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Lipid imaging for visualizing cilastatin amelioration of cisplatin-induced nephrotoxicity

2018; Elsevier BV; Volume: 59; Issue: 9 Linguagem: Inglês

10.1194/jlr.m080465

1539-7262

Estefanía Moreno-Gordaliza, Diego Esteban‐Fernández, Alberto Lázaro, Sarah Aboulmagd, Blanca Humanes, Alberto Tejedor, Michael Linscheid, M. Milagros Gómez-Gómez,

Drug Transport and Resistance Mechanisms

Nephrotoxicity is a major limitation to cisplatin antitumor therapies. Cilastatin, an inhibitor of renal dehydropeptidase-I, was recently proposed as a promising nephroprotector against cisplatin toxicity, preventing apoptotic cell death. In this work, cilastatin nephroprotection was further investigated in a rat model, with a focus on its effect on 76 renal lipids altered by cisplatin, including 13 new cisplatin-altered mitochondrial cardiolipin species. Lipid imaging was performed with MALDI mass spectrometry imaging (MALDI-MSI) in kidney sections from treated rats. Cilastatin was proved to significantly diminish the lipid distribution alterations caused by cisplatin, lipid levels being almost completely recovered to those of control samples. The extent of recovery of cisplatin-altered lipids by cilastatin turned out to be relevant for discriminating direct or secondary lipid alterations driven by cisplatin. Lipid peroxidation induced by cisplatin was also shown to be reduced when cilastatin was administered. Importantly, significant groups separation was achieved during multivariate analysis of cortex and outer-medullary lipids, indicating that damaged kidney can be discerned from the nephroprotected and healthy groups and classified according to lipid distribution. Therefore, we propose MALDI-MSI as a powerful potential tool offering multimolecule detection possibilities to visualize and evaluate nephrotoxicity and nephroprotection based on lipid analysis. Nephrotoxicity is a major limitation to cisplatin antitumor therapies. Cilastatin, an inhibitor of renal dehydropeptidase-I, was recently proposed as a promising nephroprotector against cisplatin toxicity, preventing apoptotic cell death. In this work, cilastatin nephroprotection was further investigated in a rat model, with a focus on its effect on 76 renal lipids altered by cisplatin, including 13 new cisplatin-altered mitochondrial cardiolipin species. Lipid imaging was performed with MALDI mass spectrometry imaging (MALDI-MSI) in kidney sections from treated rats. Cilastatin was proved to significantly diminish the lipid distribution alterations caused by cisplatin, lipid levels being almost completely recovered to those of control samples. The extent of recovery of cisplatin-altered lipids by cilastatin turned out to be relevant for discriminating direct or secondary lipid alterations driven by cisplatin. Lipid peroxidation induced by cisplatin was also shown to be reduced when cilastatin was administered. Importantly, significant groups separation was achieved during multivariate analysis of cortex and outer-medullary lipids, indicating that damaged kidney can be discerned from the nephroprotected and healthy groups and classified according to lipid distribution. Therefore, we propose MALDI-MSI as a powerful potential tool offering multimolecule detection possibilities to visualize and evaluate nephrotoxicity and nephroprotection based on lipid analysis. Cisplatin has been successfully used in combinational therapies for the treatment of numerous solid tumors, with spectacular cure rates (higher than 90%) for testicular cancer (1.Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar). The drug is able to be hydrolyzed in cellular media and interact with DNA nucleobases, causing nuclear (2.Kelland L. The resurgence of platinum-based cancer chemotherapy.Nat. 