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Alcohol during pregnancy can impair fetal development and result in offspring with neurodevelopmental deficits. Less is known about how low to moderate alcohol exposure can affect other organs, such as the kidney. Here, the effects of moderate ethanol exposure throughout pregnancy on kidney development were examined using a rat model. Rats were fed a liquid diet containing 6% ethanol (vol/vol) or control (0% ethanol) throughout pregnancy. Kidneys were collected at embryonic day (E) 20 or postnatal day (PN) 30 and total glomerular (nephron) number determined using unbiased stereology. Kidney function was examined in offspring at 8 and 19 months. At E20, fetuses exposed to ethanol had fewer nephrons with increased apoptosis. Alcohol exposure caused kidney dysregulation of pro- (Bax) and anti- (Bcl-2) apoptotic factors, and reduced expression of the cell proliferation marker, Ki67. Prenatal alcohol decreased expression of Gdnf and Tgfb1, important regulators of branching morphogenesis, in male fetuses. At PN30, kidney volume and nephron number were lower in offspring exposed to prenatal alcohol. Urine flow and osmolality were normal in offspring exposed to alcohol however sodium excretion tended to be lower in females prenatally exposed to alcohol. Findings suggest exposure to moderate levels of alcohol during pregnancy results in impaired kidney development and leads to a permanent nephron deficit. Although the impact on adult kidney function was relatively minor, these data highlight that even at moderate levels, alcohol consumption during pregnancy can have deleterious long-term outcomes and should be avoided.
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Apoptosis/efectos de los fármacos , Etanol/administración & dosificación , Riñón/efectos de los fármacos , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Animales , Femenino , Riñón/fisiopatología , Masculino , Nefronas/efectos de los fármacos , Nefronas/fisiopatología , Embarazo , Ratas , Factores SexualesRESUMEN
Patients with diabetic hypertensive nephropathy have accelerated disease progression. Diabetes and hypertension have both been associated with changes in renal catecholamines and reactive oxygen species. With a specific focus on renal catecholamines and oxidative stress we examined a combined model of hypertension and diabetes using normotensive BPN/3J and hypertensive BPH/2J Schlager mice. Induction of diabetes (5 × 55 mg/kg streptozotocin i.p.) did not change the hypertensive status of BPH/2J mice (telemetric 24 h avg. MAP, non-diabetic 131 ± 2 vs. diabetic 129 ± 1 mmHg, n.s at 9 weeks of study). Diabetes-associated albuminuria was higher in BPH/2J vs. diabetic BPN/3J (1205 + 196/-169 versus 496 + 67/-59 µg/24 h, p = 0.008). HPLC measurement of renal cortical norepinephrine and dopamine showed significantly greater levels in hypertensive mice whilst diabetes was associated with significantly lower catecholamine levels. Diabetic BPH/2J also had greater renal catecholamine levels than diabetic BPN/3J (diabetic: norepinephrine BPN/3J 40 ± 4, BPH/2J 91 ± 5, p = 0.010; dopamine: BPN/3J 2 ± 1; BPH/2J 3 ± 1 ng/mg total protein, p < 0.001 after 10 weeks of study). Diabetic BPH/2J showed greater cortical tubular immunostaining for monoamine oxidase A and cortical mitochondrial hydrogen peroxide formation was greater in both diabetic and non-diabetic BPH/2J. While cytosolic catalase activity was greater in non-diabetic BPH/2J it was significantly lower in diabetic BPH/2J (cytosolic: BPH/2J 127 ± 12 vs. 63 ± 6 nmol/min/ml, p < 0.001). We conclude that greater levels of renal norepinephrine and dopamine associated with hypertension, together with diabetes-associated compromised anti-oxidant systems, contribute to increased renal oxidative stress in diabetes and hypertension. Elevations in renal cortical catecholamines and reactive oxygen species have important therapeutic implications for hypertensive diabetic patients.
