La ciclosporina a origina estrés oxidativo y disfunción mitocondrial en células tubulares renales

9 p.Estudiamos el efecto de la ciclosporina A (CsA) sobre la estructura y función mitocondrial en células LLC-PK1. Las células se incubaron durante 24 horas con CsA 1 µM y se analizó la producción de anión superóxido, contenido de NAD(P)H, oxidación de cardiolipina y potencial de membrana mitocondrial; además se estudió la formación de radicales libres y el contenido de glutatión reducido intracelular. Nuestros resultados demuestran que la CsA provocó un aumento del anión superóxido mitocondrial de modo paralelo al descenso de NAD(P)H; además, se produjo oxidación de la cardiolipina de la membrana interna y un descenso del potencial de membrana mitocondrial. Finalmente, observamos un aumento de la producción de radicales libres intracelulares y un descenso del glutatión reducido. En conclusión, la CsA produce modificaciones importantes en la fisiología y estructura mitocondrial con aumento de la síntesis de especies reactivas de oxígeno y descenso de la capacidad antioxidante, hechos que podrían justificar la toxicidad celular de la droga.Junta de Comunidades de Castilla-La Manch

Vitamin E protects against the mitochondrial damage caused by cyclosporin A in LLC-PK1 cells

27 p.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 mitochondriaJunta de Comunidades de Castilla-La Manch

The role of CCR5/CXCR3 expressing CD8+ cells in liver damage and viral control during persistent hepatitis C virus infection

20 p.Background/Aims:CXCR3 and CCR5 play a major role in recruiting cytotoxic T cells (Tc) and secreting secondary type 1 cytokines (Tc1) in the liver. HCV could impair their expression as a survival mechanism. The role of these chemokine receptors on CD8+ cells in chronic hepatitis C is analysed. Methods:Serum, chemokines, peripheral blood and intrahepatic lymphocytes from chronic hepatitis C patients were studied. CXCR3 / CCR5 expressing CD8+ cells were quantified by flow-cytometry. Serum chemokines concentration (CXCL10/CCL3) was measured by ELISA. Basal data were correlated with liver inflammation. Longitudinal data were obtained during treatment and correlated with virologic response. Results:CCR5/CXCR3 expressing CD8+ cells were enriched in the liver and correlated with inflammation. Chronic HCV patients presented the same frequency of CCR5high/CXCR3high expressing CD8+ cells in peripheral blood as in healthy controls but higher serum concentration of CXCL10/CCL3. Treatment with PEG-interferon a-2b plus ribavirin increased CCR5high/CXCR3high expressing CD8+ cells frequency in peripheral blood and decreased CXCL10/CCL3 serum concentration. Increase in CXCR3high expressing CD8+ cells after 24 weeks of treatment was correlated with SVR. Conclusions:In chronic hepatitis C, anti-viral treatment induces an increase in CD8+ cells expressing chemokine receptors associated with Tc1 response and a reduction in their ligands. Achievement of viral control is associated with an increase in CXCR3high expressing CD8+ cells during treatmentSchering-Plough-SpainJunta de Comunidades de Castilla-La Manch