Cyclosporine A-induced apoptosis in renal tubular cells is related to oxidative damage and mitochondrial fission
Highlights
► CsA induced ROS synthesis and decreased reduced glutathione in LLC-PK1 cells. ► CsA modified physiology of inner and outer mitochondrial membrane. ► CsA provoked mitochondrial fission and apoptosis. ► Vit E inhibited the mitochondrial alterations and apoptosis induced by CsA.
Introduction
Cyclosporine A (CsA) is a drug used to treat many autoimmune diseases and in the prevention of transplant rejection. Nephrotoxicity constitutes its main adverse effect and can cause acute or chronic kidney damage (Naesens et al., 2009). Although they have been intensively studied, the intimal cellular mechanisms of CsA nephrotoxicity remain at present not completely elucidated (Parra Cid et al., 2003). Many studies have demonstrated a significant role for reactive oxygen species (ROS). In fact, we have shown that glomeruli of rats treated with CsA increased ROS and lipid peroxidation products (Parra et al., 1998). As the antioxidant Vitamin E (Vit E) avoided kidney failure and inhibited glomerular ROS synthesis, our data suggested that ROS have a pathogenic role in CsA nephrotoxicity (de Arriba et al., 2009, Parra Cid et al., 2003, Parra et al., 1998). These results were confirmed in cellular models, showing that CsA induced ROS synthesis in mesangial or tubular cells and the inhibition of this increase by Vit E (de Arriba et al., 2009, Parra Cid et al., 2003, Parra et al., 1998, Wang and Salahudeen, 1994).
The mitochondrial respiratory chain is responsible for most of the ROS synthesis in cells (Jackson et al., 2002, Orrenius, 2007), and we hypothesized that CsA could induce modifications in mitochondrial physiology which can trigger an increased ROS synthesis. Mitochondria are complex organelles that have an outer (OMM) and an inner membrane (IMM) that folds into a complicate network of tubules and lamellae called cristae (Jackson et al., 2002, Nunnari and Suomalainen, 2012). The IMM is the site of oxidative phosphorylation, and the electron flow through the protein complexes of the respiratory chain is coupled to the exit of protons to the intermembrane space generating a membrane potential (ΔΨm). ATP synthesis is driven by the flux of protons into the mitochondrial matrix through the ATP synthase and a decrease in ΔΨm could compromise cellular energy metabolism (Jackson et al., 2002).
Mitochondrial respiration accounts for about 90% of cellular oxygen uptake and 1–2% of the oxygen consumed is converted to ROS (Jackson et al., 2002, Jezek and Hlavata, 2005, Nunnari and Suomalainen, 2012). The main ROS produced by mitochondria is superoxide anion (O2−), a highly reactive compound. Several cellular antioxidant systems in mitochondria or cytosol inhibited its oxidant capacity (Jackson et al., 2002, Jezek and Hlavata, 2005, Nunnari and Suomalainen, 2012). Among these systems there are antioxidant enzymes like mitochondrial Mn-superoxide dismutase or cytosolic Cu–Zn-superoxide dismutase, glutathione peroxidase and catalase. Also, several non-protein antioxidants can inactivate ROS like glutathione (GSH) or vitamins C and E. The thiol-containing compound GSH acts as the primary cellular homeostatic redox buffer in cells, and when ROS are produced, the proportion of oxidized glutathione (GSSG) increases, in a reaction mediated by glutathione peroxidase.
CsA increased mitochondrial ROS and this effect could be related to an increase in ROS production, a decrease of the antioxidant systems or both (de Arriba et al., 2009, Parra Cid et al., 2003, Parra et al., 1998). The increase of ROS can induce direct consequences on mitochondrial functions. Besides contributing to cellular energy metabolism, mitochondria participate in several cellular functions like fatty acid synthesis, gluconeogenesis, intracellular calcium control and regulation of cellular apoptosis (Nunnari and Suomalainen, 2012). We and others have demonstrated that CsA induced cellular apoptosis in tubular or endothelial cells (Justo et al., 2003, Perez de Hornedo et al., 2007) and it has been hypothesized that mitochondrial ROS synthesis and apoptosis are two related phenomena. Mitochondrial injury leads to the decrease of ΔΨm, mitochondrial permeability transition pore (MPTP) formation and release of apoptotic mediators, such as cytochrome c and other proteins to cytosol, caspase activation and cellular apoptosis (de Arriba et al., 2009). As Vit E inhibited these processes, we confirmed that ROS were crucial in CsA mitochondrial effects (de Arriba et al., 2009).