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BACKGROUND: The mitochondrial unfolded protein response (UPRmt) is activated when misfolded proteins accumulate within mitochondria and leads to increased expression of mitochondrial chaperones and proteases to maintain protein quality and mitochondrial function. Cardiac mitochondria are essential for contractile function and regulation of cell viability, while mitochondrial dysfunction characterizes heart failure. The role of the UPRmt in the heart is unclear. OBJECTIVES: The purpose of this study was to: 1) identify conditions that activate the UPRmt in the heart; and 2) study the relationship among the UPRmt, mitochondrial function, and cardiac contractile function. METHODS: Cultured cardiac myocytes were subjected to different stresses in vitro. Mice were subjected to chronic pressure overload. Tissues and blood biomarkers were studied in patients with aortic stenosis. RESULTS: Diverse neurohumoral or mitochondrial stresses transiently induced the UPRmt in cultured cardiomyocytes. The UPRmt was also induced in the hearts of mice subjected to chronic hemodynamic overload. Boosting the UPRmt with nicotinamide riboside (which augments NAD+ pools) in cardiomyocytes in vitro or hearts in vivo significantly mitigated the reductions in mitochondrial oxygen consumption induced by these stresses. In mice subjected to pressure overload, nicotinamide riboside reduced cardiomyocyte death and contractile dysfunction. Myocardial tissue from patients with aortic stenosis also showed evidence of UPRmt activation, which correlated with reduced tissue cardiomyocyte death and fibrosis and lower plasma levels of biomarkers of cardiac damage (high-sensitivity troponin T) and dysfunction (N-terminal pro-B-type natriuretic peptide). CONCLUSIONS: These results identify the induction of the UPRmt in the mammalian (including human) heart exposed to pathological stresses. Enhancement of the UPRmt ameliorates mitochondrial and contractile dysfunction, suggesting that it may serve an important protective role in the stressed heart.
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Hemodinámica , Mitocondrias Cardíacas/metabolismo , Miocitos Cardíacos/metabolismo , Respuesta de Proteína Desplegada/fisiología , Animales , Estenosis de la Válvula Aórtica/metabolismo , Estenosis de la Válvula Aórtica/fisiopatología , Apoptosis , Supervivencia Celular/fisiología , Células Cultivadas , Humanos , Ratones , Contracción Miocárdica/fisiología , Transducción de SeñalRESUMEN
BACKGROUND: The sodium glucose co-transporter 2 (SGLT2) is primarily located within S1 of the renal proximal tubule being responsible for approximately 90% of glucose re-uptake in the kidney. Inhibition of SGLT2 is an exciting new pharmacological approach for the reduction of blood glucose in type 2 diabetic patients via inhibition of tubular glucose reabsorption. In addition to lowering glucose, this group of drugs has shown significant cardiovascular and renal protective effects. CONCLUSION: This review aims to outline the current state of preclinical research and clinical trials for different SGLT2 inhibitors and outline some of the proposed mechanisms of action, including possible effects on sympathetic nerve activity, which may contribute to the unexpected beneficial cardiovascular and reno-protective effects of this class of compounds.
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Enfermedades Cardiovasculares , Diabetes Mellitus Tipo 2 , Enfermedades Renales , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Glucemia , Enfermedades Cardiovasculares/complicaciones , Enfermedades Cardiovasculares/prevención & control , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Humanos , Hipoglucemiantes , Enfermedades Renales/complicaciones , Enfermedades Renales/prevención & control , Transportador 2 de Sodio-Glucosa , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéuticoRESUMEN
The heart relies on complex mechanisms that provide adequate myocardial oxygen supply in order to maintain its contractile function. At the cellular level, oxygen undergoes one electron reduction to superoxide through the action of different types of oxidases (e.g. xanthine oxidases, uncoupled nitric oxide synthases, NADPH oxidases or NOX). Locally generated oxygen-derived reactive species (ROS) are involved in various signaling pathways including cardiac adaptation to different types of physiological and pathophysiological stresses (e.g. hypoxia or overload). The specific effects of ROS and their regulation by oxidases are dependent on the amount of ROS generated and their specific subcellular localization. The NOX family of NADPH oxidases is a main source of ROS in the heart. Seven distinct Nox isoforms (NOX1-NOX5 and DUOX1 and 2) have been identified, of which NOX1, 2, 4 and 5 have been characterized in the cardiovascular system. For the purposes of this review, we will focus on the effects of NADPH oxidase 4 (NOX4) in the heart.
RESUMEN
Increasing evidence points to the fact that defects in the resolution of inflammatory pathways predisposes individuals to the development of chronic inflammatory diseases, including diabetic complications such as accelerated atherosclerosis. The resolution of inflammation is dynamically regulated by the production of endogenous modulators of inflammation, including lipoxin A4 (LXA4). Here, we explored the therapeutic potential of LXA4 and a synthetic LX analog (Benzo-LXA4) to modulate diabetic complications in the streptozotocin-induced diabetic ApoE-/- mouse and in human carotid plaque tissue ex vivo. The development of diabetes-induced aortic plaques and inflammatory responses of aortic tissue, including the expression of vcam-1, mcp-1, il-6, and il-1ß, was significantly attenuated by both LXA4 and Benzo-LXA4 in diabetic ApoE-/- mice. Importantly, in mice with established atherosclerosis, treatment with LXs for a 6-week period, initiated 10 weeks after diabetes onset, led to a significant reduction in aortic arch plaque development (19.22 ± 2.01% [diabetic]; 12.67 ± 1.68% [diabetic + LXA4]; 13.19 ± 1.97% [diabetic + Benzo-LXA4]). Secretome profiling of human carotid plaque explants treated with LXs indicated changes to proinflammatory cytokine release, including tumor necrosis factor-α and interleukin-1ß. LXs also inhibited platelet-derived growth factor-stimulated vascular smooth muscle cell proliferation and transmigration and endothelial cell inflammation. These data suggest that LXs may have therapeutic potential in the context of diabetes-associated vascular complications.
