title: Stable Isotope Labeling with Amino Acids (SILAC)-Based Proteomics of Primary Human Kidney Cells Reveals a Novel Link between Male Sex Hormones and Impaired Energy Metabolism in Diabetic Kidney Disease.
abstract: Male sex predisposes to many kidney diseases. Considering that androgens exert deleterious effects in a variety of cell types within the kidney, we hypothesized that dihydrotestosterone (DHT) would alter the biology of the renal tubular cell by inducing changes in the proteome. We employed stable isotope labeling with amino acids (SILAC) in an indirect spike-in fashion to accurately quantify the proteome in DHT- and 17β-estradiol (EST)-treated human proximal tubular epithelial cells (PTEC). Of the 5043 quantified proteins, 76 were differentially regulated. Biological processes related to energy metabolism were significantly enriched among DHT-regulated proteins. SILAC ratios of 3 candidates representing glycolysis, N-acetylglucosamine metabolism and fatty acid β-oxidation, namely glucose-6-phosphate isomerase (GPI), glucosamine-6-phosphate-N-acetyltransferase 1 (GNPNAT1), and mitochondrial trifunctional protein subunit alpha (HADHA), were verifiedin vitro. In vivo, renal GPI and HADHA protein expression was significantly increased in males. Furthermore, male sex was associated with significantly higher GPI, GNPNAT1, and HADHA kidney protein expression in two different murine models of diabetes. Enrichment analysis revealed a link between our DHT-regulated proteins and oxidative stress within the diabetic kidney. This finding was validatedin vivo, as we observed increased oxidative stress levels in control and diabetic male kidneys, compared with females. This in depth quantitative proteomics study of human primary PTEC response to sex hormone administration suggests that male sex hormone stimulation results in perturbed energy metabolism in kidney cells, and that this perturbation results in increased oxidative stress in the renal cortex. The proteome-level changes associated with androgens may play a crucial role in the development of structural and functional changes in the diseased kidney. With our findings, we propose a possible link between diabetic and non-diabetic kidney disease progression and male sex hormone levels. Data are available via ProteomeXchange (https://www.ebi.ac.uk/pride/archive/) with identifier PXD003811.
authors.affiliation: From the ‡Department of Nephrology, Hospital del Mar-Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain, 08003; sclotet88@gmail.com.|§Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada.|**Division of Nephrology, University Health Network, Toronto, Ontario M5G 2N2, Canada.
authors.forename: Sergi
authors.initials: S
authors.lastname: Clotet
authors.affiliation: From the ‡Department of Nephrology, Hospital del Mar-Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain, 08003.
authors.forename: Maria Jose
authors.initials: MJ
authors.lastname: Soler
authors.affiliation: From the ‡Department of Nephrology, Hospital del Mar-Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain, 08003.
authors.forename: Marta
authors.initials: M
authors.lastname: Riera
authors.affiliation: From the ‡Department of Nephrology, Hospital del Mar-Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain, 08003.
authors.forename: Julio
authors.initials: J
authors.lastname: Pascual
authors.affiliation: §Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Fei
authors.initials: F
authors.lastname: Fang
authors.affiliation: §Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada.
authors.forename: Joyce
authors.initials: J
authors.lastname: Zhou
authors.affiliation: ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1W7, Canada.
authors.forename: Ihor
authors.initials: I
authors.lastname: Batruch
authors.affiliation: ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1W7, Canada.|‖Department of Laboratory Medicine and Pathobiology, University of Toronto, Ontario M5S 1A8, Canada.
authors.forename: Stella K
authors.initials: SK
authors.lastname: Vasiliou
authors.affiliation: ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1W7, Canada.
authors.forename: Apostolos
authors.initials: A
authors.lastname: Dimitromanolakis
authors.affiliation: From the ‡Department of Nephrology, Hospital del Mar-Institut Hospital del Mar d'Investigacions Mèdiques, Barcelona, Spain, 08003.
authors.forename: Clara
authors.initials: C
authors.lastname: Barrios
authors.affiliation: ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1W7, Canada.
authors.forename: Eleftherios P
authors.initials: EP
authors.lastname: Diamandis
authors.affiliation: §Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada.|**Division of Nephrology, University Health Network, Toronto, Ontario M5G 2N2, Canada.
authors.forename: James W
authors.initials: JW
authors.lastname: Scholey
authors.affiliation: §Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada.|**Division of Nephrology, University Health Network, Toronto, Ontario M5G 2N2, Canada.
authors.forename: Ana
authors.initials: A
authors.lastname: Konvalinka
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism; D003928:Diabetic Nephropathies / Q000378:metabolism*; D013196:Dihydrotestosterone / Q000494:pharmacology*; D004195:Disease Models, Animal; D004734:Energy Metabolism / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D005956:Glucose-6-Phosphate Isomerase / Q000378:metabolism; D006801:Humans; D007553:Isotope Labeling / Q000379:methods; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D008297:Male; D051379:Mice; D063992:Mitochondrial Trifunctional Protein, alpha Subunit / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*; D017855:Transferases (Other Substituted Phosphate Groups) / Q000378:metabolism
chemical_list: D016207:Cytokines; D013196:Dihydrotestosterone; C576205:HADHA protein, human; D063992:Mitochondrial Trifunctional Protein, alpha Subunit; D017855:Transferases (Other Substituted Phosphate Groups); C511050:GNPTAB protein, human; C554959:GPI protein, human; D005956:Glucose-6-Phosphate Isomerase