Dataset Information

SILAC-Based Proteomics of Primary Human Kidney Cells treated with Sex Hormones.

ABSTRACT: Male sex predispose to many kidney diseases. Considering that androgens exert deleterious effects in a variety of cell types within kidney, we hypothesized that dihydrotestosterone (DHT) would impair 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), in 4 different experiments. 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 increased in males. Furthermore, male sex was associated to higher GPI, GNPNAT1 and HADHA protein expression in diabetic Akita mice. Functional group enrichment analysis revealed a link between our DHT-regulated proteins and glycosphingolipid metabolism within the male kidney. This is the most in depth quantitative proteomic study of human primary PTEC response to sex hormone treatment. In this study we open new perspectives on how to explore the molecular mechanisms responsible for the deleterious effects of androgens in the context of kidney disease, especially diabetic nephropathy.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

DISEASE(S): Disease Free

SUBMITTER: Sergi Clotet

LAB HEAD: Ana Konvalinka

PROVIDER: PXD003811 | Pride | 2017-01-17

REPOSITORIES: Pride

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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.

Clotet Sergi S Soler Maria Jose MJ Riera Marta M Pascual Julio J Fang Fei F Zhou Joyce J Batruch Ihor I Vasiliou Stella K SK Dimitromanolakis Apostolos A Barrios Clara C Diamandis Eleftherios P EP Scholey James W JW Konvalinka Ana A

Molecular & cellular proteomics : MCP 20170104 3

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 t ...[more]

PMID: 28062795

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Project description:Somatic mutation in the X-linked phosphatidylinositol glycan class A (PIG-A) gene causes glycosylphosphatidylinositol (GPI) anchor deficiency in humans with Paroxysmal Nocturnal Hemoglobinuria (PNH). Clinically, patients with PNH have intravascular hemolysis, venous thrombosis and bone marrow failure. We produced a conditional Pig-a knock-out mouse model specifically inactivating the Pig-a gene in hematopoietic cells to study the role of PIG-A deficiency in PNH pathophysiology. We used Affymetrix Mouse Genome 430 2.0 chips to investigate the gene expression pattern in the mouse model of targeted Pig-a deletion. Experiment Overall Design: We performed microarray analysis on 3 pools of sorted GPI-deficient (GPI-) and GPI normal (GPI+) bone marrow cells derived from the same Pig-a knock-out animals, and identified 1275/669 genes of the 45,101 transcripts potentially available for screening using 2-fold change, <10% false discovery rate (FDR) and percentage of present calls as selection criteria. The major representative genes belong to the category of immune response. We tested whether the molecular differences between GPI- and GPI+ bone marrow cells could be preserved when GPI- cells were transplanted in lethally-irradiated wild type mice (C57BL/6). Microarray analysis was performed using sorted bone marrow cells from 4 animals transplanted with GPI-deficient bone marrow cells (T-GPI-) from Pig-a knock-out donors, 3 animals transplanted with GPI-normal bone marrow cells from C57BL/6 donors (T-GPI+), and GPI-normal bone marrow cells from 4 wild-type C57BL/6 mice (WT-GPI+). We found 296 probesets with 2-fold change cutoff, of which T-GPI- cells had 145 up-regulated genes in comparison to WT-GPI+ cells, and had 123 genes differentially expressed when compared to T-GPI+ cells. The gene expression of the GPI-deficient cells was very similar between the two sets of microarray experiments affirming the maintenance of the phenotype before and after bone marrow cell transplantation.

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