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Antiinflamatorios no Esteroideos/uso terapéutico , Aorta/efectos de los fármacos , Aterosclerosis/tratamiento farmacológico , Diabetes Mellitus Experimental/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Lipoxinas/uso terapéutico , Animales , Antiinflamatorios no Esteroideos/farmacología , Aterosclerosis/etiología , Quimiocina CCL2/metabolismo , Citocinas/metabolismo , Diabetes Mellitus Experimental/complicaciones , Humanos , Inflamación/etiología , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Lipoxinas/farmacología , Ratones , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
Background The failure of spontaneous resolution underlies chronic inflammatory conditions, including microvascular complications of diabetes such as diabetic kidney disease. The identification of endogenously generated molecules that promote the physiologic resolution of inflammation suggests that these bioactions may have therapeutic potential in the context of chronic inflammation. Lipoxins (LXs) are lipid mediators that promote the resolution of inflammation.Methods We investigated the potential of LXA4 and a synthetic LX analog (Benzo-LXA4) as therapeutics in a murine model of diabetic kidney disease, ApoE-/- mice treated with streptozotocin.Results Intraperitoneal injection of LXs attenuated the development of diabetes-induced albuminuria, mesangial expansion, and collagen deposition. Notably, LXs administered 10 weeks after disease onset also attenuated established kidney disease, with evidence of preserved kidney function. Kidney transcriptome profiling defined a diabetic signature (725 genes; false discovery rate P≤0.05). Comparison of this murine gene signature with that of human diabetic kidney disease identified shared renal proinflammatory/profibrotic signals (TNF-α, IL-1ß, NF-κB). In diabetic mice, we identified 20 and 51 transcripts regulated by LXA4 and Benzo-LXA4, respectively, and pathway analysis identified established (TGF-ß1, PDGF, TNF-α, NF-κB) and novel (early growth response-1 [EGR-1]) networks activated in diabetes and regulated by LXs. In cultured human renal epithelial cells, treatment with LXs attenuated TNF-α-driven Egr-1 activation, and Egr-1 depletion prevented cellular responses to TGF-ß1 and TNF-αConclusions These data demonstrate that LXs can reverse established diabetic complications and support a therapeutic paradigm to promote the resolution of inflammation.
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Antiinflamatorios no Esteroideos/uso terapéutico , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/genética , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Lipoxinas/uso terapéutico , Albuminuria/etiología , Animales , Antiinflamatorios no Esteroideos/farmacología , Colágeno/metabolismo , Diabetes Mellitus Experimental , Nefropatías Diabéticas/complicaciones , Modelos Animales de Enfermedad , Regulación de la Expresión Génica/efectos de los fármacos , Mesangio Glomerular/patología , Humanos , Inyecciones Intraperitoneales , Lipoxinas/farmacología , Masculino , Ratones Noqueados para ApoE , FN-kappa B/genética , Factor de Crecimiento Derivado de Plaquetas/genética , Transcriptoma , Factor de Crecimiento Transformador beta1/genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
NADPH oxidase-derived excessive production of reactive oxygen species (ROS) in the kidney plays a key role in mediating renal injury in diabetes. Pathological changes in diabetes include mesangial expansion and accumulation of extracellular matrix (ECM) leading to glomerulosclerosis. There is a paucity of data about the role of the Nox5 isoform of NADPH oxidase in animal models of diabetic nephropathy since Nox5 is absent in the mouse genome. Thus, we examined the role of Nox5 in human diabetic nephropathy in human mesangial cells and in an inducible human Nox5 transgenic mouse exposed to streptozotocin-induced diabetes. In human kidney biopsies, Nox5 was identified to be expressed in glomeruli, which appeared to be increased in diabetes. Colocalization demonstrated Nox5 expression in mesangial cells. In vitro, silencing of Nox5 in human mesangial cells was associated with attenuation of the hyperglycemia and TGF-ß1-induced enhanced ROS production, increased expression of profibrotic and proinflammatory mediators, and increased TRPC6, PKC-α, and PKC-ß expression. In vivo, vascular smooth muscle cell/mesangial cell-specific overexpression of Nox5 in a mouse model of diabetic nephropathy showed enhanced glomerular ROS production, accelerated glomerulosclerosis, mesangial expansion, and ECM protein (collagen IV and fibronectin) accumulation as well as increased macrophage infiltration and expression of the proinflammatory chemokine MCP-1. Collectively, this study provides evidence of a role for Nox5 and its derived ROS in promoting progression of diabetic nephropathy.
