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
2017; Elsevier BV; Volume: 16; Issue: 3 Linguagem: Inglês
10.1074/mcp.m116.061903
ISSN1535-9484
AutoresSergi Clotet, María José 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,
Tópico(s)Muscle metabolism and nutrition
ResumoMale 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. 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. Chronic Kidney Disease (CKD) 1The abbreviations used are: CKD, chronic kidney disease; AR, androgen receptor; ARE, androgen response element; DHT, dihydrotestosterone; DKD, diabetic kidney disease; ERα/ERβ, estrogen receptor alpha/estrogen receptor beta; ERE, estrogen response element; EST, 17β-estradiol; FAO, fatty acid beta-oxidation; GNPNAT1, glucosamine-6-phosphate-N-acetyltransferase 1; GPER, G protein coupled estrogen receptor 1; GPI, glucose-6-phosphate isomerase; HADHA, mitochondrial trifunctional protein subunit alpha; HBP, hexosamine biosynthetic pathway; HK-2, immortalized human kidney cells; LC-MS/MS, liquid chromatography – tandem mass spectrometry; PTEC, proximal tubular epithelial cells; ROS, reactive oxygen species; SILAC, stable isotope labeling with amino acids in cell culture; TCA, tricarboxylic acid. 1The abbreviations used are: CKD, chronic kidney disease; AR, androgen receptor; ARE, androgen response element; DHT, dihydrotestosterone; DKD, diabetic kidney disease; ERα/ERβ, estrogen receptor alpha/estrogen receptor beta; ERE, estrogen response element; EST, 17β-estradiol; FAO, fatty acid beta-oxidation; GNPNAT1, glucosamine-6-phosphate-N-acetyltransferase 1; GPER, G protein coupled estrogen receptor 1; GPI, glucose-6-phosphate isomerase; HADHA, mitochondrial trifunctional protein subunit alpha; HBP, hexosamine biosynthetic pathway; HK-2, immortalized human kidney cells; LC-MS/MS, liquid chromatography – tandem mass spectrometry; PTEC, proximal tubular epithelial cells; ROS, reactive oxygen species; SILAC, stable isotope labeling with amino acids in cell culture; TCA, tricarboxylic acid. often results in irreversible deterioration of renal function that can progress to renal failure (1.Levey A.S. Eckardt K.U. Tsukamoto Y. Levin A. Coresh J. Rossert J. De Zeeuw D. Hostetter T.H. Lameire N. Eknoyan G. Definition and classification of chronic kidney disease: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO).Kidney Int. 2005; 67: 2089-2100Abstract Full Text Full Text PDF PubMed Scopus (2511) Google Scholar). Sex plays a relevant role in the progression and severity of many kidney diseases (2.Neugarten J. Golestaneh L. Gender and the prevalence and progression of renal disease.Adv. Chronic Kidney Dis. 2013; 20: 390-395Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar). At a clinical level, it is generally accepted that male sex is a risk factor for CKD (3.Neugarten J. Acharya A. Silbiger S.R. Effect of gender on the progression of nondiabetic renal disease: a meta-analysis.J. Am. Soc. Nephrol. 2000; 11: 319-329Crossref PubMed Google Scholar). 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In addition, preincubation with the antiandrogen flutamide prevented the apoptotic effects of testosterone, indicating that deleterious effects of androgens are mediated by testosterone conversion to DHT and binding to androgen receptor (AR). In turn, EST significantly attenuated the fibrotic effect of TGFβ-1 in mesangial cells (9.Silbiger S. Lei J. Ziyadeh F.N. Neugarten J. Estradiol reverses TGF-beta1-stimulated type IV collagen gene transcription in murine mesangial cells.Am. J. Physiol. 1998; 274: F1113-F1118PubMed Google Scholar). The counter-regulatory effects of androgens and estrogens have also 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 (ERα) deficiency and testosterone administration were associated with podocyte loss and augmented apoptosis in vivo (10.Doublier S. Lupia E. Catanuto P. Periera-Simon S. Xia X. Korach K. Berho M. Elliot S.J. Karl M. 