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

Sergi Clotet,Maria Jose Soler,Marta Riera,Julio Pascual,Fei Fang,Joyce Zhou,Ihor Batruch,Stella K Vasiliou,Apostolos Dimitromanolakis,Clara Barrios,Eleftherios P Diamandis,James W Scholey,Ana Konvalinka

doi:10.1074/mcp.m116.061903

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 verified in 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 validated in 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.

Highlights

From the ‡Department of Nephrology, Hospital del Mar-Institut Hospital del Mar d’Investigacions Mediques, Barcelona, Spain, 08003; §Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada; ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1W7, Canada; ʈDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Ontario M5S 1A8, Canada; **Division of Nephrology, University Health Network, Toronto, Ontario M5G 2N2, Canada
These phosphorylation events are two of many initiated by sex hormones, this was an important confirmation that proximal tubular epithelial cells (PTEC) exhibited a biological response to DHT and EST
Biological Significance: Validation of Top Candidates in the Diabetic Kidney—Because our top candidate proteins belong to biological processes critical for DKD [59], namely glycolysis, HBP and fatty acid beta-oxidation (FAO), we evaluated the effect of sex on renal HADHA, glucose-6-phosphate isomerase (GPI), and glucosamine-6-phosphate-N-acetyltransferase 1 (GNPNAT1) protein expression in female and male mice of two different models of diabetes: the STZ-induced and the Akita (Ins2WT/C96Y) models of type 1 diabetes

Summary

Introduction

From the ‡Department of Nephrology, Hospital del Mar-Institut Hospital del Mar d’Investigacions Mediques, Barcelona, Spain, 08003; §Institute of Medical Sciences, University of Toronto, Toronto, Ontario M5S 1A8, Canada; ¶Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto, Ontario M5G 1W7, Canada; ʈDepartment of Laboratory Medicine and Pathobiology, University of Toronto, Ontario M5S 1A8, Canada; **Division of Nephrology, University Health Network, Toronto, Ontario M5G 2N2, Canada. Sex plays a relevant role in the progression and severity of many kidney diseases [2]. The counter-regulatory effects of androgens and estrogens have been observed in podocytes, where EST prevented the testosterone-induced increase in the percentage of TUNEL-positive cells. In this sense, both estrogen receptor alpha (ER␣) deficiency and testosterone administration were associated with podocyte loss and augmented apoptosis in vivo [10]. Genomic actions of sex hormones are mediated by primary interactions with their specific nuclear receptors (AR for androgens and ER␣/ER␤ for estrogens) [15]. Androgens can stimulate MAPK/ERK [21] and AKT pathways [22] after interacting with the AR or the G-coupled receptor GPRC6A on the cell membrane [23, 24]

Objectives

Results

Conclusion