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Nefropatías Diabéticas/metabolismo , NADPH Oxidasas/metabolismo , Animales , Western Blotting , Línea Celular , Nefropatías Diabéticas/genética , Ensayo de Inmunoadsorción Enzimática , Humanos , Inflamación/metabolismo , Riñón/metabolismo , Glomérulos Renales/metabolismo , Células Mesangiales/metabolismo , Ratones , Ratones Transgénicos , NADPH Oxidasas/genética , Proteína Quinasa C beta/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
AIMS/HYPOTHESIS: Oxidative stress is a promising target in diabetes-associated vasculopathies, with inhibitors of NADPH oxidases (NOX), in particular isoforms 1 and 4, shown to be safe in early clinical development. We have explored a highly relevant late-stage intervention protocol using the clinically most advanced compound, the NOX1/4 inhibitor GKT137831, to determine whether end-organ damage can be reversed/attenuated when GKT137831 is administered in the setting of established diabetic complications. METHODS: GKT137831 was administered at two doses, 30 mg kg-1 day-1 and 60 mg kg-1 day-1, to ApoE -/- mice 10 weeks after diabetes induction with streptozotocin (STZ), for a period of 10 weeks. RESULTS: Consistent with Nox4 -/- mouse data, GKT137831 was protective in a model of diabetic nephropathy at both the 30 mg kg-1 day-1 and 60 mg kg-1 day-1 doses, through suppression of proinflammatory and profibrotic processes. Conversely, in diabetic atherosclerosis, where Nox1 -/y and Nox4 -/- mice have yielded qualitatively opposing results, the net effect of pharmacological NOX1/4 inhibition was protection, albeit to a lower extent and only at the lower 30 mg kg-1 day-1 dose. CONCLUSIONS/INTERPRETATION: As dose-dependent and tissue-specific effects of the dual NOX1/4 inhibitor GKT137831 were observed, it is critical to define in further studies the relative balance of inhibiting NOX4 vs NOX1 in the micro- and macrovasculature in diabetes.
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Complicaciones de la Diabetes/metabolismo , Complicaciones de la Diabetes/prevención & control , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas/metabolismo , Pirazoles/uso terapéutico , Piridinas/uso terapéutico , Animales , Aterosclerosis/metabolismo , Aterosclerosis/prevención & control , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/prevención & control , Ratones , Ratones Noqueados , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/deficiencia , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasa 4 , NADPH Oxidasas/antagonistas & inhibidores , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Estrés Oxidativo/efectos de los fármacos , Pirazolonas , PiridonasRESUMEN
Smooth muscle cell (SMC) proliferation and fibrosis contribute to the development of advanced atherosclerotic lesions. Oxidative stress caused by increased production or unphysiological location of reactive oxygen species (ROS) is a known major pathomechanism. However, in atherosclerosis, in particular under hyperglycaemic/diabetic conditions, the hydrogen peroxide-producing NADPH oxidase type 4 (NOX4) is protective. Here we aim to elucidate the mechanisms underlying this paradoxical atheroprotection of vascular smooth muscle NOX4 under conditions of normo- and hyperglycaemia both in vivo and ex vivo. Following 20-weeks of streptozotocin-induced diabetes, Apoe(-/-) mice showed a reduction in SM-alpha-actin and calponin gene expression with concomitant increases in platelet-derived growth factor (PDGF), osteopontin (OPN) and the extracellular matrix (ECM) protein fibronectin when compared to non-diabetic controls. Genetic deletion of Nox4 (Nox4(-/)(-)Apoe(-/-)) exacerbated diabetes-induced expression of PDGF, OPN, collagen I, and proliferation marker Ki67. Aortic SMCs isolated from NOX4-deficient mice exhibited a dedifferentiated phenotype including loss of contractile gene expression, increased proliferation and ECM production as well as elevated levels of NOX1-associated ROS. Mechanistic studies revealed that elevated PDGF signalling in NOX4-deficient SMCs mediated the loss of calponin and increase in fibronectin, while the upregulation of NOX1 was associated with the increased expression of OPN and markers of proliferation. These findings demonstrate that NOX4 actively regulates SMC pathophysiological responses in diabetic Apoe(-/-) mice and in primary mouse SMCs through the activities of PDGF and NOX1.
