title: Mapping cyclosporine-induced changes in protein secretion by renal cells using stable isotope labeling with amino acids in cell culture (SILAC).
abstract: Nephrotoxicity is an adverse event that strongly limits the use of the immunosuppressant cyclosporine in solid organ transplantation and the precise molecular mechanisms underlying this toxicity remain unclear. MS-based proteomic analysis of the secretome of HEK-293 renal cells exposed to cyclosporine was performed to identify changes in protein secretion, as a first step to discover potential biomarkers of such nephrotoxicity. To detect and quantify the perturbed proteins in the culture medium we used SILAC and nano-scale liquid chromatography followed by MALDI-TOF/TOF mass spectrometry. Among 106 proteins identified, 80 were quantified in both forward/reverse SILAC experiments and quantitative proteomic analysis revealed altered levels of expression for 24 secreted proteins. These included the down-regulation of a number of extracellular matrix/cell adhesion components, and the up-regulation of secreted cyclophilins A and B, macrophage inhibition factor and phosphatidylethanolamine-binding protein 1. These changes in protein secretion were not prevented by co-incubation with the antioxidant N-acetylcysteine, suggesting that they were not triggered by cyclosporine-induced oxidative stress. The results from the present study provide important new knowledge to gain insights into the molecular mechanisms of cyclosporine-related toxicity. Some of the proteins identified here should be tested as potential biomarkers of cyclosporine nephrotoxicity in subsequent clinical studies.
authors.affiliation: INSERM, UMR-S850, Limoges, France. fabien.lamoureux@chu-rouen.fr
authors.forename: F
authors.initials: F
authors.lastname: Lamoureux
authors.forename: L N
authors.initials: LN
authors.lastname: Gastinel
authors.forename: E
authors.initials: E
authors.lastname: Mestre
authors.forename: P
authors.initials: P
authors.lastname: Marquet
authors.forename: M
authors.initials: M
authors.lastname: Essig
mesh_terms: D000596:Amino Acids / Q000493:pharmacokinetics; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002478:Cells, Cultured; D021984:Cyclophilin A / Q000378:metabolism; D021983:Cyclophilins / Q000378:metabolism; D016572:Cyclosporine / Q000633:toxicity*; D015536:Down-Regulation; D016326:Extracellular Matrix Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D007553:Isotope Labeling / Q000379:methods; D007668:Kidney / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D020543:Proteome / Q000378:metabolism*; D015854:Up-Regulation
chemical_list: D000596:Amino Acids; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D007166:Immunosuppressive Agents; D020543:Proteome; C013065:cell aggregation factors; C081786:cyclophilin B; D016572:Cyclosporine; D021984:Cyclophilin A; D021983:Cyclophilins