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Am J Physiol Renal Physiol 293: F428-F437, 2007. First published April 11, 2007; doi:10.1152/ajprenal.00367.2006
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INNOVATIVE METHODOLOGY

Profiling of the renal kinome: a novel tool to identify protein kinases involved in angiotensin II-dependent hypertensive renal damage

Martin H. de Borst,1,2 Sander H. Diks,3 Juliane Bolbrinker,4 Mark W. Schellings,5 Mario B. A. van Dalen,1 Maikel P. Peppelenbosch,3 Reinhold Kreutz,4 Yigal M. Pinto,5 Gerjan Navis,2 and Harry van Goor1

Departments of 1Pathology, 2Nephrology, and 3Cell Biology, University of Groningen and University Medical Center Groningen, Groningen; 4Department of Clinical Pharmacology, Charité-University Medicine Berlin, Campus Benjamin Franklin, Berlin, Germany; and 5Department of Experimental and Molecular Cardiology, Cardiovascular Research Institute Maastricht, University of Maastricht, Maastricht, The Netherlands

Submitted 12 September 2006 ; accepted in final form 6 April 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Regulation of protein kinase activities is crucial in both physiology and disease, but analysis is hampered by the multitude and complexity of kinase networks. We used novel peptide array chips containing 1,152 known kinase substrate sequences to profile different kinase activities in renal lysates from homozygous Ren2 rats, a model characterized by hypertension and angiotensin II (ANG II)-mediated renal fibrosis, compared with Sprague-Dawley (SD) control rats and Ren2 rats treated with an angiotensin-converting enzyme inhibitor (ACEi). Five-wk-old homozygous Ren2 rats were left untreated or treated with the ACEi ramipril (1 mg·kg–1·day–1) for 4 wk; age-matched SD rats served as controls (n = 5 each). Peptide array chips were incubated with renal cortical lysates in the presence of radioactively labeled ATP. Radioactivity incorporated into the substrate motifs was measured to quantify kinase activity. A number of kinases with modulated activities, which might contribute to renal damage, were validated by Western blotting, immunoprecipitation, and immunohistochemistry. Relevant kinases identified by the peptide array and confirmed using conventional techniques included p38 MAP kinase and PDGF receptor-beta, which were increased in Ren2 and reversed by ACEi. Furthermore, insulin receptor signaling was reduced in Ren2 compared with control rats, and G protein-coupled receptor kinase (GRK) activity decreased in Ren2 + ACEi compared with untreated Ren2 rats. Array-based profiling of tissue kinase activities in ANG II-mediated renal damage provides a powerful tool for identification of relevant kinase pathways in vivo and may lead to novel strategies for therapy.

protein kinase; signal transduction; array; Ren2

PHOSPHORYLATION BY PROTEIN KINASES is among the most studied signaling mechanisms in biomedical research. Yet the study of protein kinase signaling is limited by the extensive number of kinases and substrates in the mammalian proteome, the enormous complexity of signaling pathways, and cross talk between signaling pathways. The recent development of kinase arraying technology similar to gene and protein arrays forms a first step toward interpretation of the mammalian kinome, i.e., the complete set of kinases (15).

Kinase array technology can contribute to the identification of novel signaling pathways, as demonstrated in human peripheral blood mononuclear cells (PBMC), T cells, and adipocytes (3T3-L1) (10, 21). However, the application of kinase array technology covering a significant part of the mammalian kinome is thus far limited to studies in cultured cells. Simultaneous analysis of a large number of kinases in normal and diseased tissues would provide a variety of novel information on kinase signaling in physiology and pathophysiology.

In the kidney, kinase activation is pivotal in both physiology and disease (9). Both animals and patients demonstrate increased activation of numerous kinases [e.g., mitogen-activated protein (MAP) kinases] in renal disease compared with healthy subjects (1, 22). Moreover, specific pharmacological inhibition of MAP kinases reduces renal fibrosis in experimental models (8, 34). Yet kinase inhibition can also deteriorate renal disease, presumably through induction of alternative signaling pathways (26). Thus it is very important to discover specific targets for intervention appropriate under particular conditions, as well as to study the effect of intervention on specific kinases in signaling pathways.