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To enhance our understanding of sex effects on CKD progression, we aimed to perform an in-depth analysis of the sex hormone-regulated proteome in human proximal tubular epithelial cells (PTEC) after stimulation with DHT or EST. We hypothesized that male sex hormones induce changes in the proteome of the renal tubular cell in a more detrimental manner than estrogens. Quantitative proteomics is widely considered as an invaluable approach to explore biological systems in vitro and in vivo (25.Ong S.E. Blagoev B. Kratchmarova I. Kristensen D.B. Steen H. Pandey A. Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics.Mol. Cell. Proteomics. 2002; 1: 376-386Abstract Full Text Full Text PDF PubMed Scopus (4569) Google Scholar, 26.Mann M. Functional and quantitative proteomics using SILAC.Nat. Rev. Mol. Cell Biol. 2006; 7: 952-958Crossref PubMed Scopus (759) Google Scholar, 27.Lamond A.I. Uhlen M. Horning S. Makarov A. 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Furthermore, the same standard can be used for multiple samples and similar cell lines, and there is therefore no need to label all of them (29.Geiger T. Wisniewski J.R. Cox J. Zanivan S. Kruger M. Ishihama Y. Mann M. Use of stable isotope labeling by amino acids in cell culture as a spike-in standard in quantitative proteomics.Nat. Protoc. 2011; 6: 147-157Crossref PubMed Scopus (225) Google Scholar). Single cell line spike-in standard has been previously and efficiently used when multiple samples with high similarity were studied, or in cases where growing the cells in SILAC media was challenging (30.Zanivan S. Maione F. Hein M.Y. Hernandez-Fernaud J.R. Ostasiewicz P. Giraudo E. Mann M. SILAC-based proteomics of human primary endothelial cell morphogenesis unveils tumor angiogenic markers.Mol. Cell. Proteomics. 2013; 12: 3599-3611Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). Because SILAC-labeling of primary PTEC is not trivial and the several passages needed for the full incorporation of the labeled amino acids may lead to cell culture-induced loss of differentiation, we have employed spike-in SILAC to accurately quantify their proteome after sex hormone incubation. In particular, we SILAC-labeled immortalized human kidney HK-2 cells, and used their "heavy" proteome as internal standard to quantify the treated proteome from PTEC. Both PTEC and HK-2 are epithelial cells and originate from human renal proximal tubule. Thus, we reasoned that HK-2 proteome would allow us to accurately quantify a large proportion of PTEC proteome. In addition, HK-2 cells have been reported to show excellent labeling efficiency (31.Quan H. Peng X. Liu S. Bo F. Yang L. Huang Z. Li H. Chen X. Di W. Differentially expressed protein profile of renal tubule cell stimulated by elevated uric acid using SILAC coupled to LC-MS.Cell Physiol. 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Sex hormones differentially modulate STAT3-dependent antioxidant responses during oxidative stress in renal proximal tubule cells.In Vivo. 2014; 28: 1097-1100PubMed Google Scholar). Among all different cell types in the kidney, we performed our study in proximal tubular cells because tubules constitute most of the renal parenchymal mass, and tubular atrophy with interstitial fibrosis carries prognostic significance, and represents the common final pathway of most causes of CKD (36.Hodgkins K.S. Schnaper H.W. Tubulointerstitial injury and the progression of chronic kidney disease.Pediatr. Nephrol. 2012; 27: 901-909Crossref PubMed Scopus (146) Google Scholar, 37.Zoja C. Abbate M. Remuzzi G. Progression of renal injury toward interstitial inflammation and glomerular sclerosis is dependent on abnormal protein filtration.Nephrol. Dial. Transplant. 2015; 30: 706-712Crossref PubMed Scopus (71) Google Scholar). To our knowledge, the effect of sex hormones on the proteome of human kidney cells has not been previously explored. In addition, we are the first to use a spike-in SILAC quantitative proteomic approach between two different renal cell lines. In this discovery-based study, we provide a detailed portrait of the key biological processes impaired by DHT and not EST. By performing bioinformatics analyses and the corresponding verification and validation experiments, we demonstrate: (1) a sex-specific regulation of three candidate proteins related to energy metabolism; and (2) an association between male sex and increased oxidative stress levels within the kidney. In this article we suggest for the