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Bladder dysfunction in diabetes progresses gradually over time. However, the mechanisms of the development are not clear. We tested the hypothesis that oxidative stress plays a key role in the development of diabetic bladder dysfunction using an inducible smooth muscle (SM)-specific superoxide dismutase 2 (Sod2) gene knockout (SM-Sod2 KO) mouse model. Eight-week-old male Sod2lox/lox, SM-CreERT2(ki)Cre/+ mice and wildtype mice were assigned to diabetic or control groups. 4-Hydroxytamoxifen was injected into Sod2lox/lox, SM-CreERT2(ki)Cre/+ mice to activate CreERT2-mediated deletion of Sod2. Diabetes was induced by injection of streptozotocin, whereas control mice were injected with vehicle. Nine weeks later, bladder function was evaluated, and bladders were harvested for immunoblot analysis. Wild-type diabetic mice presented compensated bladder function along with increased nitrotyrosine and MnSOD in detrusor muscle. Induction of diabetes in SM-Sod2 KO mice caused deteriorated bladder function and even greater increases in nitrotyrosine compared with wild-type diabetic mice. Expression levels of apoptosis regulator Bax and cleaved caspase-3 were increased, but apoptosis regulator Bcl-2 expression was decreased in detrusor muscle of both diabetic groups, with more pronounced effects in SM-Sod2 KO diabetic mice. Our findings demonstrate that exaggerated oxidative stress can accelerate the development of bladder dysfunction in diabetic mice and the enhanced activation of apoptotic pathways in the bladder may be involved in the process. [ABSTRACT FROM AUTHOR]

It is now accepted that nutrient abundance in the blood, especially glucose, leads to the generation of reactive oxygen species (ROS), ultimately leading to increased oxidative stress in a variety of tissues. In the absence of an appropriate compensatory response from antioxidant mechanisms, the cell, or indeed the tissue, becomes overwhelmed by oxidative stress, leading to the activation of intracellular stress-associated pathways. Activation of the same or similar pathways also appears to play a role in mediating insulin resistance, impaired insulin secretion, and late diabetic complications. The ability of antioxidants to protect against the oxidative stress induced by hyperglycemia and elevated free fatty acid (FFA) levels in vitro suggests a causative role of oxidative stress in mediating the latter clinical conditions. In this review, we describe common biochemical processes associated with oxidative stress driven by hyperglycemia and/or elevated FFA and the resulting clinical outcomes: β-cell dysfunction and peripheral tissue insulin resistance. [ABSTRACT FROM AUTHOR]

Excessive generation of superoxide and mitochondrial dysfunction has been described as being important events during ischemia-reperfusion (I/R) injury. Our laboratory has demonstrated that manganese superoxide dismutase (MnSOD), a major mitochondrial antioxidant that eliminates superoxide, is inactivated during renal transplantation and renal I/R and precedes development of renal failure. We hypothesized that MnSOD knockdown in the kidney augments renal damage during renal I/R. Using newly characterized kidney-specific MnSOD knockout (KO) mice the extent of renal damage and oxidant production after I/R was evaluated. These KO mice (without I/R) exhibited low expression and activity of MnSOD in the distal nephrons, had altered renal morphology, increased oxidant production, but surprisingly showed no alteration in renal function. After I/R the MnSOD KO mice showed similar levels of injury to the distal nephrons when compared with wild-type mice. Moreover, renal function, MnSOD activity, and tubular cell death were not significantly altered between the two genotypes after I/R. Interestingly, MnSOD KO alone increased autophagosome formation, mitochondrial biogenesis, and DNA replication/repair within the distal nephrons. These findings suggest that the chronic oxidative stress as a result of MnSOD knockdown induced multiple coordinated cell survival signals including autophagy and mitochondrial biogenesis, which protected the kidney against the acute oxidative stress following I/R. [ABSTRACT FROM AUTHOR]

