 1). Lanes 1
through 3 show amplification products from normal kidney cDNA,
and lanes 4 through 6 show amplification products from diabetic
kidney cDNA. The arrow highlights a differentially expressed 500-bp
cDNA that was increased in all three of the diabetic kidney samples.
This amplified cDNA fragment (RK15) is complementary to mouse
serum glucocorticoid-regulated kinase (Sgk) mRNA. (B) Northern
blot analysis of normal and diabetic db mouse kidney probed with
RK15. Each lane contained 10 mg of total RNA. The RK15 probe
recognized an mRNA of 2.4 kb. The blot was then stripped and
reprobed with GAPDH.
REC were treated in triplicate with 0.1, 1.0, and 10 nM
thrombin (Figure 4). Treatment with 0.1 nM thrombin resulted
in a 1.2-fold induction of Sgk mRNA, whereas 1.0 and 10 nM
thrombin increased Sgk mRNA twofold and fivefold, respec-
tively (Figure 4). Sgk message levels were also examined in rat
glomerular mesangial cells. However, Sgk mRNA was only
minimally induced by thrombin treatment at 10 nM (data not Figure 3. Sgk message levels in normal versus diabetic human kid-
neys as well as in a kidney tumor and polycystic kidney. (Top) Total
shown). Thus, thrombin regulation of Sgk mRNA may be
RNA from the renal cortex of two nondiabetic patients (N), two
selective for epithelial cells. Therefore, further experiments diabetic patients (D), a kidney tumor (T), and a polycystic kidney
were done with REC. (PKD) was probed with RK15. An arrow highlights Sgk and GAPDH
To determine whether the effect of thrombin is mediated mRNA. (Bottom) A histogram showing the relative abundance of the
through the PAR1 thrombin receptor, peptides corresponding Sgk message after correction for loading and transfer with GAPDH.
to the tethered ligand of the activated PAR1 thrombin receptor
were examined. The active 6 amino acid peptide SFLLRN
increased Sgk levels two- to threefold compared with the that within 15 min of thrombin treatment, Sgk is induced
inactive 5-mer FLLRN (Figure 5). The active peptide produced nearly twofold (Figure 6). Message levels continue to rise at 30
an increase in Sgk mRNA similar to that observed with throm- min and are maximally increased at 1 h of treatment (seven-
bin. fold). Sgk message levels are maintained at fourfold induction
A time course study was done to determine the rate of Sgk for up to 24 h (Figure 6).
induction in response to thrombin. Northern analysis indicates Previous reports have indicated that induction of Sgk mes-J Am Soc Nephrol 10: 2488 –2494, 1999 Expression of Sgk in Kidney 2491
Figure 6. Temporal expression of Sgk and rpL32 mRNA in response
to thrombin treatment by Northern blot. REC were treated with 10 nM
thrombin for the times indicated at the top of each lane. Cells were
serum-starved for 24 h. All cells were kept in culture for the same
amount of time.
Figure 4. Relative levels of Sgk expression in response to increasing
concentrations of thrombin in rat epithelial cells (REC) by Northern
blot analysis. Before treatment, these cells were placed in serum-free
media for 24 h. They then received either no thrombin (0) or thrombin
at 0.1, 1.0, or 10 nM for 16 h. The histogram shows the levels of Sgk
mRNA in response to thrombin treatment after correcting for loading
and transfer with rpL23 mRNA (n 5 3). Below is a representative
Northern blot for Sgk and rpL32 mRNA.
Figure 7. Stability of Sgk message in the presence of thrombin (10
nM) was investigated by treating REC with actinomycin D. The cells
were then exposed to actinomycin D (5 mg/ml) for times indicated.
Figure 5. Sgk message levels in REC after treatment with peptides
corresponding to the tethered ligand of activated thrombin receptor. C,
control; 6, SFLLRN (10 mM); 5, FLLRN (10 mM); T, thrombin (10 mRNA at the transcriptional level. The induction of Sgk mes-
nM). Cells were treated with peptides or thrombin for 2 h. sage by thrombin is not affected by the protein synthesis
inhibitor cycloheximide (Figure 8). Thrombin increased Sgk
message four- to fivefold in the presence or absence of cyclo-
heximide. Thus, thrombin is another factor that positively
sage by agents such as serum, glucocorticoids, and follicle-
stimulating hormone, as well as by changes in cell volume, is regulates Sgk expression through immediate transcriptional
an immediate-early transcriptional response that does not re- activation.