Hypertension is an important and widely prevalent risk factor for the development of chronic kidney disease (CKD), which may progress to end-stage renal disease. Despite current regimens [e.g., angiotensin-converting enzyme (ACE) inhibitors, angiotensin II type 1 (AT1) receptor blockers] that may halt or even reverse renal disease progression, the prevalence of end-stage renal disease is increasing (23, 29). Thus it is crucial to identify novel targets for intervention in hypertensive renal damage.

In the present study we screened renal kinase activities in homozygous Ren2 rats, a model of angiotensin II-mediated hypertensive renal damage, control rats, and Ren2 rats treated with an ACE inhibitor, a well-defined intervention to reduce renal fibrosis (24). Our primary target was to investigate whether this novel kinase array technology could indeed detect relevant protein kinases involved in hypertensive renal damage.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals. Homozygous male TGR(mRen2)27 (Ren2) rats were obtained from the Campus Benjamin Franklin (Berlin, Germany), and male Sprague-Daley (SD) rats were purchased from Charles River Laboratories (Sulzfeld, Germany). Animals were studied in compliance with institutional regulations. Rats were grouped (n = 5 each) under conditions of regular 12-h diurnal cycles and climate-controlled conditions at 22°C, received standard diet, and had free access to food and water. Ren2 rats were randomly assigned to treatment groups at birth. After weaning at the age of 4 wk, homozygous Ren2 rats were treated with the ACE inhibitor (ACEi) ramipril (1 mg·kg–1·day–1) in drinking water (Ren2+ACEi) (Aventis Pharma, Frankfurt am Main, Germany) (20) or left untreated. Age-matched SD rats served as controls.

Systolic blood pressure (SBP) was measured in 8-wk-old male Ren2, Ren2+ACEi, and SD rats under slight ether anesthesia using the tail-cuff method. Animals were placed in metabolic cages for 24 h to collect urine samples for urinary albumin excretion (UAE) measurement.

At the age of 8 wk, rats were killed. The kidneys were rapidly excised, rinsed, and dried. A cross section of the left kidney was fixed in methacarn solution (60% methanol, 30% chloroform, and 10% acetic acid) for 24 h, which was then changed to 80% ethanol until further dehydration and embedding in paraffin. The rest of the kidney was separated into cortex and medulla, immediately frozen in liquid nitrogen, and stored at –80°C until further analysis.

Antibodies. For Western blotting and immunoprecipitation, we used primary antibodies against phospho-p38 MAP kinase [pp38(Tyr180/Thr182), no. 9211, Cell Signaling Technology, Danvers, MA], platelet-derived growth factor receptor-beta (PDGFRbeta; no. 3162, Cell Signaling Technology), and phosphorylated insulin receptor substrate-1 [p-IRS-1(Ser636/639), no. 2388, Cell Signaling Technology]. For immunostaining, we used separate antibodies against pp38 (no. 4631, Cell Signaling Technology, {alpha}-smooth muscle actin ({alpha}-SMA; clone 1A4, Sigma, St. Louis, MO), and PDGFRbeta (sc-432, Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies against housekeeping proteins beta-actin and GAPDH were purchased from Santa Cruz Biotechnology.

Renal morphology. Midcoronal sections of kidneys, routinely stained with periodic acid-Schiff, were scored for glomerular mesangial matrix expansion (MME) and focal glomerular sclerosis (FGS) as described previously (6). Briefly, MME was scored positive if broadening of mesangial areas was two to three times that of the mesangial width seen in glomeruli of control renal tissue. FGS was scored positive if collapse of capillary lumina, MME, hyalinosis, and adhesion of the glomerular tuft to Bowman's capsule were simultaneously present. Glomeruli were scored for MME and FGS as follows: unaffected glomeruli were scored as 0, a score of 1 was given if one glomerular quadrant was affected, two quadrants affected was scored as 2, three quadrants affected was scored as 3, and a score of 4 was given if all quadrants were positive for MME or FGS. Mean glomerular MME or FGS scores were calculated. Interstitial fibrosis was scored positive when tubular atrophy and broadening of the peritubular compartment were simultaneously present. Scores of 0–4 were assigned: a score of 0 indicated no interstitial fibrosis, a score of 1 indicated 0–25% involvement of the total interstitial surface of the biopsy, a score of 2 indicated 25–50% involvement, a score of 3 indicated 50–75% involvement, and a score of 4 indicated 75–100% involvement.