Nicorandil exhibits a protective effect in the vascular system, which is thought to be due to vasodilatation from opening ATP-dependent potassium channels and donation of nitric oxide. Recently, nicorandil was shown to be renoprotective in models of acute kidney injury and glomerulonephritis. However, the specific mechanisms of renoprotection are unclear. We evaluated the effect of nicorandil on the rat remnant kidney model of chronic kidney disease. Blood pressure was unchanged by a 10-wk course of nicorandil, while albuminuria was significantly reduced. Glomerular injury and tubulointerstitial injury were also ameliorated by nicorandil. Oxidative stress, as noted by renal nitrotyrosine level and urine 8-hydroxy-2=-deoxyguanosine, were elevated in this model and was significantly reduced by nicorandil treatment. Treatment was associated with maintenance of the mitochondrial antioxidant, manganese SOD, in podocytes and with suppression of xanthine oxidase expression in infiltrating macrophages. Interestingly, these two cell types express sulfonylurea receptor 2 (SUR2), a binding site of nicorandil in the ATP-dependent K channel. Consistently, we found that stimulating SUR2 with nicorandil prevented angiotensin II-mediated upregulation of xanthine oxidase in the cultured macrophage, while xanthine oxidase expression was rather induced by blocking SUR2 with glibenclamide. In conclusion, nicorandil reduces albuminuria and ameliorates renal injury by blocking oxidative stress in chronic kidney disease. [ABSTRACT FROM AUTHOR]

Our previous study has shown that chronic heart failure (CHF) reduces expression and activation of voltage-gated sodium (Nav) channels in baroreceptor neurons, which are involved in the blunted baroreceptor neuron excitability and contribute to the impairment of baroreflex in the CHF state. The present study examined the role of mitochondriaderived superoxide in the reduced Nav channel function in coronary artery ligation-induced CHF rats. CHF decreased the protein expression and activity of mitochondrial complex enzymes and manganese SOD (MnSOD) and elevated the mitochondria-derived superoxide level in the nodose neurons compared with those in sham nodose neurons. Adenoviral MnSOD (Ad.MnSOD) gene transfection (50 multiplicity of infection) into the nodose neurons normalized the MnSOD expression and reduced the elevation of mitochondrial superoxide in the nodose neurons from CHF rats. Ad.MnSOD also partially reversed the reduced protein expression and current density of the Nav channels and the suppressed cell excitability (the number of action potential and the current threshold for inducing action potential) in aortic baroreceptor neurons from CHF rats. Data from the present study indicate that mitochondrial dysfunction, including decreased protein expression and activity of mitochondrial complex enzymes and MnSOD and elevated mitochondria-derived superoxide, contributes to the reduced Nav channel activation and cell excitability in the aortic baroreceptor neurons in CHF rats. [ABSTRACT FROM AUTHOR]

Studies to quantify the protein acetylome show that lysine-residue acetylation rivals phosphorylation in prevalence as a posttranslational modification. Interesting, this posttranslational modification is modified by nutrient flux and by redox stress and targets the vast majority of metabolic pathway proteins in the mitochondria. Furthermore, the mitochondrial deacetylase enzyme SIRT3 appears to be regulated by exercise in skeletal muscle and in response to pressure overload in the heart. The alteration of protein lysine residues by acetylation and the enzymes controlling deacetylation are beginning to be explored as important regulatory events in the control of mitochondrial function and homeostasis. This review focuses on the mitochondrial targets of SIRT3 that are functionally implicated in heart biology and pathology and on the direct cardiac consequences of the genetic manipulation of SIRT3. As therapeutic modulators of other SIRT isoforms have been identified, the longer-term objective of our understanding of this biology would be to identify SIRT3 modulators as putative cardiac therapeutic agents. [ABSTRACT FROM AUTHOR]