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Aterosclerosis/enzimología , Diabetes Mellitus Experimental/enzimología , Miocitos del Músculo Liso/fisiología , NADPH Oxidasa 4/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Animales , Aorta/metabolismo , Aorta/patología , Aterosclerosis/etiología , Aterosclerosis/patología , Becaplermina , Proliferación Celular , Células Cultivadas , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Fibrosis , Masculino , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , NADPH Oxidasa 1/metabolismo , NADPH Oxidasa 4/genética , Osteopontina/genética , Osteopontina/metabolismo , Proteínas Proto-Oncogénicas c-sis/genética , Proteínas Proto-Oncogénicas c-sis/metabolismo , Superóxidos/metabolismoRESUMEN
Oxidative stress and inflammation are central mediators of atherosclerosis particularly in the context of diabetes. The potential interactions between the major producers of vascular reactive oxygen species (ROS), NADPH oxidase (NOX) enzymes and immune-inflammatory processes remain to be fully elucidated. In the present study we investigated the roles of the NADPH oxidase subunit isoforms, NOX4 and NOX1, in immune cell activation and recruitment to the aortic sinus atherosclerotic plaque in diabetic ApoE(-/-) mice. Plaque area analysis showed that NOX4- and NOX1-derived ROS contribute to atherosclerosis in the aortic sinus following 10 weeks of diabetes. Immunohistochemical staining of the plaques revealed that NOX4-derived ROS regulate T-cell recruitment. In addition, NOX4-deficient mice showed a reduction in activated CD4(+) T-cells in the draining lymph nodes of the aortic sinus coupled with reduced pro-inflammatory gene expression in the aortic sinus. Conversely, NOX1-derived ROS appeared to play a more important role in macrophage accumulation. These findings demonstrate distinct roles for NOX4 and NOX1 in immune-inflammatory responses that drive atherosclerosis in the aortic sinus of diabetic mice.
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Aortitis/enzimología , Apolipoproteínas E/deficiencia , Aterosclerosis/enzimología , Diabetes Mellitus Experimental/enzimología , Inmunidad Celular , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasas/metabolismo , Seno Aórtico/enzimología , Animales , Aortitis/genética , Aortitis/inmunología , Aortitis/patología , Apolipoproteínas E/genética , Aterosclerosis/genética , Aterosclerosis/inmunología , Aterosclerosis/patología , Linfocitos T CD4-Positivos/enzimología , Linfocitos T CD4-Positivos/inmunología , Quimiotaxis de Leucocito , Citocinas/inmunología , Citocinas/metabolismo , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/inmunología , Diabetes Mellitus Experimental/patología , Predisposición Genética a la Enfermedad , Mediadores de Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Macrófagos/enzimología , Macrófagos/inmunología , Ratones Noqueados , NADH NADPH Oxidorreductasas/deficiencia , NADH NADPH Oxidorreductasas/genética , NADPH Oxidasa 1 , NADPH Oxidasa 4 , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Estrés Oxidativo , Fenotipo , Placa Aterosclerótica , Especies Reactivas de Oxígeno/metabolismo , Transducción de Señal , Seno Aórtico/inmunología , Seno Aórtico/patologíaRESUMEN
OBJECTIVE: Oxidative stress is considered a hallmark of atherosclerosis. In particular, the superoxide-generating type 1 NADPH oxidase (NOX1) has been shown to be induced and play a pivotal role in early phases of mouse models of atherosclerosis and in the context of diabetes mellitus. Here, we investigated the role of the most abundant type 4 isoform (NOX4) in human and mouse advanced atherosclerosis. APPROACH AND RESULTS: Plaques of patients with cardiovascular events or established diabetes mellitus showed a surprising reduction in expression of the most abundant but hydrogen peroxide (H2O2)-generating type 4 isoform (Nox4), whereas Nox1 mRNA was elevated, when compared with respective controls. As these data suggested that NOX4-derived reactive oxygen species may convey a surprisingly protective effect during plaque progression, we examined a mouse model of accelerated and advanced diabetic atherosclerosis, the streptozotocin-treated ApoE(-/-) mouse, with (NOX4(-/-)) and without genetic deletion of Nox4. Similar to the human data, advanced versus early plaques of wild-type mice showed reduced Nox4 mRNA expression. Consistent with a rather protective role of NOX4-derived reactive oxygen species, NOX4(-/-) mice showed increased atherosclerosis when compared with wild-type mice. Deleting NOX4 was associated with reduced H2O2 forming activity and attenuation of the proinflammatory markers, monocyte chemotratic protein-1, interleukin-1ß, and tumor necrosis factor-α, as well as vascular macrophage accumulation. Furthermore, there was a greater accumulation of fibrillar collagen fibres within the vascular wall and plaque in diabetic Nox4(-/-)ApoE(-/-) mice, indicative of plaque remodeling. These data could be replicated in human aortic endothelial cells in vitro, where Nox4 overexpression increased H2O2 and reduced the expression of pro-oxidants and profibrotic markers. Interestingly, Nox4 levels inversely correlated with Nox2 gene and protein levels. Although NOX2 is not constitutively active unlike NOX4 and forms rather superoxide, this opens up the possibility that at least some effects of NOX4 deletion are mediated by NOX2 activation. CONCLUSIONS: Thus, the appearance of reactive oxygen species in atherosclerosis is apparently not always a nondesirable oxidative stress, but can also have protective effects. Both in humans and in mouse, the H2O2-forming NOX4, unlike the superoxide-forming NOX1, can act as a negative modulator of inflammation and remodeling and convey atheroprotection. These results have implications on how to judge reactive oxygen species formation in cardiovascular disease and need to be considered in the development of NOX inhibitory drugs.