To adjudge interstitial myofibroblast transformation, an early event in interstitial fibrosis, paraffin sections were immunohistochemically stained for {alpha}-SMA as described below. The percentage of {alpha}-SMA-positive staining per tubulointerstitial field was quantified using computerized morphometry (8).

Kinase array. For kinase array experiments, PepChip kinase arrays (Pepscan Systems, Lelystad, The Netherlands; www.pepscan.nl) were used. Renal cortical tissue was lysed in cell lysis buffer (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, 1 mM Na2EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM MgCl2, 1 mM beta-glycerophosphate, 1 mM Na3VO4, 1 mM NaF, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM PMSF) using a Dounce homogenizer. After lysis, the volume of the cell lysate was equalized with lysis buffer. The kinase array was then performed according to the supplied protocol (http://pepscan.nl/pdf/Manual%20PepChip%20Kinase%200203.pdf). In short, the cell lysates were spun down and cleared on a 0.22-µm filter. Ten microliters of peptide array incubation mix [50% glycerol, 50 µM ATP, 0.05% (vol/vol) Brij-35, 0.25 mg/ml bovine serum albumin, and [{gamma}-33P]ATP (1 MBq)] was added to the lysate. Next, the peptide array mix was loaded onto the chip and allowed to phosphorylate the substrates for 90 min at 37°C in a humidified stove. Subsequently, the peptide array was washed twice with Tris-buffered saline (TBS) with 0.1% Triton X-100, twice in 2 M NaCl, and twice in demineralized H2O and then air-dried. The experiments were performed three times in duplicate. The amount of incorporated activity on the chip was detected using a STORM 860 (General Electric) scanner and analyzed with array software (ScanAlyze, Eisen Software).

A number of kinases with markedly altered activities among the studied groups were selected to be studied in more detail. Since not all substrates spotted on the chip correspond to known rat protein sequences, we selected only rat substrate sequences using NCBI BLAST for further analysis (2).

Immunoprecipitation. Renal cortical tissue samples were lysed and incubated with an immobilized phosphotyrosine antibody (1:10; Cell Signaling Technology) at 4°C overnight. Subsequently, the samples were centrifuged at 2,000 rpm, 4°C, and washed three times with RIPA buffer. Finally, the samples were prepared for Western blotting as described below.

Western blotting. Renal cortical tissue (n = 3 per group) was lysed in ice-cold RIPA buffer with 10 µg/ml aprotinin, 1 mM orthovanadate, 10 mM NaF, and PMSF (10 mg/ml in isopropanol). Protein quantity was measured using the pyrogallol red-molybdate method to obtain similar protein loads per lane. Tissue lysates were separated on a 10% polyacrylamide gel and electroblotted onto a nitrocellulose membrane; proteins were visualized with Ponceau-S (Pharmacia, Uppsala, Sweden), which confirmed similar protein amounts per lane. Blots were incubated for 60 min in blocking buffer [TBST (TBS and 0.05% Tween 20, pH 7.6) with 5% skimmed milk], washed for 30 min in TBST, and incubated overnight at 4°C with primary antibodies. Immunostaining was amplified by incubation with horseradish peroxidase-conjugated antibodies for 60 min. Blots were washed, and immunoluminescence was detected with LumiGlo (Upstate, Charlottesville, VA). Band staining intensities were quantified by densitometry.

Immunohistochemistry. Four-micrometer paraffin sections were dewaxed; endogenous peroxidase was blocked by incubation with 0.3% H2O2 in PBS for 30 min. Sections were then incubated with primary antibodies for 60 min. Binding was detected by sequential incubation with peroxidase-labeled secondary antibodies. Peroxidase activity was visualized using 3,3'-diaminobenzidene tetrahydrochloride (DAB; DAKO), counterstained with hematoxilin, and mounted with Kaiser's glycerin gelatin. For phosphorylated p38 (pp38) immunostaining, a slightly different protocol was used. Sections were washed in TBST and blocked in TBST plus 1% BSA for 60 min before incubation with the primary antibody.

