Profiling of the renal kinome: a novel tool to identify protein kinases involved in angiotensin II-dependent hypertensive renal damage
AJP - Renal Physiology
de Borst MH, Diks SH, Bolbrinker J, Schellings MW, van Dalen MB, Peppelenbosch MP, Kreutz R, Pinto YM, Navis G, van Goor H. Profiling of the renal kinome: a novel tool to identify protein kinases involved in angiotensin II-dependent hypertensive renal damage. 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
... ile 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 ⅐ kg Ϫ1 ⅐ day Ϫ1 ) for 4 wk; age-matched SD rats served as controls (n ϭ 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-␤, 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 ϩ 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).