quire de novo protein synthesis (14,15,23,30). Therefore, the Thrombin receptor activation has been shown to involve
effect of thrombin on the mRNA half-life of Sgk was deter- certain second-messenger pathways including protein kinase C
mined in the presence of the RNA synthesis inhibitor actino- (31). The protein kinase C second-messenger system was in-
mycin D (Figure 7). Sgk message levels decrease within 15 vestigated to determine whether it plays a role in thrombin
min of actinomycin D treatment. The half-life of Sgk is ap- receptor-mediated induction of Sgk with the PKC inhibitor
proximately 30 min, which is similar to previous reports (30). RO-32-0432. As shown in Figure 9, the combined treatment of
This experiment indicates that thrombin does not affect Sgk RO-32-0432 and thrombin abolished the induction observed
mRNA stability and suggests that thrombin regulates Sgk with thrombin treatment alone.2492 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2488 –2494, 1999
ogy of the db/db mouse has been shown to resemble the
glomerular basement membrane expansion and accumulation
of matrix components in human diabetic kidney (36). A recent
study demonstrated that progression of renal disease as mea-
sured by creatinine clearance and albumin excretion in the
db/db mice resembles renal functional changes seen human
diabetes (13).
The Sgk transcripts were expressed at low levels in kidneys
of both normal db/wt mice and healthy patients, whereas Sgk
mRNA was elevated in obese db/db kidneys and human dia-
betic kidneys. A significant observation was that Sgk mRNA
did not increase in the brain, liver, or heart of the db/db mouse
and that Sgk message was not elevated in polycystic kidney
disease and cancer of patients. The increase in Sgk mRNA was
Figure 8. Sgk and rpL32 mRNA in response to thrombin (Thr.) seen only in diabetic kidney, which is particularly susceptible
treatment in the presence or absence of cycloheximide (CHX). REC to diabetes-induced damage and suggests that Sgk plays a
were serum-starved for 24 h. Subsequently, some of the cells were specific role in diabetes-induced renal disease.
pretreated with 10 mg/ml cycloheximide for 2 h. Thrombin (10 nM) Several factors have been shown to be involved in diabetic
was then added to certain flasks for 2 h, after which RNA was
nephropathy including thrombin. The expression and function
extracted from all of the cells.
of the thrombin receptor in the human kidney has been impli-
cated as an important regulatory component of glomerular and
vascular effects within the normal and diseased kidney (29).
Because renal thrombin activity is increased in patients with
diabetic nephropathy (25,26), thrombin may influence the ex-
pression of Sgk in renal disease. In this study, we demonstrate
that thrombin increased Sgk mRNA in glomerular epithelial
cells within 15 min and that thrombin does not affect Sgk
mRNA stability. Furthermore, de novo synthesis of protein is
not required for thrombin-induced increase in Sgk mRNA.
This strongly suggests that thrombin regulates Sgk mRNA
levels at the transcriptional level.
Thrombin acts on the thrombin receptor by cleavage of the
N-terminal domain of the receptor, thus unmasking a peptide
Figure 9. Thrombin-induced Sgk expression is influenced by an
sequence on the receptor, which can then act as a tethered
inhibitor of PKC. REC were serum-starved for 24 h. Four flasks were ligand to activate the thrombin receptor (37). It has been
then pretreated with RO-32-0432 (RO) at 1 mM for 4 h. Two of those determined that the 6 amino-terminal peptide sequence
flasks and two additional flasks received thrombin (Thr., 10 nM) for SFLLRN of the thrombin receptor PAR1 that is exposed after
2 h. thrombin cleavage is sufficient and necessary for thrombin
receptor PAR1 activation. Furthermore, the amino-terminal
serine is essential for the activity of this peptide. Our data
Discussion indicate that the 6-mer peptide corresponding to the sequence
In this study, we identified a 500-bp mouse sequence with of the tethered ligand can also increase Sgk mRNA. This
90% homology to the previously described rat and human Sgk, response was dependent on the correct sequence and the pres-
which was increased in obese db/db mouse kidneys by DDPCR ence of the amino-terminal serine. Thus, thrombin rapidly
using primers designed to conserved regions of kinase catalytic increased Sgk mRNA by direct stimulation of the thrombin
domains, to increase the chance of identifying kinases involved receptor PAR-1. Whether thrombin activation of Sgk transcrip-
in diabetic nephropathy. The deduced amino acid sequence of tion is a compensatory mechanism in renal disease or contrib-
Sgk predicts that it is a kinase with specificity toward serine/ utes to the progression of renal disease in diabetes remains to
threonine substrates. Although neither the kinase activity nor be determined.