Statistical analysis. Data are presented as means ± 1 standard deviation. Kinase array data are presented as differential ({Delta}) kinase activity of untreated Ren2 vs. SD or Ren2+ACEi vs. untreated Ren2 rats, indicated in percentages. Statistical differences between groups were calculated using the nonparametric Kruskal-Wallis test. Intra- and interindividual variances were calculated by dividing the intra- or interindividual standard deviation, respectively, by the average of the individual measurements or the studied group, respectively.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
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Physiological and renal structural parameters. SBP and UAE were increased in untreated Ren2 rats compared with SD controls (Fig. 1). Treatment with the ACEi ramipril strongly reduced SBP and UAE. Furthermore, both glomerular (FGS, MME) and tubular (interstitial fibrosis, interstitial {alpha}-SMA expression) parameters of fibrosis were increased in Ren2 rats compared with SD controls and ameliorated by ACEi (Fig. 2).


Figure 1
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  		Fig. 1. Systolic blood pressure (SBP) and urinary albumin excretion (UAE) in 8-wk-old untreated homozygous Ren2 rats, age-matched Sprague-Dawley (SD) controls, and age-matched Ren2 rats treated with the angiotensin-converting enzyme inhibitor (ACEi) ramipril (1 mg·kg–1·day–1) for 4 wk. Both SBP and UAE were increased in untreated Ren2 rats, in Ren2 rats treated with ramipril (Ren2+ACEi); SBP and UAE were strongly decreased compared with untreated Ren2 rats.

 

Figure 2
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  		Fig. 2. Focal glomerulosclerosis (FGS), mesangial matrix expansion (MME), interstitial fibrosis (IF), and interstitial {alpha}-smooth muscle actin (SMA) expression are depicted for all 3 groups. Data indicate significantly increased FGS, MME, IF, and {alpha}-SMA in untreated Ren2 rats compared with SD controls and significant reduction of all parameters in Ren2+ACEi compared with untreated Ren2 rats.

 
Kinase activities. A general overview of the kinase array data is presented in Table 1. In the Ren2 untreated group, activities of 89 kinases were more than two standard deviations above the average of the SD group. All of these were reduced in Ren2 rats treated with ACE inhibition. On the other hand, 106 kinases show decreased activities in Ren2 rats compared with SD animals. Of these, 16 kinases were increased upon ACE inhibition, whereas no kinases decreased further in Ren2+ACEi rats. In total, ACE inhibition increased 97 kinase activities and decreased 135 compared with untreated Ren2 rats.


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  		Table 1. General overview of kinase array results

 
The interindividual variation was calculated, both per group and overall: SD, 9.8%, Ren2, 10.7%; Ren2+ACEi, 11.5%; overall, 10.7%. The intraindividual variation was lower: SD, 9.2%; Ren2, 8.9%; Ren2+ACEi, 10.3%; overall, 9.4%.

p38 MAP kinase activation in Ren2 rats is reduced by ACEi. First, to validate our peptide array data, we studied the activity of a relatively well-defined protein kinase, namely, p38 MAP kinase, whose activity has been associated with renal damage. As illustrated in Fig. 3, top left, the array data indicated increased p38 MAP kinase in Ren2 rats compared with SD controls and a reduction in Ren2+ACEi compared with untreated Ren2 rats. These findings could be confirmed by immunohistochemistry, revealing strong induction of Tyr180/Thr182-phosphorylated p38 expression by tubular epithelial cells in areas of tubulointerstitial damage in untreated Ren2 rats. In SD controls or Ren2+ACEi rats, only very few tubular epithelial cells showed pp38-positive nuclei. Similar results were obtained by pp38 Western blotting as illustrated in Fig. 3, bottom left.


Figure 3
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  		Fig. 3. Phosphorylation of p38 MAP kinase (pp38) in cortical renal lysates of SD, Ren2, and Ren2+ACEi rats. Top left: kinase array data indicating increased kinase activity of pp38 MAP kinase in untreated Ren2 rats compared with SD controls, which was reversed by ACEi. Right: immunohistochemistry (IHC) for pp38 shows few pp38-positive tubular epithelial cells in SD controls (top) and Ren2+ACEi rats (bottom) and strong induction of nuclear pp38 expression in areas of tubulointerstitial damage in untreated Ren2 rats (middle). Bottom left: Western blotting (WB) for pp38 on cortical lysates confirmed the increased presence of pp38 in Ren2 rats compared with SD controls and a reduction in Ren2+ACEi rats. *P < 0.05 vs. SD. #P < 0.05 vs. Ren2 untreated.