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Aorta/enzimología , Enfermedades de la Aorta/prevención & control , Aterosclerosis/prevención & control , Peróxido de Hidrógeno/metabolismo , Inflamación/prevención & control , NADPH Oxidasas/metabolismo , Estrés Oxidativo , Placa Aterosclerótica , Remodelación Vascular , Animales , Aorta/patología , Enfermedades de la Aorta/enzimología , Enfermedades de la Aorta/genética , Enfermedades de la Aorta/patología , Apolipoproteínas E/deficiencia , Apolipoproteínas E/genética , Aterosclerosis/enzimología , Aterosclerosis/genética , Aterosclerosis/patología , Estudios de Casos y Controles , Células Cultivadas , Citocinas/metabolismo , Modelos Animales de Enfermedad , Células Endoteliales/enzimología , Células Endoteliales/patología , Colágenos Fibrilares/metabolismo , Humanos , Inflamación/enzimología , Inflamación/genética , Inflamación/patología , Mediadores de Inflamación/metabolismo , Masculino , Glicoproteínas de Membrana/metabolismo , Ratones Noqueados , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 2 , NADPH Oxidasa 4 , NADPH Oxidasas/deficiencia , NADPH Oxidasas/genética , Interferencia de ARN , Transducción de Señal , Superóxidos/metabolismo , Factores de Tiempo , TransfecciónRESUMEN
AIMS/HYPOTHESIS: Changes in podocyte morphology and function are associated with albuminuria and progression of diabetic nephropathy. NADPH oxidase 4 (NOX4) is the main source of reactive oxygen species (ROS) in the kidney and Nox4 is upregulated in podocytes in response to high glucose. We assessed the role of NOX4-derived ROS in podocytes in vivo in a model of diabetic nephropathy using a podocyte-specific NOX4-deficient mouse, with a major focus on the development of albuminuria and ultra-glomerular structural damage. METHODS: Streptozotocin-induced diabetes-associated changes in renal structure and function were studied in male floxedNox4 and podocyte-specific, NOX4 knockout (podNox4KO) mice. We assessed albuminuria, glomerular extracellular matrix accumulation and glomerulosclerosis, and markers of ROS and inflammation, as well as glomerular basement membrane thickness, effacement of podocytes and expression of the podocyte-specific protein nephrin. RESULTS: Podocyte-specific Nox4 deletion in streptozotocin-induced diabetic mice attenuated albuminuria in association with reduced vascular endothelial growth factor (VEGF) expression and prevention of the diabetes-induced reduction in nephrin expression. In addition, podocyte-specific Nox4 deletion reduced glomerular accumulation of collagen IV and fibronectin, glomerulosclerosis and mesangial expansion, as well as glomerular basement membrane thickness. Furthermore, diabetes-induced increases in renal ROS, glomerular monocyte chemoattractant protein-1 (MCP-1) and protein kinase C alpha (PKC-α) were attenuated in podocyte-specific NOX4-deficient mice. CONCLUSIONS/INTERPRETATION: Collectively, this study shows the deleterious effect of Nox4 expression in podocytes by promoting podocytopathy in association with albuminuria and extracellular matrix accumulation in experimental diabetes, emphasising the role of NOX4 as a target for new renoprotective agents.
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Citoprotección/genética , Diabetes Mellitus Experimental/complicaciones , Nefropatías Diabéticas/genética , NADPH Oxidasas/genética , Podocitos/metabolismo , Albuminuria/genética , Albuminuria/patología , Animales , Diabetes Mellitus Experimental/inducido químicamente , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/patología , Eliminación de Gen , Riñón/metabolismo , Riñón/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , NADPH Oxidasa 4 , NADPH Oxidasas/metabolismo , Especificidad de Órganos/genética , Podocitos/patología , EstreptozocinaRESUMEN
The family of NADPH oxidase (Nox) proteins plays an integral role in the homeostatic functions of the cell, including gene expression, cell migration, proliferation, senescence and inflammation. There are currently 4 isoforms (Nox1, 2, 4 and 5) that are expressed across all cell types of the vascular system and play an important role in many physiological processes such as endothelial function, vascular tone and angiogenesis. The balance between Nox derived reactive oxygen species production and their elimination by dismutase enzymes is a critical finely tuned process. It is when this balance is shifted in disease states, either leading to an over- or under-production of reactive oxygen species that vascular injury develops. To date, Nox isoforms have been linked to the development of many vascular diseases including hypertension, atherosclerosis and stroke. The contribution of each isoform to the pathophysiology of vascular disease appears to be a matter of debate with most studies suggesting that Nox1 oxidase and Nox2 oxidase play deleterious roles, whereas Nox4 oxidase potentially plays a protective role in the vasculature. This review will discuss the current knowledge on the role of Nox derived oxidative stress in the pathophysiology of various vascular diseases including hypertension and atherosclerosis.