Mitochondria are organelles that form elongated tubules that continuosly divide and fuse to form a dynamic interconnecting network (Berman et al., 2008, Mannella, 2008). The two opposing processes are called fission and fusion and their regulation is dependent on a complex interplay of several proteins (Hoppins et al., 2007, Soubannier and McBride, 2009, Westermann, 2008). Under physiological conditions, mitochondria are elongated. Upon stress stimulation, they become fragmented, and this process may contribute to mitochondrial permeabilization and the release of apoptotic factors (Hoppins et al., 2007, Soubannier and McBride, 2009, Westermann, 2008). However, it is not defined whether CsA induces mitochondrial dynamics modifications and their possible relationship with cellular apoptosis.
Our aim was to analyze the mitochondrial structural and functional alterations that CsA induced in LLC-PK1 cells. We evaluated the role of CsA in ROS synthesis and mitochondrial membrane potential (ΔΨm), events mainly related to the IMM physiology. We also studied the effect of CsA on OMM, analyzing the influence of CsA on membrane permeabilization. Furthermore, we examined the role of CsA in mitochondrial dynamics and the expression of the main proteins involved in the mechanisms of mitochondrial fission and fusion, and its relationship with cellular apoptosis. Finally, to establish the possible role of ROS in these processes we tested the effect of the antioxidant Vit E.
Section snippets
Materials
LLC-PK1 cells were obtained from ATCC (Rockville, MD, USA). CsA, ethanol, RPMI 1640 medium, Hank's Balanced salt sodium, bongkrekic acid, ionomycin calcium salt, cobalt (II) chloride hexahydrate (CoCl2), cytochrome c (rabbit monoclonal IgG, C5723), FITC goat anti-rabbit IgG (F0382), anti-actin (A-2066), DMSO (D2650), propidium iodide (PI) (P417), NP-40 (174385), PBS (4417), Triton X-100 and RNAase (R7884) were purchased from Sigma–Aldrich (St. Louis, MO, USA). d-l-Alpha-tocopheril acetate (Vit
CsA increased cellular ROS and decreased reduced GSH, which is prevented by Vit E in LLC-PK1 cells
Our experiments with confocal microscopy showed that DCFH-DA fluorescence increased in cells treated with CsA respect to control cells. However, cells pretreated with Vit E emitted less fluorescence than control cells (Fig. 1A).
The amount of GSH measured as mBBr fluorescence, was higher in control cells than in CsA-treated cells. Vit E preincubation inhibited the fluorescence intensity decrease induced by CsA (Fig. 1B).
CsA allowed the opening of mitochondrial pores
The experiments with confocal microscope showed that CsA decreased
Discussion
CsA increases ROS production in several cellular models, modifying the oxidative balance, decreasing antioxidant levels and inducing lipid peroxidation (Parra Cid et al., 2003, Redondo-Horcajo et al., 2010). These alterations were related to cellular apoptosis. Treatment of cells with antioxidants inhibited ROS production and apoptosis suggesting that both phenomena are associated (de Arriba et al., 2009, Parra Cid et al., 2003).
The immediate consequence of ROS overproduction is the decrease of
Conclusion
Our data support that CsA modifies mitochondrial structure and function through the synthesis of ROS (Fig. 6). CsA altered IMM provoking CL oxidation and decrease in the expression of Opa1, originating cristae remodeling and facilitating the liberation of cytochrome c into the intermembrane space. CsA also induces MPTP, with the consequent decrease of ΔΨm. CsA also modified the OMM causing Bax-induced pore formation that mediates the liberation of several proteins involved in apoptosis like
Conflict of interest statement
All authors declare that there are no conflicts of interest in this study.
Acknowledgments
This work was supported by a grant from the Fundación para la Investigación Sanitaria en Castilla-La Mancha – FISCAM, Junta de Comunidades de Castilla-La Mancha [PI-2006-02]. M. Calvino was supported by a Contrato de Apoyo a la Investigación en el SNS from Instituto de Salud Carlos III [CA07/00157] and Fundación de Investigación Biomédica del Hospital La Princesa de Madrid. S. Benito was supported by an Ayuda para la Incorporación de Jóvenes Investigadores a Grupos de Investigación de
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