 
PDGFRbeta phosphorylation is increased in Ren2 rats and reduced by ACEi. Our experiments also indicated increased activity of PDGFRbeta in untreated Ren2 rats. A statistically significant increase was observed for the ESSNYMAPY motif, containing the Tyr770 and Tyr774 regions of the protein (Fig. 4, top). Various PDGFRbeta motifs also showed reduced activity in Ren2+ACEi compared with untreated Ren2 rats. Furthermore, we found increased phosphorylation of two SHP-2 motifs in Ren2 rats and reduction in Ren2+ACEi rats. SHP-2 is a well-known PDGFR substrate (3). Immunoprecipitation confirmed an increased phosphorylated PDGFRbeta/total PDGFRbeta ratio in untreated Ren2 rats and a clear reduction of this ratio in Ren2+ACEi rats (Fig. 4, bottom left). Moreover, immunohistochemistry showed increased PDGFRbeta protein expression by fibroblasts in areas of tubulointerstitial damage in untreated Ren2 rats but not in SD controls and Ren2+ACEi rats (Fig. 4, bottom right).


Figure 4
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  		Fig. 4. Kinase array data indicate increased activity of PDGF receptor-beta (PDGFRbeta) in untreated Ren2 rats (top left) and reduction upon ACE inhibition (top right). Furthermore, phosphorylation of two SHP-2 (a PDGFRbeta substrate) motifs was similarly increased in Ren2 rats and reversed in Ren2+ACEi rats (middle left and middle right). These data suggest increased PDGFRbeta activity in Ren2 rats and reduction by ACE inhibition. This was confirmed by immunoprecipitation (bottom left). Control was rat cardiac fibroblast lysate. IHC shows increased PDGFR expression by interstitial fibroblasts in areas of renal fibrosis in Ren2 rats but not in SD or Ren2+ACEi rats. *P ≤ 0.05 vs. SD. #P ≤ 0.05 vs. Ren2 untreated rats.

 
ACE inhibition reduces GRK activity in Ren2 rats. All GRK substrate sequences that were spotted on the array and are known to be expressed in rat (n = 20) are presented in Fig. 5. In Ren2 rats compared with SD controls, the number of increased and decreased activities for GRK substrates was about similar. Strikingly, when Ren2+ACEi rats were compared with untreated Ren2 rats, most GRK substrates displayed reduced activities in Ren2 rats treated with ACEi. Kinase activity for phosphorylation of the muscarinic acetylcholine receptor M2 protein at the ENTVSTSLG motif (Fig. 5, substrate 17) was significantly increased in untreated Ren2 rats compared with SD controls and decreased in untreated Ren2 compared with Ren2+ACEi rats (both P < 0.05).


Figure 5
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  		Fig. 5. Overview of all G protein-coupled receptor kinase (GRK) substrates spotted on the microchip and their respective kinase activities. Graphs represent relative ({Delta}) insulin receptor (IR) kinase activities in Ren2 vs. SD (top) and Ren2+ACEi vs. Ren2 untreated rats (bottom). In Table 2, corresponding motif sequences, respective substrates, and potential phosphorylation sites are presented. Numbers in the graphs refer to respective substrate numbers in Table 2 and in text. There was no clear difference between Ren2 rats and SD controls in GRK signaling. However, in Ren2+ACEi rats there was a clear reduction in GRK activity compared with untreated Ren2 rats, reaching statistical significance in 4 substrates. *P < 0.05.

 
Insulin receptor signaling is reduced in Ren2 rats. Another consistent finding in the peptide array was reduction of insulin receptor (IR) signaling in Ren2 rats compared with SD controls, as indicated in Fig. 6. In total, 24 rat sequences from known IR substrates (e.g., insulin receptor substrate-1, IRS-1) were spotted on the peptide array. Of these 24 peptide sequences, 20 showed decreased kinase activity in Ren2 compared with controls, whereas only 4 were increased (Fig. 6, top). Peptide sequences from insulin receptor (IR; autophosphorylation), IRS-1, beta2-adrenergic receptor, phosphatidylinositol 3-kinase (PI3-kinase) p85-{alpha}, and eukaryotic translation initiation factor 4E, all known insulin receptor substrates, displayed reduced activities in lysates from Ren2 rats compared with SD controls. These findings implicate impaired renal insulin receptor signaling in homozygous Ren2 rats. Furthermore, array data for Ren2+ACEi rats indicate that renal insulin receptor signaling was not improved by ACEi in Ren2 rats. These findings were confirmed by Western blot analysis for Ser636/639 phospho-IRS-1 (Fig. 7). Renal IRS-1 phosphorylation was decreased in untreated Ren2 rats compared with SD controls, whereas ACE inhibition did not improve IR signaling.