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Aterosclerosis/metabolismo , Hipertensión/metabolismo , NADPH Oxidasas/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Accidente Cerebrovascular/metabolismo , Animales , Aterosclerosis/enzimología , Aterosclerosis/fisiopatología , Humanos , Hipertensión/enzimología , Hipertensión/fisiopatología , Estrés Oxidativo , Accidente Cerebrovascular/enzimología , Accidente Cerebrovascular/fisiopatologíaRESUMEN
Current treatments for diabetic nephropathy (DN) only result in slowing its progression, thus highlighting a need to identify novel targets. Increased production of reactive oxygen species (ROS) is considered a key downstream pathway of end-organ injury with increasing data implicating both mitochondrial and cytosolic sources of ROS. The enzyme, NADPH oxidase, generates ROS in the kidney and has been implicated in the activation of protein kinase C (PKC), in the pathogenesis of DN, but the link between PKC and Nox-derived ROS has not been evaluated in detail in vivo. In this study, global deletion of a NADPH-oxidase isoform, Nox4, was examined in mice with streptozotocin-induced diabetes (C57Bl6/J) in order to evaluate the effects of Nox4 deletion, not only on renal structure and function but also on the PKC pathway and downstream events. Nox4 deletion attenuated diabetes-associated increases in albuminuria, glomerulosclerosis, and extracellular matrix accumulation. Lack of Nox4 resulted in a decrease in diabetes-induced renal cortical ROS derived from the mitochondria and the cytosol, urinary isoprostanes, and PKC activity. Immunostaining of renal cortex revealed that major isoforms of PKC, PKC-α and PKC-ß1, were increased with diabetes and normalized by Nox4 deletion. Downregulation of the PKC pathway was observed in tandem with reduced expression of vascular endothelial growth factor (VEGF), transforming growth factor (TGF)-ß1 and restoration of the podocyte slit pore protein nephrin. This study suggests that deletion of Nox4 may alleviate renal injury via PKC-dependent mechanisms, further strengthening the view that Nox4 is a suitable target for renoprotection in diabetes.
RESUMEN
RAGE (receptor for advanced glycation end-products) is expressed on multiple cell types implicated in the progression of atherosclerosis and plays a role in DAA (diabetes-associated atherosclerosis). The aim of the present study was to determine the relative role of either BM (bone marrow)- or non-BM-derived RAGE in the pathogenesis of STZ (streptozotocin)-induced DAA. Male ApoE (apolipoprotein E)-null (ApoE-/-:RAGE+/+) and ApoE:RAGE-null (ApoE-/-:RAGE-/-) mice at 7 weeks of age were rendered diabetic with STZ. At 8 weeks of age, ApoE-/- and ApoE-/-:RAGE-/- control and diabetic mice received BM from either RAGE-null or RAGE-bearing mice, generating various chimaeras. After 10 and 20 weeks of diabetes, mice were killed and gene expression and atherosclerotic lesion formation were evaluated respectively. Deletion of RAGE in either the BM cells or non-BM cells both resulted in a significant attenuation in DAA, which was associated with reduced VCAM-1 (vascular cell adhesion molecule-1) expression and translated into reduced adhesion in vitro. In conclusion, the results of the present study highlight the importance of both BM- and non-BM-derived RAGE in attenuating the development of DAA.