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Cisplatin particularly accumulates in the S3 segment of the proximal tubule (9.Moreno-Gordaliza E. Giesen C. Lazaro A. Esteban-Fernandez D. Humanes B. Canas B. Panne U. Tejedor A. Jakubowski N. Gomez-Gomez M.M. Elemental bioimaging in kidney by LA-ICP-MS as a tool to study nephrotoxicity and renal protective strategies in cisplatin therapies.Anal. Chem. 2011; 83: 7933-7940Crossref PubMed Scopus (119) Google Scholar), leading to cell death and renal dysfunction. In such nonproliferative cells, OS [involving reactive oxygen species generation and consequent damage of lipids, proteins, or DNA (10.Humanes B. Lazaro A. Camano S. Moreno-Gordaliza E. Lazaro J.A. Blanco-Codesido M. Lara J.M. Ortiz A. Gomez-Gomez M.M. Martin-Vasallo P. et al.Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.Kidney Int. 2012; 82: 652-663Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar)], nitrosative stress, and mitochondrial damage are particularly involved, with extrinsic and intrinsic apoptotic pathways taking place (3.Karasawa T. Steyger P.S. An integrated view of cisplatin-induced nephrotoxicity and ototoxicity.Toxicol. Lett. 2015; 237: 219-227Crossref PubMed Scopus (306) Google Scholar). Numerous nephroprotective approaches based on different molecular targets have been tested in vivo and in vitro for cisplatin (1.Pabla N. Dong Z. Cisplatin nephrotoxicity: mechanisms and renoprotective strategies.Kidney Int. 2008; 73: 994-1007Abstract Full Text Full Text PDF PubMed Scopus (1345) Google Scholar, 11.Yang Y. Song M. Liu Y. Liu H. Sun L. Peng Y. Liu F. Venkatachalam M.A. Dong Z. 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Tejedor A. Jakubowski N. Gomez-Gomez M.M. Elemental bioimaging in kidney by LA-ICP-MS as a tool to study nephrotoxicity and renal protective strategies in cisplatin therapies.Anal. Chem. 2011; 83: 7933-7940Crossref PubMed Scopus (119) Google Scholar, 10.Humanes B. Lazaro A. Camano S. Moreno-Gordaliza E. Lazaro J.A. Blanco-Codesido M. Lara J.M. Ortiz A. Gomez-Gomez M.M. Martin-Vasallo P. et al.Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.Kidney Int. 2012; 82: 652-663Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar) models without affecting the antitumor effect of the drug. Its ability to inhibit apoptosis and OS and to decrease inflammatory response in renal proximal tubule epithelial cells (RPTECs) has been demonstrated (10.Humanes B. Lazaro A. Camano S. Moreno-Gordaliza E. Lazaro J.A. Blanco-Codesido M. Lara J.M. Ortiz A. Gomez-Gomez M.M. 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Schultz J.A. Balaban C.D. Woods A.S. Lipid imaging within the normal rat kidney using silver nanoparticles by matrix-assisted laser desorption/ionization mass spectrometry.Kidney Int. 2015; 88: 186-192Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 38.Marsching C. Eckhardt M. Gröne H-J. Sandhoff R. Hopf C. Imaging of complex sulfatides SM3 and SB1a in mouse kidney using MALDI-TOF/TOF mass spectrometry.Anal. Bioanal. Chem. 2011; 401: 53-64Crossref PubMed Scopus (47) Google Scholar), which can be expected, considering the structural and functional differences found along the nephron. On the other hand, under certain nephropathies (39.Grove K.J. Voziyan P.A. Spraggins J.M. Wang S. Paueksakon P. Harris R.C. Hudson B.G. Caprioli R.M. Diabetic nephropathy induces alterations in the glomerular and tubule lipid profiles.J. Lipid Res. 2014; 55: 1375-1385Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 40.Kaneko Y. Obata Y. Nishino T. Kakeya H. Miyazaki Y. 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Aboulmagd S. Tejedor A. Linscheid M.W. Gómez-Gómez M.M. MALDI-LTQ-Orbitrap mass spectrometry imaging for lipidomic analysis in kidney under cisplatin chemotherapy.Talanta. 