title: Characterization of calcium oxalate crystal-induced changes in the secretome of U937 human monocytes.
abstract: In kidney stone disease, migratory monocytes have been found to mediate progressive renal inflammation through the secretion of numerous inflammatory mediators. However, whether calcium oxalate monohydrate (COM), which is the major crystalline compound of kidney stones, has any effects on proteins secreted from monocytes remained largely unknown. The present study aimed to characterize changes in the secretome of U937 human monocytes induced by COM crystals. The viability of cells in serum/protein-free medium was serially evaluated and the data revealed that an exposure time of 16 h was optimal for this study, whereas prolonged incubation for 24 h resulted in declined cell viability. Using this optimal time-point, the secreted proteins recovered from serum/protein-free culture supernatants of controlled and COM-treated cells were resolved in 2-DE and stained with Deep Purple fluorescent dye. Quantitative intensity analysis revealed statistically significant changes in levels of 18 secreted proteins (14 increased and 4 decreased) from COM-treated cells. These significantly altered secreted proteins were then identified by Q-TOF MS and/or MS/MS analyses. Among these, the increased levels of secreted heat shock protein 90 (HSP90), HSP70 and β-actin were confirmed by Western blot analysis. The increased level of extracellular HSP90 was confirmed on the COM-treated cell surface by the immunofluorescence study, whereas the increased secretion of IFN-α was validated by ELISA. Global protein network analysis, literature search and bioinformatics revealed that these significantly altered secreted proteins were involved mainly in immune response and cell survival. Therefore, changes in the secretome of monocytes induced by COM crystals may be related, at least in part, to progressive renal inflammation found in kidney stone disease.
authors.affiliation: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, 6th Floor SiMR Building, 2 Wanglang Road, Bangkoknoi, Bangkok, Thailand. thongboonkerd@dr.com vthongbo@yahoo.com and Department of Immunology and Immunology Graduate Program, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Kitisak
authors.initials: K
authors.lastname: Sintiprungrat
authors.affiliation: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, 6th Floor SiMR Building, 2 Wanglang Road, Bangkoknoi, Bangkok, Thailand. thongboonkerd@dr.com vthongbo@yahoo.com and Department of Immunology and Immunology Graduate Program, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok, Thailand and Center for Research in Complex Systems Science, Mahidol University, Bangkok, Thailand.
authors.forename: Nilubon
authors.initials: N
authors.lastname: Singhto
authors.affiliation: Medical Proteomics Unit, Office for Research and Development, Faculty of Medicine Siriraj Hospital, Mahidol University, 6th Floor SiMR Building, 2 Wanglang Road, Bangkoknoi, Bangkok, Thailand. thongboonkerd@dr.com vthongbo@yahoo.com and Center for Research in Complex Systems Science, Mahidol University, Bangkok, Thailand.
authors.forename: Visith
authors.initials: V
authors.lastname: Thongboonkerd
mesh_terms: D015153:Blotting, Western; D002129:Calcium Oxalate / Q000494:pharmacology*; D002470:Cell Survival / Q000187:drug effects; D003460:Crystallization; D015180:Electrophoresis, Gel, Two-Dimensional; D004797:Enzyme-Linked Immunosorbent Assay; D005455:Fluorescent Antibody Technique; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D016898:Interferon-alpha / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics; D015203:Reproducibility of Results; D055571:Secretory Pathway / Q000187:drug effects; D020298:U937 Cells
chemical_list: D018841:HSP90 Heat-Shock Proteins; D016898:Interferon-alpha; D020543:Proteome; D002129:Calcium Oxalate