J Am Soc Nephrol 10: 2488–2494, 1999 Sgk, a Putative Serine/Threonine , 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 including J-mϩ/ϩLeprdb, six animals per group (Jackson Laboratories, Bar 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- threonine kinases have been described (7–12). These kinases nol chloroform extraction (16). 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 ␮M mice, an animal model of select aspects of diabetic nephropa- dNTP, and 1 ␮Mofa3Ј 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 (DDPCR). This study found that serum glucocorticoid-regu- (17,18). The primers used for cDNA synthesis and PCR were de- 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 ␮M dNTP, 1 ␮M of each 5Ј and 3Ј primer (Table 1), 0.2 ␮Ci of [␣-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: [email protected] 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 system J Am Soc Nephrol 10: 2488–2494, 1999 Expression of Sgk in Kidney 2489

Table 1. Sequence of primers used in differential display PCR analysis

5Ј Specific Primers 3Ј Specific Primers

#1 5Ј-CAC TAA AGA GCG TGC #6 5Ј-CAG TAT ACA GGT CTC #2 5Ј-CCT TAA GAA AAC CAC #7 5Ј-TTA GCT TGT ACA GCT #3 5Ј-TGC TAG GAA CGT CCT #8 5Ј-CCA TCA GTA TAC AGG #4 5Ј-GGA AGC AGC TTG CAT #9 5Ј-CAG TAT ACA GGT TGT #5 5Ј-AAC CCG TAC ATC GTG #10 5Ј-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 whose levels increased in all three diabetic kidneys sampled (19). Lyophilized human plasma thrombin (average activity 1000 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 ␮g/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 ␮M for 2 h. REC were pretreated with message was specific to the kidney, RNA from other organs RO-32-0432 at 1 ␮Mfor4h. 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 ␮g 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 ␮g 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- shown). Thus, thrombin regulation of Sgk mRNA may be neys as well as in a kidney tumor and polycystic kidney. (Top) Total 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 at1hoftreatment (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 ϭ 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 ␮g/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 ␮M); 5, FLLRN (10 ␮M); 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 g/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 ␮M 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 2h. 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 (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 has J 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: elevated by both glucocorticoids and serum, the subcellular Identification and cloning of a protein kinase-encoding mouse gene, Plk, related to the polo gene of Drosophila. 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