2017; 164: 16-26Crossref PubMed Scopus (34) Google Scholar). Therefore, this suggests MALDI-MSI as a potentially useful tool for identifying renal damage beyond traditional methods. Herein, we have further investigated the nephroprotective effect of cilastatin in a cisplatin-treated rat model, with a special focus in kidney lipid distribution. Moreover, we have explored the possibility of using MALDI-MSI as an alternative tool for nephroprotection and nephrotoxicity evaluation in kidney sections with promising results. Cisplatin was obtained from Pharmacia Nostrum (Madrid, Spain). Cilastatin was kindly offered by Merck Sharp and Dohme S.A. (Madrid, Spain). Both drugs were dissolved in 0.9% NaCl solution (Braun Medical S.A, Barcelona, Spain) for administration. The 2,5-dihydroxybenzoic acid (DHB) and 9-aminoacrydine (9-AA), used as MALDI matrices, and methanol (MeOH), acetonitrile (ACN), 2-propanol (iPrOH), and trifluoroacetic acid (TFA), used as matrix solvents, were all purchased from Sigma-Aldrich (Steinheim, Germany). Female, 7 week old Wistar rats (WKY, Criffa, Barcelona, Spain) were treated at the animal facilities of the Instituto de Investigación Sanitaria Gregorio Marañón (IiSGM, Madrid, Spain). The animals were fed and provided water ad libitum and kept under controlled light (12 h light/dark cycle), temperature, and humidity conditions. All the procedures were approved by the Ethics Committee on Animal Experimentation from the IiSGM, and animals were treated in accordance with Directive 2010/63/EU and of Spanish Royal Decree 53/2013 on the protection of animals used for experimentation and other scientific purposes. Four groups of rats were administered treatments by intraperitoneal injection (i.p.) as previously described (9.Moreno-Gordaliza E. Giesen C. Lazaro A. Esteban-Fernandez D. Humanes B. Canas B. Panne U. Tejedor A. Jakubowski N. Gomez-Gomez M.M. Elemental bioimaging in kidney by LA-ICP-MS as a tool to study nephrotoxicity and renal protective strategies in cisplatin therapies.Anal. Chem. 2011; 83: 7933-7940Crossref PubMed Scopus (119) Google Scholar, 10.Humanes B. Lazaro A. Camano S. Moreno-Gordaliza E. Lazaro J.A. Blanco-Codesido M. Lara J.M. Ortiz A. Gomez-Gomez M.M. Martin-Vasallo P. et al.Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.Kidney Int. 2012; 82: 652-663Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar): 1) control rats injected a 0.9% NaCl solution in the same doses and regimes of groups 3 and 4 (n = 4); 2) cilastatin-treated rats [150 mg·kg−1 of body weight (bw) per day, n = 4]; 3) cisplatin-treated rats (single dose of 5 mg·kg−1 bw at day 0, i.p., n = 4); and 4) cisplatin (as in group 3) plus cilastatin (as in group 2) treated rats (n = 4). After 5 days of treatment, animals were anesthetized and euthanized. Prior to the euthanization, urine from each animal was collected for 24 h in metabolic cages. Serum was obtained by centrifugation of blood samples. Kidneys were perfused with saline solution at 4°C, removed, decapsulated, and wrapped in aluminum foil, followed by snap-freezing in liquid N2 and stored at −80°C. Sagittal sections of 5 or 10 µm thickness were obtained with a cryostat (Thermo Fisher Scientific, model no. HM525 NX) at −20°C, thaw-mounted onto Superfrost Plus slides (Thermo Fisher Scientific, Braunschweig, Germany), and stored at −80°C. The 5 µm sagittal rat kidney sections were stained with hematoxylin–eosin (HE) (Sigma-Aldrich). Microphotographs were taken from the sections for histological examination using an inverted IX70 microscope (Olympus, Hamburg, Germany) with 20× and 60× magnification. Blood urea nitrogen (BUN) and creatinine were determined in serum using a modular AutoAnalyzer Cobas 711 (Roche, Basel, Switzerland). Glomerular filtration rate (GFR) was calculated by using creatinine clearance rate. The sulfosalicylic acid method (43.Gallego-Delgado J. Lazaro A. Gomez-Garre D. Osende J.I. Gonzalez-Rubio M.L. Herraiz M. Manzarbeitia F. Fortes J. Fernandez-Cruz A. Egido J. Long-term organ protection by doxazosin and/or quinapril as antihypertensive therapy.J. Nephrol. 2006; 19: 588-598PubMed Google Scholar) was used for total protein analysis in urine. Immunohistochemistry was carried out on 5 µm tissue sections as previously described (10.Humanes B. Lazaro A. Camano S. Moreno-Gordaliza E. Lazaro J.A. Blanco-Codesido M. Lara J.M. Ortiz A. Gomez-Gomez M.M. Martin-Vasallo P. et al.Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.Kidney Int. 2012; 82: 652-663Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Polyclonal anti-Kidney Injury Molecule-1 (anti-KIM-1) (R&D Systems; dilution 1:20) and monoclonal anti-4-hydroxy-2-nonenal (anti-4-HNE) (Oxis International Inc., Foster City, CA; dilution 1:75) were used as primary antibodies. The specificity of the antibodies was verified by controls lacking the primary antibody, producing no background. The surface area labeled by the antibodies was evaluated by quantitative image analysis (10.Humanes B. Lazaro A. Camano S. Moreno-Gordaliza E. Lazaro J.A. Blanco-Codesido M. Lara J.M. Ortiz A. Gomez-Gomez M.M. Martin-Vasallo P. et al.Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.Kidney Int. 2012; 82: 652-663Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). DNA fragmentation, an apoptosis indicator, was determined by TUNEL assay in 5 µm kidney tissue sections using a Fluorescein FragEL DNA Fragmentation Detection Kit (Calbiochem, San Diego, CA), as previously described (10.Humanes B. Lazaro A. Camano S. Moreno-Gordaliza E. Lazaro J.A. Blanco-Codesido M. Lara J.M. Ortiz A. Gomez-Gomez M.M. Martin-Vasallo P. et al.Cilastatin protects against cisplatin-induced nephrotoxicity without compromising its anticancer efficiency in rats.Kidney Int. 2012; 82: 652-663Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). A confocal microscope (Leica-SP2, Leica Microsystems, Heidelberg, Germany) was employed for visualization of TUNEL-positive cells. For each renal section, cells undergoing apoptosis were quantified in a blinded manner by counting all positive apoptotic cells in eight nonoverlapping random fields viewed at 20× magnification. First, 10 µm kidney slices were thawed inside a desiccator. Either DHB (60 mg·ml−1 in 70% MeOH with 0.1% TFA) or 9-AA (60 mg·ml−1 in 70% iPrOH/ACN (60:40) with 0.1% TFA) were used as matrices for positive or negative ion mode analysis, respectively. Each matrix was applied on the tissues with an airbrush in 15 cycles comprising deposition for 20 s followed by drying for 40 s, as previously described (42.Moreno-Gordaliza E. Esteban-Fernández D. Lázaro A. Humanes B. Aboulmagd S. Tejedor A. Linscheid M.W. Gómez-Gómez M.M. MALDI-LTQ-Orbitrap mass spectrometry imaging for lipidomic analysis in kidney under cisplatin chemotherapy.Talanta. 2017; 164: 16-26Crossref PubMed Scopus (34) Google Scholar). Data were acquired using a MALDI LTQ Orbitrap XL Pro mass spectrometer (Thermo Scientific, San José, CA), and laser intensity was adjusted to 22.5 µJ for DHB and 25–30 µJ for 9-AA. Imaging was performed in rastering mode, with a spatial resolution of 100 µm. Mass spectra were registered in full-scan mode at m/z 200–2,000 and 2 microscans per step either in positive or negative ion mode, with a 60,000 mass resolution on the Fourier Transform MS analyzer. Data processing was carried out using ImageQuest software (Thermo Scientific), MS images were extracted with 0.008 Da tolerance, and total ion current (TIC) normalization was applied. MSI raw files were converted to imzML (44.Schramm T. Hester A. Klinkert I. Both J-P. Heeren R.M.A. Brunelle A. Laprévote O. Desbenoit N. Robbe M-F. Stoeckli M. et al.imzML—a common data format for the flexible exchange and processing of mass spectrometry imaging data.J. Proteomics. 2012; 75: 5106-5110Crossref PubMed Scopus (219) Google Scholar) using ImageQuest and further processed with MSiReader (45.Robichaud G. Garrard K.P. Barry J.A. Muddiman D.C. MSiReader: an open-source interface to view and analyze high resolving power MS imaging files on Matlab platform.J. Am. Soc. Mass Spectrom. 2013; 24: 718-721Crossref PubMed Scopus (276) Google Scholar) for tissue comparison in substructural renal features. Data were extracted from six regions of interest (ROIs) per treatment type and substructure, each ROI comprising 200 data points. For exact mass identification, a database search in LipidMaps (46.Fahy E. Cotter D. Sud M. Subramaniam S. Lipid classification, structures and tools.Biochim. Biophys. Acta. 2011; 1811: 637-647Crossref PubMed Scopus (325) Google Scholar) and LipidBlast (47.Kind T. Liu K-H. Lee D.Y. DeFelice B. 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