Figure 6
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  		Fig. 6. Overview of all IR substrates that were spotted on the microchip and their respective kinase activities. Graphs represent {Delta}IR kinase activities in Ren2 vs. SD (top) and Ren2+ACEi vs. Ren2 untreated rats (bottom). In Table 3, corresponding motif sequences, respective substrates, and potential phosphorylation sites are presented. Numbers in the graphs refer to respective substrate numbers in Table 3 and in text. In Ren2 rats compared with SD controls, phosphorylation of most substrates was decreased, indicating reduced IR kinase activity in the renal cortex of Ren2 rats. Statistical significance was reached in 9 substrates. *P < 0.05. Upon ACE inhibition, there was no clear recovery of IR signaling.

 

Figure 7
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  		Fig. 7. Western blot for phosphorylated insulin receptor substrate (IRS)-1 at Ser636/639 showing decreased IRS-1 phosphorylation in Ren2 rats compared with SD controls, which was not affected by treatment with an ACEi. *P < 0.05 vs. SD.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In this study we successfully applied a novel kinase array technology enabling simultaneous analysis of a large number of kinases in angiotensin II-driven renal disease. This array, containing over 1,100 known peptide sequences from mammalian protein kinase substrates, has been used previously to study cultured cells, but not lysates from complex animal tissue.

The kinase array data suggest that p38 MAP kinase activation is involved in angiotensin II-dependent hypertensive renal damage. These findings are in line with studies showing increased p38 phosphorylation in both experimental (33) and human (35) renal disease. Furthermore, p38 inhibition is renoprotective in homozygous Ren2 rats (8) and in other models (34).

In addition, the array data indicated increased kinase activity of the PDGFRbeta in Ren2 rats compared with SD controls and reduced activity in Ren2+ACEi compared with untreated rats. Using immunoprecipitation, we confirmed this pattern of tyrosine phosphorylation of the PDGFR. Immunohistochemistry revealed increased PDGFRbeta expression by fibroblasts in areas of tubulointerstitial damage in Ren2 rats. Increased phosphorylation of PDGFRbeta in untreated Ren2 rats can be partly explained by increased protein expression, but the increased phosphorylated PDGFRbeta/total PDGFRbeta ratio in immunoprecipitation indicates that this pathway is activated in Ren2 rats compared with SD controls and reduced by ACEi, thus confirming the array data. Importantly, it was recently shown that in the Ren2 model, imatinib treatment reduced renal perivascular fibrosis and microvascular hypertrophy (32). Imatinib reduces tyrosine kinase activity of bcr/abl but also of other tyrosine kinases, including PDGFR{alpha}, PDGFRbeta, and c-kit (31). Thus, since pharmacological inhibition of p38 or PDGFR is renoprotective in the homozygous Ren2 model, we conclude that the array identified kinases relevant to renal injury. Our array data also indicated increased phosphorylation of SHP-2 at Tyr546 and Tyr584 in Ren2 rats vs. SD controls, which was reduced by ACE inhibition. Phosphorylation of SHP2 at Tyr546 or Tyr584 is controlled by some (fibroblast growth factor and PDGF) but not all (epidermal growth factor and insulin-like growth factor) growth factors and is crucially involved in sustained ERK activation (3). Since our group has previously shown that ERK inhibition is renoprotective in the homozygous Ren2 model (8), we postulate that in this model the PDGFR plays a role in renal damage through SHP-2-mediated sustained ERK activation.

There is now growing evidence that GRK signaling plays a pivotal role in the development and maintenance of hypertension (7). In animal models of hypertension, a generalized defect in vascular GRK2 protein expression was found that could be an important factor in the impairment of beta-adrenergic-mediated vasodilation (11). Furthermore, in transgenic mice targeted to overexpress GRK2 in cardiac myocytes, a reduced responsiveness to beta-adrenergic receptor (betaAR) agonists and angiotensin II was observed, whereas mice overexpressing a natural GRK2 inhibitor exhibited increased sensitivity to betaAR agonists (13, 17). We found increased GRK2 activity in Ren2 rats at the Glu305-Gly313 motif from M2 muscarinic ACh receptors (M2 mAChR), which was reduced by ACE inhibition (Fig. 5, substrate 17). Mutation studies of M2 mAChR residues Thr307-Ser311 revealed a role for this motif in receptor internalization (27). Reduced GRK activity in Ren2+ACEi rats may also reflect reduced renal AT1 receptor signaling, since the AT1 receptor is also a G protein-coupled receptor kinase.