Reactive oxygen species (ROS), particularly superoxide (O2-), have been identified as key signaling intermediates in ANG Il-induced neuronal activation and sympathoexcitation associated with cardiovascular diseases, such as hypertension and heart failure. Studies of the central nervous system have identified NADPH oxidase as a primary source of O2- in ANG 11-stimulated neurons; however, additional sources of O2-, including mitochondria, have been mostly overlooked. Here, we tested the hypothesis that ANG II increases mitochondria-produced O2- in neurons and that increased scavenging of mitochondria-produced O2-' attenuates ANG Il-dependent intraneuronal signaling. Stimulation of catecholaminergic (CATH.a) neurons with ANG H (100 nM) increased mitochondria-localized O2- levels, as measured by MitoSOX Red flu- orescence. This response was significantly attenuated in neurons overex- pressing the mitochondria-targeted O2- -scavenging enzyme Mn-SOD. To examine the biological significance of the ANG 11-mediated increase in mitochondria-produced O2-, we used the whole cell configuration of the patch-clamp technique to record the well-characterized ANG II- induced inhibition of voltage-gated K+ current (IKv) in neurons. Adeno- virus-mediated Mn-SOD overexpression or pretreatment with the cell- permeable antioxidant tempol (1 nM) significantly attenuated ANG Il-induced inhibition of IKv. In contrast, pretreatment with extracellular SOD protein (400 U/mI) had no effect. Mn-SOD overexpression also inhibited ANG 11-induced activation of Ca2+/calmodulin kinase H, a redox-sensitive protein known to modulate 1Kv. These data indicate that ANG 11 increases mitochondrial O2-, which mediates, at least in part, ANG Il-induced activation of Ca2+/calmodulin kinase II and inhibition of IKv in neurons. [ABSTRACT FROM AUTHOR]

One of the obstacles in irradiation therapy is cytoresistance, acquired by activation of self-defense systems, such as antioxidant or molecular chaperone systems, to cope with stress. We investigated whether irradiation preconditioning (IP) rendered resistance of the kidney against subsequent ischemia-reperfusion (I/R) and attempted tel elucidate any such protective mechanisms. Mice were irradiated with a total of 4, 6, or 8 Gy using a cesium-137 source irradiator and then, 6 days later, were subjected to 28 mm of bilateral renal ischemia followed by reperfusion. Eight Gy of IP significantly attenuated the increases in plasma creatinine (PCr) and blood urea nitrogen (RUN) concentration, structural damage, lipid peroxidation, superoxide formation, expression and activity of NADPH oxidase (NOX)-2, nitrotyrosine level, and hydrogen peroxide production after I/R in kidney tissues, indicating that IP protects the kidneys from I/R injury. IP markedly increased the activity of NOX, resulting in increased superoxide formation, manganese superoxide dismutase (MnSOD) activity and expression, and heat shock protein (HSP)-27 expression in kidneys. However, it did not change expressions of catalase, copper-zinc superoxide dismutase (CuZnSOD), and HSP-72. To investigate whether the protection afforded by IP was associated with increases in MnSOD and HSP-27 expression triggered by increased superoxide formation after IP, we administered manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin, a superoxide scavenger, to IP mice. This administration blocked superoxide formation and subsequent increases in MnSOD and HSP-27 expression and accelerated the post-I/R increases in PCr and BUN. In conclusion, IP renders kidney resistance to I/R injury, and this resistance is mediated by increased superoxide formation, which activates MnSOD activity and expression as well as HSP-27 expression. [ABSTRACT FROM AUTHOR]