first time that male sex hormones induce perturbations in the metabolism of the tubular cell that may ultimately lead to impaired oxidative stress and hypertrophy in the kidney and consequently increase the susceptibility to renal disease progression in males, especially in the context of diabetes. With our findings, we provide a novel link between male sex hormones and impaired energy metabolism in the kidney that may shed new light on how to explore the mechanisms involved in the more rapid progression of CKD ascribed to male sex. To cover the intrinsic variability associated to cell culture across passages, sex hormone stimulation experiments in PTEC were conducted in two different passages (passage 4 and 5). In each passage, incubation of PTEC with sex hormones or ethanol was performed in two separate experiments, aiming to obtain biological replicates that allowed us to achieve adequate reliability and consistency in further quantitative proteomics profiling. PTEC were purchased from Lonza Walkersville Inc (Walkersville, MD). They were cultured in custom-made Dulbecco's modified Eagle's medium (DMEM), and supplemented with 10% v/v dialyzed fetal bovine serum (FBS), 10 ng/ml EGF, 5 μg/ml transferrin, 5 μg/ml insulin, 0.05 μm hydrocortisone, 50 units/ml penicillin, and 50 μg/ml streptomycin, as previously described (38.Konvalinka A. Zhou J. Dimitromanolakis A. Drabovich A.P. Fang F. Gurley S. Coffman T. John R. Zhang S.L. Diamandis E.P. Scholey J.W. Determination of an angiotensin II-regulated proteome in primary human kidney cells by stable isotope labeling of amino acids in cell culture (SILAC).J. Biol. Chem. 2013; 288: 24834-24847Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). Cells were serum starved for 18 h and treated with 100 nm DHT (n = 4) or EST (n = 3) for 10min (aiming to detect AKT and ERK phosphorylation as control experiments) or 8h (aiming to activate both nongenomic and genomic signaling for proteomic analysis). Ethanol treated cells were used as controls (CONT, n = 4). After stimulation, cells were washed three times with PBS, harvested with trypsin, and snap-frozen at −80 °C until further analysis. HK-2 cells used for SILAC labeling were provided by Dr. López Novoa (Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain). They were cultured in DMEM/F12 (1:1) free of arginine, lysine, methionine and leucine (AthenaES, Baltimore, MD), and supplemented with 10% v/v dialyzed FBS, 50units/ml penicillin, 50 μg/ml streptomycin, 2 mm glutamine, 5 μg/μl transferrin, 5 μg/μl insulin, 0.05 μm hydrocortisone, 1 nm T3 hormone, 10 ng/ml EGF, 147.5 mg/L heavy arginine (13C6), 91.25 mg/L heavy lysine (13C615N2), 17.24 mg/L light methionine, and 59.05 mg/L light leucine. After 5, 6, 8 and 10 cell population doublings of SILAC labeling, HK-2 cells were serum starved for 18h and cell pellets were collected as mentioned above for further spike-in experiments. At each point, 1×105-2×105 cells were separated and collected as a different pellet for determination of labeling efficiency. Percent label incorporation for individual peptides was calculated manually using the equation (Intensity H/Total Intensity) * 100. 100% labeling efficiency was considered for peptides with intensity equal to 0 in the light form. Results from labeling efficiency experiments are summarized in supplemental Table S1. Both PTEC and HK-2 cells were cultured in a humidified incubator at 37 °C and 5% CO2. All media were freshly made and filtered using a 0.22 μm syringe filter. Cell pellets from treated PTEC (light) and labeled HK-2 cells (heavy) were thawed on ice, resuspended in 200 μl of 0.1% w/v acid-labile detergent RapiGest SF (Waters, Milford, MA) in 25 mm ammonium bicarbonate, vortexed, and sonicated three times for 30 s. All lysates were centrifuged for 20 min at 15,000 × g at 4 °C. Total protein concentration was measured using a Coomassie (Bradford) protein assay reagent (Pierce, Waltham, MA). One hundred and fifty micrograms of protein from heavy HK-2 cells were spiked to 150 μg of each sample from light treated PTEC (1:1 mixing ratio) (Fig. 1). Proteins in detergent solution were denatured at 60 °C, and the disulfide bonds were reduced with 10 mm dithiothreitol. Following