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RAGE-induced cytosolic ROS promote mitochondrial superoxide generation in diabetes.

ABSTRACT: Damaged mitochondria generate an excess of superoxide, which may mediate tissue injury in diabetes. We hypothesized that in diabetic nephropathy, advanced glycation end-products (AGEs) lead to increases in cytosolic reactive oxygen species (ROS), which facilitate the production of mitochondrial superoxide. In normoglycemic conditions, exposure of primary renal cells to AGEs, transient overexpression of the receptor for AGEs (RAGE) with an adenoviral vector, and infusion of AGEs to healthy rodents each induced renal cytosolic oxidative stress, which led to mitochondrial permeability transition and deficiency of mitochondrial complex I. Because of a lack of glucose-derived NADH, which is the substrate for complex I, these changes did not lead to excess production of mitochondrial superoxide; however, when we performed these experiments in hyperglycemic conditions in vitro or in diabetic rats, we observed significant generation of mitochondrial superoxide at the level of complex I, fueled by a sustained supply of NADH. Pharmacologic inhibition of AGE-RAGE-induced mitochondrial permeability transition in vitro abrogated production of mitochondrial superoxide; we observed a similar effect in vivo after inhibiting cytosolic ROS production with apocynin or lowering AGEs with alagebrium. Furthermore, RAGE deficiency prevented diabetes-induced increases in renal mitochondrial superoxide and renal cortical apoptosis in mice. Taken together, these studies suggest that AGE-RAGE-induced cytosolic ROS production facilitates mitochondrial superoxide production in hyperglycemic environments, providing further evidence of a role for the advanced glycation pathway in the development and progression of diabetic nephropathy.

SUBMITTER: Coughlan MT

PROVIDER: S-EPMC2663823 | biostudies-literature | 2009 Apr

REPOSITORIES: biostudies-literature

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RAGE-induced cytosolic ROS promote mitochondrial superoxide generation in diabetes.

Coughlan Melinda T MT Thorburn David R DR Penfold Sally A SA Laskowski Adrienne A Harcourt Brooke E BE Sourris Karly C KC Tan Adeline L Y AL Fukami Kei K Thallas-Bonke Vicki V Nawroth Peter P PP Brownlee Michael M Bierhaus Angelika A Cooper Mark E ME Forbes Josephine M JM

Journal of the American Society of Nephrology : JASN 20090121 4

Damaged mitochondria generate an excess of superoxide, which may mediate tissue injury in diabetes. We hypothesized that in diabetic nephropathy, advanced glycation end-products (AGEs) lead to increases in cytosolic reactive oxygen species (ROS), which facilitate the production of mitochondrial superoxide. In normoglycemic conditions, exposure of primary renal cells to AGEs, transient overexpression of the receptor for AGEs (RAGE) with an adenoviral vector, and infusion of AGEs to healthy rodent ...[more]

PMID: 19158353

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Project description:Background:Upon vascular damage, the exposed subendothelial matrix recruits circulating platelets to site of injury while inducing their firm adhesion mainly via GPVI-collagen interaction. GPVI also supports aggregatory and pro-coagulant functions in arterial shear rate even on the matrix other than collagen. Reactive oxygen species (ROS) modulate these stages of thrombosis; however augmented oxidant stress also disturbs platelet functions. Stored-dependent platelet lesion is associated with the increasing levels of ROS. Whether ROS accumulation is also relevant to collagen-dependent platelet dysfunction is the main interest of this study. Methods:Fresh PRP-PCs (platelet concentrates) were either stimulated with potent ROS-inducers PMA and CCCP or stored for 5?days. Intra-platelet superoxide (O2 --) or mitochondrial-ROS and GPVI expression were detected by flowcytometery. GPVI shedding, platelet aggregation and spreading/adhesion to collagen were analyzed by western blot, aggregometry and fluorescence-microscopy, respectively. Results:Mitochondrial-ROS levels in 5?days-stored PCs were comparable to those induced by mitochondrial uncoupler, CCCP while O2 -- generations were higher than those achieved by PMA. Shedding levels in 5?days-stored PCs were higher than those induced by these potent stimuli. GPVI expressions were reduced comparably in CCCP treated and 5?days-stored PCs. Platelet adhesion was also diminished during storage while demonstrating significant reverse correlation with GPVI shedding. However, only firm adhesion (indicated by platelets spreading or adhesion surface area) was relevant to GPVI expression. Platelet adhesion and aggregation also showed reverse correlations with both O2-- and mitochondrial-ROS formations; nonetheless mitochondrial-ROS was only relevant to firm adhesion. Conclusion:As a sensitive indicator of platelet activation, GPVI shedding was correlated with either simple adhesion or spreading to collagen, while GPVI expression was only relevant to platelet spreading. Thereby, if the aim of GPVI evaluation is to examine platelet firm adhesion, expression seems to be a more specific choice. Furthermore, the comparable levels of ROS generation in 5?days-stored PCs and CCCP treated platelets, indicated that these products are significantly affected by oxidative stress. Reverse correlation of accumulating ROS with collagen-dependent platelet dysfunction is also a striking sign of an oxidant-induced lesion that may raise serious question about the post-transfusion quality and competence of longer-stored platelet products.

Project description:Skeletal muscle generation of reactive oxygen species (ROS) is increased following contractile activity and these species interact with multiple signaling pathways to mediate adaptations to contractions. The sources and time course of the increase in ROS during contractions remain undefined. Confocal microscopy with specific fluorescent probes was used to compare the activities of superoxide in mitochondria and cytosol and the hydrogen peroxide content of the cytosol in isolated single mature skeletal muscle (flexor digitorum brevis) fibers prior to, during, and after electrically stimulated contractions. Superoxide in mitochondria and cytoplasm were assessed using MitoSox red and dihydroethidium (DHE) respectively. The product of superoxide with DHE, 2-hydroxyethidium (2-HE) was acutely increased in the fiber cytosol by contractions, whereas hydroxy-MitoSox showed a slow cumulative increase. Inhibition of nitric oxide synthases increased the contraction-induced formation of hydroxy-MitoSox only with no effect on 2-HE formation. These data indicate that the acute increases in cytosolic superoxide induced by contractions are not derived from mitochondria. Data also indicate that, in muscle mitochondria, nitric oxide (NO) reduces the availability of superoxide, but no effect of NO on cytosolic superoxide availability was detected. To determine the relationship of changes in superoxide to hydrogen peroxide, an alternative specific approach was used where fibers were transduced using an adeno-associated viral vector to express the hydrogen peroxide probe, HyPer within the cytoplasmic compartment. HyPer fluorescence was significantly increased in fibers following contractions, but surprisingly followed a relatively slow time course that did not appear directly related to cytosolic superoxide. These data demonstrate for the first time temporal and site specific differences in specific ROS that occur in skeletal muscle fibers during and after contractile activity.

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RAGE-induced cytosolic ROS promote mitochondrial superoxide generation in diabetes.

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RAGE-induced cytosolic ROS promote mitochondrial superoxide generation in diabetes.

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