Regulation of coronary function in diabetic hearts is an important component in preventing ischemic cardiac events but remains poorly studied. Exercise is recommended in the management of diabetes, but its effects on diabetic coronary function are relatively unknown. We investigated coronary artery myogenic tone and endothelial function, essential elements in maintaining vascular fluid dynamics in the myocardiurn. We hypothesized that exercise reduces pressure-induced myogenic constriction of coronary arteries while improving endothelial function in db/db mice, a model of type 2 diabetes. We used pressurized mouse coronary arteries isolated from hearts of control and db/db mice that were sedentary or exercised for 1 h/day on a motorized exercise-wheel system (set at 5.2 m/day, 5 days/wk). Exercise caused a ~10% weight loss in db/db mice and decreased whole body oxidative stress, as measured by plasma 8-isoprostane levels, but failed to improve hyperglycemia or plasma insulin levels. Exercise did not alter myogenic regulation of arterial diameter stimulated by increased transmural pressure, nor did it alter smooth muscle responses to U-46619 (a thromboxane agonist) or sodium nitroprusside (an endothelium-independent dilator). Moderate levels of exercise restored ACh-simulated, endothelium-dependent coronary artery vasodilation in db/db mice and increased expression of Mn SOD and decreased nitrotyrosine levels in hearts of db/db mice. We conclude that the vascular benefits of moderate levels of exercise were independent of changes in myogenic tone or hyperglycemic status and primarily involved increased nitric oxide bioavailability in the coronary microcirculation. [ABSTRACT FROM AUTHOR]

Advanced glycation end products (AGEs) promote reactive oxygen species (ROS) formation and oxidant stress (OS) in diabetes and aging-related diseases. AGE-induced OS is suppressed by AGER1, an AGE-receptor that counteracts receptor for advanced glycation end products (RAGE) and epidermal growth factor receptor (EGFR)- mediated Shc/Ras signal activation, resulting in decreased OS, Akt, FKHRL1, and antioxidants; e.g., MnSOD, regulate OS. Serine phosphorylation of p66shc also promotes OS. We examined the effects of two defined AGEs Nϵ-carboxy-methyl-lysine (CML) and methyl-glyoxal derivatives (MG) on these cellular pathways and their functional relationship to AGER1 in human embryonic kidney cells (HEK293). Stimulation of HEK293 cells with either AGE compound increased phosphorylation of Akt and FKHRL1 by approximately threefold in a redox-dependent manner. The use of p66shc mutants showed that the AGE-induced effects required Ser-36 phosphorylation of p66shc. AGE-induced phosphorylation of FKHRL1 led to a 70% downregulation of MnSOD, an effect partially blocked by a phosphatidylinositol 3-kinase inhibitor (LY-294002) and strongly inhibited by an antioxidant (N-acetylcysteine). These pro-oxidant responses were suppressed in AGER1 overexpressing cells and reappeared when AGER1 expression was reduced by small interfering RNA (siRNA). These studies point to a new pathway for the induction of OS by AGEs involving FKHRL1 inactivation and MnSOD suppression via Ser-36 phosphorylation of p66shc in human kidney cells. This represents a key mechanism by which AGER1 maintains cellular resistance against OS. Thus the decrease of AGER1 noted in aging and diabetes may further enhance OS and reduce innate antioxidant defenses. [ABSTRACT FROM AUTHOR]

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG 11 with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to amino- tyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites. [ABSTRACT FROM AUTHOR]

Manganese superoxide dismutase (MnSOD) is one of the main antioxidant enzymes that protects the heart against ischemia-reperfusion (I/R) injury. Ischemic preconditioning (IPC) is a short period of ischemia-reperfusion that reduces subsequent prolonged I/R injury. Although MnSOD localizes in mitochondria, the immediate subcellular distribution of MnSOD in heart after IPC and I/R has not been studied. In a Langendorff mouse heart model, IPC significantly improved cardiac function and reduced the infarction size induced by I/R. Immunoblotring and double immunostaining in fresh preparations revealed that I/R resulted in an increase in cytosolic MnSOD content accompanied by the release of cytochrome c. In contrast, IPC increased mitochondrial MnSOD and reduced cytosolic MnSOD and cytochrome c release induced by I/R. We found that compared with freshly prepared fractions, the freeze-thaw approach results in mitochondrial integrity disruption and release of large amounts of MnSOD into the cytosol along with mitochondrial markers even in the absence of I/R. In contrast, fresh preparations exhibit early MnSOD release into the cytosol after I/R that is prevented by IPC and cyclosporin A administration. [ABSTRACT FROM AUTHOR]