[Cyclosporin A causes oxidative stress and mitochondrial dysfunction in renal tubular cells]. / La ciclosporina A origina estrés oxidativo y disfunción mitocondrial en células tubulares renales.

Reactive oxygen species (ROS) have been implicated in cyclosporin A (CsA) nephrotoxicity. As mitochondria are one of the main sources of ROS in cells, we evaluated the role of CsA in mitochondrial structure and function in LLC-PK1 cells. We incubated cells with CsA 1 microM for 24 hours and studies were performed with flow citometry and confocal microscopy. We studied mitochondrial NAD(P)H content, superoxide anion (O2.-) production (MitoSOX Red), oxidation of cardiolipin of inner mitochondrial membrane (NAO) and mitochondrial membrane potential (DIOC2(3)). Also we analyzed the intracellular ROS synthesis (H2DCF-DA) and reduced glutation (GSH) of cells. Our results showed that CsA decreased NAD(P)H and membrane potential, and increased O2.- in mitochondria. CsA also provoked oxidation of cardiolipin. Furthermore, CsA increased intracellular ROS production and decreased GSH content. These results suggest that CsA has crucial effects in mitochondria. CsA modified mitochondrial physiology through the decrease of antioxidant mitochondrial compounds as NAD(P)H and the dissipation of mitochondrial membrane potential and increase of oxidants as O2.-. Also, CsA alters lipidic structure of inner mitochondrial membrane through the oxidation of cardiolipin. These effects trigger a chain of events that favour intracellular synthesis of ROS and depletion of GSH that can compromise cellular viability. Nephrotoxic cellular effects of CsA can be explained, at least in part, through its influence on mitochondrial functionalism.

[All-trans retinoic acid induces apoptosis in human mesangial cells: involvement of stress activated p38 kinase]. / El ácido retinoico todo-trans induce apoptosis en células mesangiales humanas: implicación de la quinasa activada por estrés p38.

All-trans retinoic acid (AR-t) is used for treating acute promyelocytic leukemia and renal cell carcinoma and it also has therapeutic value in several animal models of renal disease. Among its renal targets, mesangial cells have been widely studied: they have both retinoic acid receptors (RAR) and retinoid X receptors (RXR) and the cell growth is inhibited when human mesangial cells are incubated with 1-10 microM AR-t. Although his effect has been related with the antiproliferative action of AR-t, there are no studies on the involvement of apoptosis in AR-t induced cell growth when higher concentrations of retinoid are used. Our studies show that 25 microM AR-t triggers mesangial cell apoptosis assessed by light and fluorescence microscopy (Giemsa stain and acridine orange stain, respectively), DNA electrophoresis, flow cytometry (annexin-V) and immunocytochemistry (TUNEL). AR-t induced apoptosis was not inhibited by preincubation with the RXR pan-antagonist HX531 nor with the RAR pan-antagonist AGN 193109, this suggesting RAR and RXIR are not involved in AR-t induced cell death. Previous results of our group showed that ERK (extracellular regulated kinase) and INK (c-Jun kinase), two members of the MAP (mitogen activated protein) kinase family, are involved in non apoptotic effects of AR-t on mesangial cells. Therefore we focussed on the stress activated p38 kinase, the third member of the MAPK family, to investigate its involvement in AR-t induced apoptosis. The results confirmed a role of p38 since: 1) preincubation with B5203589, a p38 inhibitor, inhibited ARA induced apoptosis; 2) incubation with AR-t induced p38 phosphorilation after few minutes and p38 remained phosphorilated for at least 8 hours and 3) AR-t induced p38 phosphorilation was inhibited by SB203589. These data suggest that AR-t might have toxic side effects on the kidney but also suggest that AR-t could be an useful inhibitor of pathological mesangial cell expansion.

Flow cytometric analysis of the in situ hybridization of cyclooxygenase isoforms in mesangial cells treated with cyclosporine A.

BACKGROUND: Cyclosporine A increases oxidative stress in kidney and we hypothesized that cyclooxygenase (COX) may be involved in this effect. MATERIAL AND METHODS: Mesangial cells of Cyclosporine A-treated (4, 7 or 10 days) rats were obtained to evaluate mRNA expression of COX-isoforms (COX-1, constitutive and COX-2, inducible) by "in situ" hybridization. Probes were labelled using "Gene Image Random Prime Labelling Protocol" and COX expression was measured by flow cytometry. RESULTS AND DISCUSSION: "In situ" hybridization by flow cytometry is an useful method to detect mRNA. We observed an increased COX-2 expression in a time-dependent manner in parallel with Reactive Oxygen Species synthesis. COX-1 expression increased only at 10 days.