Asunto(s)
Aterosclerosis/genética , Diabetes Mellitus Experimental/genética , Receptores Inmunológicos/fisiología , Animales , Aterosclerosis/complicaciones , Aterosclerosis/patología , Médula Ósea/inmunología , Médula Ósea/metabolismo , Adhesión Celular/genética , Diabetes Mellitus Experimental/complicaciones , Diabetes Mellitus Experimental/patología , Eliminación de Gen , Regulación de la Expresión Génica , Masculino , Metaloproteinasas de la Matriz/metabolismo , Ratones , Ratones Transgénicos , Receptor para Productos Finales de Glicación Avanzada , Receptores Inmunológicos/genética , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
With the increasing incidence of obesity and type 2 diabetes, it is predicted that more than half of Americans will have diabetes or pre-diabetes by 2020. Diabetic patients develop vascular complications at a much faster rate in comparison to non-diabetic individuals, and cardiovascular risk is increased up to tenfold. With the increasing incidence of diabetes across the world, the development of vascular complications will become an increasing medical burden. Diabetic vascular complications affect the micro- and macro-vasculature leading to kidney disease often requiring dialysis and transplantation or cardiovascular disease increasing the risk for myocardial infarction, stroke and amputations as well as leading to premature mortality. It has been suggested that many complex pathways contribute to the pathobiology of diabetic complications including hyperglycaemia itself, the production of advanced glycation end products (AGEs) and interaction with the receptors for AGEs such as the receptor for advanced glycation end products (RAGE), as well as the activation of vasoactive systems such as the renin-angiotensin aldosterone system (RAAS) and the endothelin system. More recently, it has been hypothesised that reactive oxygen species derived from NAD(P)H oxidases (Nox) may represent a common downstream mediator of vascular injury in diabetes. Current standard treatment of care includes the optimization of blood glucose and blood pressure usually including inhibitors of the renin-angiotensin system. Although these interventions are able to delay progression, they fail to prevent the development of complications. Thus, there is an urgent medical need to identify novel targets in diabetic vascular complications which may include the blockade of Nox-derived ROS formation, as well as blockade of AGE formation and inhibitors of RAGE activation. These strategies may provide superior protection against the deleterious effects of diabetes on the vasculature.
Asunto(s)
Enfermedades Cardiovasculares/etiología , Diabetes Mellitus Tipo 2/complicaciones , Angiopatías Diabéticas/complicaciones , Enfermedades Renales/etiología , Animales , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/patología , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Angiopatías Diabéticas/metabolismo , Angiopatías Diabéticas/patología , Productos Finales de Glicación Avanzada/metabolismo , Humanos , Enfermedades Renales/metabolismo , Enfermedades Renales/patología , NADPH Oxidasas/metabolismo , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Sistema Renina-AngiotensinaRESUMEN
Diabetic nephropathy may occur, in part, as a result of intrarenal oxidative stress. NADPH oxidases comprise the only known dedicated reactive oxygen species (ROS)-forming enzyme family. In the rodent kidney, three isoforms of the catalytic subunit of NADPH oxidase are expressed (Nox1, Nox2, and Nox4). Here we show that Nox4 is the main source of renal ROS in a mouse model of diabetic nephropathy induced by streptozotocin administration in ApoE(-/-) mice. Deletion of Nox4, but not of Nox1, resulted in renal protection from glomerular injury as evidenced by attenuated albuminuria, preserved structure, reduced glomerular accumulation of extracellular matrix proteins, attenuated glomerular macrophage infiltration, and reduced renal expression of monocyte chemoattractant protein-1 and NF-κB in streptozotocin-induced diabetic ApoE(-/-) mice. Importantly, administration of the most specific Nox1/4 inhibitor, GKT137831, replicated these renoprotective effects of Nox4 deletion. In human podocytes, silencing of the Nox4 gene resulted in reduced production of ROS and downregulation of proinflammatory and profibrotic markers that are implicated in diabetic nephropathy. Collectively, these results identify Nox4 as a key source of ROS responsible for kidney injury in diabetes and provide proof of principle for an innovative small molecule approach to treat and/or prevent chronic kidney failure.
Asunto(s)
Diabetes Mellitus Experimental , Nefropatías Diabéticas , NADPH Oxidasas/antagonistas & inhibidores , Podocitos/enzimología , Pirazoles/farmacología , Piridinas/farmacología , Albuminuria/tratamiento farmacológico , Albuminuria/enzimología , Albuminuria/genética , Animales , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Línea Celular Transformada , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/genética , Nefropatías Diabéticas/tratamiento farmacológico , Nefropatías Diabéticas/enzimología , Nefropatías Diabéticas/genética , Modelos Animales de Enfermedad , Inhibidores Enzimáticos/farmacología , Matriz Extracelular/metabolismo , Silenciador del Gen , Glucosa/farmacología , Humanos , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , NADH NADPH Oxidorreductasas/antagonistas & inhibidores , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADPH Oxidasa 1 , NADPH Oxidasa 4 , NADPH Oxidasas/genética , NADPH Oxidasas/metabolismo , Podocitos/citología , Pirazolonas , Piridonas , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The development of macro- and microvascular complications is accelerated in diabetic patients. While some therapeutic regimes have helped in delaying progression of complications, none have yet been able to halt the progression and prevent vascular disease, highlighting the need to identify new therapeutic targets. Increased oxidative stress derived from the NADPH oxidase (Nox) family has recently been identified to play an important role in the pathophysiology of vascular disease. In recent years, specific Nox isoforms have been implicated in contributing to the development of atherosclerosis of major vessels, as well as damage of the small vessels within the kidney and the eye. With the use of novel Nox inhibitors, it has been demonstrated that these complications can be attenuated, indicating that targeting Nox derived oxidative stress holds potential as a new therapeutic strategy.