function of this protein has been conclusively shown, this is the However, the function of Sgk in cellular processes is un-
first demonstration that Sgk is associated with diabetes–in- known. Sgk shares best homology with protein kinase B (Akt),
duced renal disease. which inhibits apoptosis by phosphorylating forkhead tran-
Kidneys from the db/db diabetic obese mouse and normal scription factor (38). If Sgk also shares functional similarity,
nondiabetic littermates were used to identify genes potentially then Sgk may play a role in regulation of cell survival. Some
involved in renal disease. The db/db mouse is a genetically evidence suggests that Sgk may play a role in mitogenic
diabetic animal characterized by obesity and hyperglycemia response in the G0-G1 transition in quiescent Rat2 fibroblasts
associated with insulin resistance (4,32–35). The renal pathol- because it is induced in response to serum stimulation and hasJ Am Soc Nephrol 10: 2488 –2494, 1999 Expression of Sgk in Kidney 2493
a rapid half-life like other immediate-early response genes targeted differential display of an immediate early gene encoding
(30). In mammary tumor cells, serum also increased Sgk a putative serine/threonine kinase. J Biol Chem 270: 10351–
mRNA, and dexamethasone paradoxically increased Sgk levels 10357, 1995
while suppressing proliferation (39). Although Sgk mRNA is 8. Clay FJ, McEwen SJ, Bertoncello I, Wilks AF, Dunn AR:
Identification and cloning of a protein kinase-encoding mouse
elevated by both glucocorticoids and serum, the subcellular
gene, Plk, related to the polo gene of Drosophila. Proc Natl Acad
localization of Sgk changes from a cytoplasmic distribution
Sci USA 90: 4882– 4886, 1993
during a growth-arrested state to a nuclear localization when 9. Golsteyn RM, Schultz SJ, Bartek J, Ziemiecki A, Ried T, Nigg
cells are proliferating and BrdUrd-positive (39). This strongly EA: Cell cycle analysis and chromosomal localization of human
suggests that Sgk is involved in growth control. In addition, the Plk1, a putative homologue of the mitotic kinases Drosophila
interplay of steroid hormones and p53 regulating its expression polo and Saccharomyces cerevisiae Cdc5. J Cell Sci 107: 1509 –
also indicate a possible role in regulation of proliferation (40). 1517, 1994
Moreover, thrombin, which causes renal cells to proliferate 10. Hamanaka R, Maloid S, Smith MR, O’Connell CD, Longo DL,
(27,41), also increased Sgk mRNA in cultured renal cells. Ferris DK: Cloning and characterization of human and murine
These data support a role for Sgk during repair processes when homologues of the Drosophila polo serine-threonine kinase. Cell
cells within injured tissues proliferate. Consistent with this Growth Differ 5: 249 –257, 1994
hypothesis, Sgk was found to be strongly expressed in glial 11. Holtrich U, Wolf G, Brauninger A, Karn T, Bohme B, Rubsamen
WH, Strebhardt K: Induction and down-regulation of PLK, a
cells and oligodendrocytes surrounding lesions in the injured
human serine/threonine kinase expressed in proliferating cells
rat brain (24). Therefore, Sgk may be involved in remodeling and tumors. Proc Natl Acad Sci USA 91: 1736 –1740, 1994
of injured and diseased tissues by regulating cell proliferation. 12. Simmons DL, Neel BG, Stevens R, Evett G, Erikson RL: Iden-
Renal failure is accompanied by a loss of electrolyte ho- tification of an early-growth-response gene encoding a novel
meostasis, which may act as a signal to initiate compensatory putative protein kinase. Mol Cell Biol 12: 4164 – 4169, 1992
cellular and molecular mechanism including the activation of 13. Cohen MP, Clements RS, Hud E, Cohen JA, Ziyadeh FN:
Sgk. Although Sgk transcript levels were found to be influ- Evolution of renal function abnormalities in the db/db mouse that
enced by alterations in anisotonic and isotonic conditions (23), parallels the development of human diabetic nephropathy. Exp