reduction, the samples were alkylated with 20 mm iodoacetamide. Proteins were then digested overnight at 37 °C with sequencing grade modified trypsin (Promega, Madison, WI). A trypsin/total protein ratio of 1:50 (w/w) was used. After digestion, RapiGest SF detergent was cleaved with trifluoroacetic acid, 1% (v/v) final concentration, and samples were centrifuged for 15 min at 15,000 rpm at 4 °C. Upon removal of Rapigest, tryptic peptides were diluted to 500 μl with strong cationic exchange (SCX) mobile phase A (0.26 m formic acid in 5% v/v acetonitrile; pH2) and loaded directly onto a 500 μl loop connected to a PolySULFOETHYL A column (2.1-mm inner diameter × 200 mm, 5 μm, 200 Å, The Nest Group Inc. (Southborough, MA)). The SCX chromatography and fractionation were performed on an HPLC system (Agilent 1100) using a 60 min two-step gradient. An elution buffer that contained all components of mobile phase A with the addition of 1 m ammonium formate was introduced at 10min and increased to 20% at 30 min and then to 100% at 45 min. Fractions were collected every 3 min from the 20 min time point onward. The resulting 10 fractions (600 μl each) corresponding to chromatographic peaks of eluting peptides were collected. Peptides in each fraction were identified by LC-MS/MS as described previously (39.Makawita S. Smith C. Batruch I. Zheng Y. Ruckert F. Grutzmann R. Pilarsky C. Gallinger S. Diamandis E.P. Integrated proteomic profiling of cell line conditioned media and pancreatic juice for the identification of pancreatic cancer biomarkers.Mol. Cell. Proteomics. 2011; 10Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). Briefly, peptides were extracted with OMIX C18 MB tips (Agilent Technologies, Lake Forest, CA), eluted in 3 μl of 65% v/v acetonitrile, diluted to 41 μl with 0.1% v/v formic acid in pure water, and loaded onto a 3.3 cm C18 pre-analytical column (IntegraFrit capillary, New Objective; inner diameter of 150 μm; 5 μm bead size; Agilent Pursuit C18, Agilent Technologies, Santa Clara, CA). Eluted peptides from the pre-analytical column were loaded onto a resolving analytical column with dimensions 15 cm × 75 μm ID (PicoTip emitter, 8 μm tip, New Objective Agilent Pursuit C18, 3 μm bead size). The trap and analytical columns were operated on the EASY-nLC1000 system (Thermo Fisher Scientific, San Jose, CA), and this liquid chromatography setup was coupled on line to a Q Exactive Plus hybrid quadrupole-Orbitrap mass spectrometer (Thermo Fisher Scientific, San Jose, CA) using a nano-ESI source (Proxeon Biosystems, Odense, Denmark). Each fraction was run using a 60 min gradient, and analyzed in a data-dependent mode in which a full MS1 scan acquisition from 400–1500 m/z with resolution 70,000 was acquired. This was followed by MS2 scan acquisition of the top 12 parent ions with resolution 17,500. The following parameters were enabled: monoisotopic precursor selection, charge state screening, and dynamic exclusion (enabled for 45 s), MS1 target value of 3e6 and maximum injection time (IT) of 100 ms, MS2 target value of 5e4 with maximum IT of 50 ms, isolation window of 1.6 Da, normalized collision energy (NCE) of 27, peptide match set to preferred, underfill ratio set to 2% (underfill ratio of 2e4), exclude isotopes set to ON. In addition, charge states of +1, >4, and unassigned charge states were not subjected to MS2 fragmentation. For protein identification and data analysis, Xcalibur software (version 3.0.63; Thermo Fisher) was utilized to generate RAW files of each MS run. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (1.Levey A.S. Eckardt K.U. Tsukamoto Y. Levin A. Coresh J. Rossert J. De Zeeuw D. Hostetter T.H. Lameire N. Eknoyan G. Definition and classification of chronic kidney disease: a position statement from Kidney Disease: Improving Global Outcomes (KDIGO).Kidney Int. 2005; 67: 2089-2100Abstract Full Text Full Text PDF PubMed Scopus (2511) Google Scholar) partner repository (https://www.ebi.ac.uk/pride/archive/) with the data set identifier PXD003811. The MS files were processed with the MaxQuant software version 1.5.2.8 (40.Cox J. Mann M. MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification.Nat. B
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