Vitamin E protects against the mitochondrial damage caused by cyclosporin A in LLC-PK1 cells.
Cyclosporin A (CsA) has nephrotoxic effects known to involve reactive oxygen species (ROS), since antioxidants prevent the kidney damage induced by this drug. Given that mitochondria are among the main sources of intracellular ROS, the aims of our study were to examine the mitochondrial effects of CsA in the porcine renal endothelial cell line LLC-PK1 and the influence of the antioxidant Vitamin E (Vit E). Following the treatment of LLC-PK1 cells with CsA, we assessed the mitochondrial synthesis of superoxide anion, permeability transition pore opening, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release and cellular apoptosis, using flow cytometry and confocal microscopy procedures. Similar experiments were done after Vit E preincubation of cells. CsA treatment increased superoxide anion in a dose-dependent way. CsA opened the permeability transition pores, caused Bax migration to mitochondria, and decreased mitochondrial membrane potential and cardiolipin content. Also CsA released cytochrome c into cytosol and provoked cellular apoptosis. Vit E pretreatment inhibited the effects that CsA induced on mitochondrial structure and function in LLC-PK1 cells and avoided apoptosis. CsA modifies mitochondrial LLC-PK1 cell physiology with loss of negative electrochemical gradient across the inner mitochondrial membrane and increased lipid peroxidation. These features are related to apoptosis and can explain the cellular damage that CsA induces. As Vit E inhibited these effects, our results suggest that they were mediated by an increase in ROS production by mitochondria.
de Arriba
G
G
Unidad de Investigación, Hospital Universitario de Guadalajara, Spain. garribad@sescam.jccm.es
de Hornedo
J Pérez
JP
Rubio
S Ramírez
SR
Fernández
M Calvino
MC
Martínez
S Benito
SB
Camarero
M Maiques
MM
Cid
T Parra
TP
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002308:Cardiolipins / Q000378:metabolism; D053178:Caspase 6 / Q000378:metabolism; D018929:Cell Culture Techniques; D016572:Cyclosporine / Q000633:toxicity*; D045304:Cytochromes c / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism; D005434:Flow Cytometry; D018374:LLC-PK1 Cells; D015227:Lipid Peroxidation / Q000187:drug effects; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000187:drug effects* / Q000378:metabolism; D033681:Mitochondrial Membrane Transport Proteins / Q000378:metabolism; D000083162:Mitochondrial Permeability Transition Pore; D008933:Mitochondrial Swelling / Q000187:drug effects; D017382:Reactive Oxygen Species / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013552:Swine; D014810:Vitamin E / Q000494:pharmacology*; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D000975:Antioxidants; D002308:Cardiolipins; D033681:Mitochondrial Membrane Transport Proteins; D000083162:Mitochondrial Permeability Transition Pore; D017382:Reactive Oxygen Species; D051028:bcl-2-Associated X Protein; D014810:Vitamin E; D016572:Cyclosporine; D045304:Cytochromes c; D053178:Caspase 6