Profiling of the renal kinome: a novel tool to identify protein kinases involved in angiotensin II-dependent hypertensive renal damage.
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.
de Borst
Martin H
MH
Dept. of Pathology, University of Groningen, University Medical Center Groningen, PO Box 30.001, 9700 RB Groningen, The Netherlands. m.h.de.borst@path.umcg.nl
Diks
Sander H
SH
Bolbrinker
Juliane
J
Schellings
Mark W
MW
van Dalen
Mario B A
MB
Peppelenbosch
Maikel P
MP
Kreutz
Reinhold
R
Pinto
Yigal M
YM
Navis
Gerjan
G
van Goor
Harry
H
D000804:Angiotensin II / Q000502:physiology*; D000806:Angiotensin-Converting Enzyme Inhibitors / Q000494:pharmacology; D000818:Animals; D001794:Blood Pressure / Q000502:physiology; D015153:Blotting, Western; D066246:ErbB Receptors / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology; D016678:Genome; D006977:Hypertension, Renal / Q000201:enzymology / Q000473:pathology*; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D007668:Kidney / Q000378:metabolism* / Q000473:pathology*; D008297:Male; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011972:Receptor, Insulin / Q000502:physiology; D020797:Receptor, Platelet-Derived Growth Factor beta / Q000378:metabolism; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D015398:Signal Transduction; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000806:Angiotensin-Converting Enzyme Inhibitors; D043562:Receptors, G-Protein-Coupled; D000804:Angiotensin II; D011494:Protein Kinases; D066246:ErbB Receptors; D011972:Receptor, Insulin; D020797:Receptor, Platelet-Derived Growth Factor beta; D048051:p38 Mitogen-Activated Protein Kinases