Sgk can in turn also affect sodium transport by activating the Nephrol 4: 166 –171, 1996
epithelial sodium channel in vitro (42). Moreover, aldosterone 14. Webster MK, Goya L, Ge Y, Maiyar AC, Firestone GL: Char-
increases the expression of Sgk mRNA in the rat kidney (42). acterization of sgk, a novel member of the serine/threonine
protein kinase gene family which is transcriptionally induced by
Taken together, these data suggest that Sgk is part of the
glucocorticoids and serum. Mol Cell Biol 13: 2031–2040, 1993
mechanism involved in proliferative responses to injury in
15. Alliston TN, Maiyar AC, Buse P, Firestone GL, Richards JS:
addition to contributing to sodium retention and mediation of Follicle stimulating hormone-regulated expression of serum/glu-
aldosterone regulation of sodium transport in renal disease. cocorticoid-inducible kinase in rat ovarian granulosa cells: A
Clearly, additional studies are required to determine the con- functional role for the Sp1 family in promoter activity. Mol
sequence of increased Sgk activity in renal disease and the Endocrinol 11: 1934 –1949, 1997
cellular signaling processes that influence Sgk activity. 16. Chomczynski P, Sacchi N: Single step method for RNA isolation
by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem 162: 156 –159, 1987
Acknowledgment 17. Liang P, Pardee AB: Differential display of eukaryotic messen-
We thank Sue Tirri for exceptional assistance in the preparation of
ger RNA by means of the polymerase chain reaction. Science
this manuscript.
257: 967–971, 1992
18. Liang P, Averboukh L, Pardee AB: Distribution and cloning of
References eukaryotic mRNAs by means of differential display: Refine-
1. Klahr S, Schreiner G, Ichikawa I: The progression of renal ments and optimization. Nucleic Acids Res 21: 3269 –3275, 1993
disease. N Engl J Med 318: 1657–1666, 1988 19. Laping NJ, Olson BA, Short B, Albrightson CR: Thrombin
2. Striker LJ, Doi T, Elliot S, Striker GE: The contribution of increases clusterin mRNA in glomerular epithelial and mesangial
glomerular mesangial cells to progressive glomerulosclerosis. cells. J Am Soc Nephrol 8: 906 –914, 1997
Semin Nephrol 9: 318 –328, 1989 20. Birchall AM, Bishop J, Bradshaw D, Coine A, Coffey J, Elliott
3. Ebihara I, Suzuki S, Nakamura T, Fukui M, Yaguchi Y, Tomino LH, Gibson VM, Greeham A, Hallam TJ, Harris W, Hill CH,
Y, Koide H: Extracellular matrix component mRNA expression Hutchings A, Lamont AG, Lawton G, Lewis EJ, Maw A, Nixon
in glomeruli in experimental focal glomerulosclerosis. J Am Soc JS, Pole D, Wadsworth J, Wikinson SE: Ro 32– 0432, a selective
Nephrol 3: 1387–1397, 1993 and orally active inhibitor of protein kinase C prevents T-cell
4. Hummel KP, Dickie MM, Coleman DL: Diabetes, a new muta- activation. J Pharmacol Exp Ther 268: 922–929, 1994
tion in the mouse. Science 153: 1127–1128, 1966 21. Dietrich JM, Jiang W, Bond JS: A novel meprin b mRNA in
5. Sharma K, Ziyadeh FN: Biochemical events and cytokine inter- mouse embryonal and human colon carcinoma cells. J Biol Chem
actions linking glucose metabolism to the development of dia- 271: 2271–2278, 1996
betic nephropathy. Semin Nephrol 17: 80 –92, 1997 22. Wang X, Yue T-L, Barone FC, Feuerstein GZ: Demonstration of
6. Ganz MB, Abu-Nader R, Saxena R, Grond J: Protein kinase Cb increased endothelial-leukocyte adhesion molecule-1 mRNA ex-
II isoform is upregulated in human proliferative glomerulone- pression in rat ischemic cortex. Stroke 26: 1665–1669, 1995
phritis. Exp Nephrol 5: 225–232, 1997 23. Waldegger S, Barth P, Raber G, Lang F: Cloning and character-
7. Donohue PJ, Alberts GF, Guo Y, Winkles JA: Identification by ization of a putative human serine/threonine protein kinase tran-2494 Journal of the American Society of Nephrology J Am Soc Nephrol 10: 2488 –2494, 1999
scriptionally modified during anisotonic and isotonic alterations 34. Chick WL, Like AA: Studies in the diabetic mutant mouse. II.
