title: Determination of an angiotensin II-regulated proteome in primary human kidney cells by stable isotope labeling of amino acids in cell culture (SILAC).
abstract: Angiotensin II (AngII), the major effector of the renin-angiotensin system, mediates kidney disease progression by signaling through the AT-1 receptor (AT-1R), but there are no specific measures of renal AngII activity. Accordingly, we sought to define an AngII-regulated proteome in primary human proximal tubular cells (PTEC) to identify potential AngII activity markers in the kidney. We utilized stable isotope labeling with amino acids (SILAC) in PTECs to compare proteomes of AngII-treated and control cells. Of the 4618 quantified proteins, 83 were differentially regulated. SILAC ratios for 18 candidates were confirmed by a different mass spectrometry technique called selected reaction monitoring. Both SILAC and selected reaction monitoring revealed heme oxygenase-1 (HO-1) as the most significantly up-regulated protein in response to AngII stimulation. AngII-dependent regulation of the HO-1 gene and protein was further verified in PTECs. To extend these in vitro observations, we overlaid a network of significantly enriched gene ontology terms from our AngII-regulated proteins with a dataset of differentially expressed kidney genes from AngII-treated wild type mice and AT-1R knock-out mice. Five gene ontology terms were enriched in both datasets and included HO-1. Furthermore, HO-1 kidney expression and urinary excretion were reduced in AngII-treated mice with PTEC-specific AT-1R deletion compared with AngII-treated wild-type mice, thus confirming AT-1R-mediated regulation of HO-1. Our in vitro approach identified novel molecular markers of AngII activity, and the animal studies demonstrated that these markers are relevant in vivo. These interesting proteins hold promise as specific markers of renal AngII activity in patients and in experimental models.
authors.affiliation: Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Ana
authors.initials: A
authors.lastname: Konvalinka
authors.forename: Joyce
authors.initials: J
authors.lastname: Zhou
authors.forename: Apostolos
authors.initials: A
authors.lastname: Dimitromanolakis
authors.forename: Andrei P
authors.initials: AP
authors.lastname: Drabovich
authors.forename: Fei
authors.initials: F
authors.lastname: Fang
authors.forename: Susan
authors.initials: S
authors.lastname: Gurley
authors.forename: Thomas
authors.initials: T
authors.lastname: Coffman
authors.forename: Rohan
authors.initials: R
authors.lastname: John
authors.forename: Shao-Ling
authors.initials: SL
authors.lastname: Zhang
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
authors.forename: James W
authors.initials: JW
authors.lastname: Scholey
mesh_terms: D000804:Angiotensin II / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D002460:Cell Line; D051547:Heme Oxygenase-1 / Q000096:biosynthesis / Q000235:genetics; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D007687:Kidney Tubules, Proximal / Q000166:cytology / Q000378:metabolism*; D008565:Membrane Proteins / Q000096:biosynthesis / Q000235:genetics; D051379:Mice; D018345:Mice, Knockout; D020543:Proteome / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects / Q000235:genetics; D014662:Vasoconstrictor Agents / Q000378:metabolism / Q000494:pharmacology
chemical_list: D015415:Biomarkers; D008565:Membrane Proteins; D020543:Proteome; D014662:Vasoconstrictor Agents; D000804:Angiotensin II; C496541:HMOX1 protein, human; D051547:Heme Oxygenase-1; C496542:Hmox1 protein, mouse
keywords: Angiotensin II; Heme Oxygenase; Kidney; Proteomics; Proximal Tubule Epithelial cells; Renin Angiotensin System; SILAC