Reduced phosphorylation of numerous IR substrate motifs was found in Ren2 rats. Western blot analysis confirmed reduced phosphorylation of IRS-1 at the important insulin sensitivity-regulating residues (Ser636/639) in Ren2 compared with SD animals (36). Using the array, we found reduced phosphorylation of IRS-1 at Tyr608 and Tyr612. IRS-1 phosphorylation at these peptides creates a docking site for PI3-kinase, enhancing IRS-1 activity. Furthermore, other motifs including IR (Tyr1362), an IR autophosphorylation motif involved in IR activation (18), were also strongly reduced in Ren2 rats compared with SD controls. Concurrently, various studies show that angiotensin II induces insulin resistance in vivo (25, 28). Ren2 rats also display insulin resistance (12, 16), which can be improved by AT1 receptor blocker (5). Although in our Ren2 animals ACEi did not clearly improve renal insulin receptor activity (as demonstrated by both the array and Western blotting), insulin sensitivity might have been restored in other tissues. Of interest, a recent publication demonstrates impaired muscular IR/IRS-1/PI3-K/Akt signaling in a model of chronic kidney disease, which implies that renal disease can affect insulin signaling throughout the body (4). This is in line with our results in the Ren2 model.

Since epidermal growth factor receptor (EGFR) transactivation plays a role in angiotensin II-mediated pathophysiology, we also checked activation of EGFR substrates (19). Although we found no clear induction in Ren2 rats compared with control rats, ACE inhibition reduced EGFR kinase activity. In particular, we found significantly reduced phosphorylation of substrates containing EGFR residues 1,068 and 1,194, known autophosphorylation residues. Phosphorylation of the EGFR at Tyr1068 is crucial to the EGF-induced Ras/MAP kinase pathway (30).

Kinase arraying technology may well become a powerful tool with numerous applications. An important advantage of our array is that it analyzes kinase activities "in silico." Various arrays used to study the kinome have been limited to determining protein phosphorylation statuses, which imply but do not provide direct evidence of protein kinase activity (15). Another advantage of this array technology is that multiple motifs per substrate are incorporated on the chip, increasing specificity and allowing identification of specific phospho-specific sites relevant under the studied conditions. Intra- and interindividual variation in vivo are reasonable (~10%), underlining the robustness of the array. On the other hand, the presence of various cell types within a renal lysate may reduce the sensitivity. In addition, our kinase activity array does not detect changes in kinase protein expression, which may be relevant to discriminate between translational and posttranslational causes of modified kinase activity. A suitable future strategy may thus be to apply this technique on selected renal structures, such as glomeruli, or even specific cell types, by using laser dissection microscopy, and to combine the kinase array with a protein expression array.

With the rapidly extending use of gene expression arrays in biomedical research, the methodology for data analysis is also improving. In this study we used conventional statistics to identify kinases of interest, although alternatives including distribution-free tests or linear models with Bayesian corrections could also be considered (14). In addition, functional clustering of kinases with altered expression among the studied groups could be a powerful approach, but the fact that many kinases have multiple functions in various cell types limits this type of analysis.

The most relevant property of this kinase array is its hypothesis-free character. This allows selection of a limited number of kinases with markedly altered activities under particular experimental conditions, which can then be studied in more detail. Ultimately, analysis of the renal kinome may provide novel targets for intervention to combat chronic renal disease.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was financially supported by the graduate school GUIDE (Groningen University Institute for Drug Exploration) and the Jan Kornelis De Cock Foundation, Groningen, The Netherlands.


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  		Table 2. GRK substrates present on the kinase array

 

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  		Table 3. Insulin receptor substrates present on the kinase array

 

   ACKNOWLEDGMENTS
 
Part of this work was presented at the 38th Annual Meeting of the American Society of Nephrology in Philadelphia, PA, in November 2005.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. H. de Borst, Dept. of Pathology and Laboratory Medicine, Univ. of Groningen and Univ. Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands (e-mail: m.h.de.borst{at}path.umcg.nl)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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