in cell volume. Proc Natl Acad Sci USA 94: 4440 – 4445, 1997 Physiological factors associated with alterations in beta cell
24. Imaizumi K, Tsuda M, Wanaka A, Tohyama M, Takagi T: proliferation. Diabetologia 6: 243–251, 1970
Differential expression of sgk mRNA, a member of the Ser/Thr 35. Coleman DL, Hummel KP: Hyperinsulinemia in pre-weaning
protein kinase gene family, in rat brain after CNS injury. Mol diabetic mice. Diabetologia 10: 607– 610, 1974
Brain Res 26: 189 –196, 1994 36. Like AA, Lavine RL, Poffenbarger PL, Chick DL: Studies in the
25. Tada H, Kuboki K, Shima Y, Takeuchi S, Sogai S: Increased diabetic mutant mouse. VI. Evolution of glomerular lesions and
intraglomerular thrombin formation in diabetic microangiopathy. associated proteinuria. Am J Pathol 66: 193–204, 1972
Diabetes Res Clin Pract 7: 121–125, 1998 37. Vu TK, Hung DT, Wheaton VI, Coughlin SR: Molecular cloning
26. Matsuo T, Koide M, Kario K, Matsuo M: Extrinsic coagulation of a functional thrombin receptor reveals a novel proteolytic
factors and tissue factor pathway inhibitor in end-stage chronic mechanism of receptor activation. Cell 64: 1057–1068, 1991
renal failure. Haemostasis 27: 163–167, 1997 38. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS,
27. Albrightson CR, Nambi P, Zabko-Potapovich B, Dytko G, Anderson MJ, Arden KC, Blenis J, Greenberg ME: Akt promotes
Groom T: Effect of thrombin on proliferation, contraction and cell survival by phosphorylating and inhibiting a forkhead tran-
prostaglandin production of rat glomerular mesangial cells in
scription factor. Cell 96: 857– 868, 1999
culture. J Pharmacol Exp Ther 263: 404 – 412, 1992
39. Buse P, Tran SH, Luther E, Phu PT, Aponte GW, Firestone GL:
28. Kanfer A: Coagulation factors in nephrotic syndrome. Am J
Cell cycle and hormonal control of nuclear-cytoplasmic local-
Nephrol 10[Suppl 1]: 63– 68, 1990
ization of the serum- and glucocorticoid-inducible protein kinase,
29. Xu Y, Zacharias U, Peraldi MN, He CJ, Lu C, Sraer JD, Brass
Sgk, in mammary tumor cells: A novel convergence point of
LF, Rondeau E: Constitutive expression and modulation of the
anti-proliferative and proliferative cell signaling pathways. J Biol
functional thrombin receptor in the human kidney. Am J Pathol
146: 101–110, 1995 Chem 274: 7253–7263, 1999
30. Webster MK, Goya L, Firestone GL: Immediate-early transcrip- 40. Maiyar AC, Huang AJ, Phu PT, Cha HH, Firestone GL: p53
tional regulation and rapid mRNA turnover of a putative serine/ stimulates promoter activity of the sgk serum/glucocorticoid-
threonine protein kinase. J Biol Chem 268: 11482–11485, 1993 inducible serine/threonine protein kinase gene in rodent mam-
31. He C-J, Peraldi MN, Adida C, Rebibou JM, Meulders Q, Sraer mary epithelial cells. J Biol Chem 271: 12414 –12422, 1996
JD, Rondeau E: Thrombin signal transduction mechanisms in 41. He CJ, Rondeau E, Medcalf RL, Lacave R, Schleuning WD,
human glomerular epithelial cells. J Cell Physiol 150: 475– 483, Sraer JD: Thrombin increases proliferation and decreases fi-
1992 brinolytic activity of kidney glomerular epithelial cells. J Cell
32. Coleman DL, Hummel KP: Studies with the mutation, diabetes, Physiol 146: 131–140, 1991
in the mouse. Diabetologia 3: 238 –248, 1967 42. Chen S, Bhargava A, Mastroberardino L, Meijer OC, Wang J,
33. Like AA, Chick WL: Studies in the diabetic mutant mouse. I. Buse P, Firestone GL, Verrey F, Pearce D: Epithelial sodium
Light microscopy and radioautography of pancreatic islets. Dia- channel regulated by aldosterone-induced protein sgk. Proc Natl
betologia 6: 207–215, 1970 Acad Sci USA 96: 2514 –2519, 1999You can also read
