NADPH Oxidase Isoforms Research Articles

NADPH oxidase isoform expression is temporally regulated and may contribute to microglial/macrophage polarization after spinal cord injury.

Spinal cord injury (SCI) results in both acute and chronic inflammation, as a result of activation of microglia, invasion of macrophages and activation of the NADPH oxidase (NOX) enzyme. The NOX enzyme is a primary source of reactive oxygen species (ROS) and is expressed by microglia and macrophages after SCI. These cells can assume either a pro- (M1) or anti-inflammatory (M2) polarization phenotype and contribute to tissue response to SCI. However, the contribution of NOX expression and ROS production to this polarization and vice versa is currently undefined. We therefore investigated the impact of SCI on NOX expression and microglial/macrophage polarization over time in a mouse model of contusion injury. Adult C57Bl/6 mice were exposed to a moderate T9 contusion SCI and tissue was assessed at acute, sub-acute and chronic time points for NOX isoform expression and co-expression with M1 and M2 microglia/macrophage polarization markers. Two NOX isoforms were increased after injury and were associated with both M1 and M2 markers, with an M1 preference for NOX2 acutely and NOX4 chronically. M2 cells were primarily found at acute time points only; the peak of NOX2 expression was associated with the decline in M2 polarization. In vitro, NOX2 inhibition shifted microglial polarization toward the M2 phenotype. These results now show that microglial/macrophage expression of NOX isoforms is independent of polarization state, but that NOX activity can influence subsequent polarization. These data can contribute to the therapeutic targeting of NOX as a therapy for SCI.

Salvianolic acid B improves vascular endothelial function in diabetic rats with blood glucose fluctuations via suppression of endothelial cell apoptosis

Vascular endothelial cell injury is an initial event in atherosclerosis. Salvianolic acid B (Sal B), a main bioactive component in the root of Salvia miltiorrhiza, has vascular protective effect in diabetes, but the underlying mechanisms remain unclear. The present study investigated the effect of Sal B on vascular endothelial function in diabetic rats with blood glucose fluctuations and the possible mechanisms implicated. The results showed that diabetic rats developed marked endothelial dysfunction as exhibited by impaired acetylcholine induced vasodilation. Supplementation with Sal B resulted in an evident improvement of endothelial function. Phosphorylation (Ser 1177) of endothelial nitric oxide synthase (eNOS) was significantly restored in Sal B treated diabetic rats, accompanied by an evident recovery of NO metabolites. Sal B effectively reduced vascular endothelial cell apoptosis, with Bcl-2 protein up-regulated and Bax protein down-regulated markedly. Treatment with Sal B led to an evident amelioration of oxidative stress in diabetic rats as manifested by enhanced antioxidant capacity and decreased contents of malondialdehyde in aortas. Protein levels of NOX2 and NOX4, two main isoforms of NADPH oxidase known as the major source of reactive oxygen species in the vasculature, were markedly decreased in Sal B treated groups. In addition, treatment with Sal B led to an evident decrease of serum lipids. Taken together, this study indicates that Sal B is capable of improving endothelial function in diabetic rats with blood glucose fluctuations, of which the underlying mechanisms might be related to suppression of endothelial cell apoptosis and stimulation of eNOS phosphorylation (Ser 1177).

PP.03.16] INCREASED ROS GENERATION INVOLVES MITOCHONDRIA IN MR-OVEREXPRESSING ADIPOCYTES – IMPACT ON VASCULAR FUNCTION.

Objective: Visceral adipose tissue (VAT) influences vascular function through aldosterone and mineralocorticoid receptors (MR). We demonstrated that adipocyte MR over-activation in mice leads to metabolic changes. Whether this is associated with vascular dysfunction remains unclear. Design and method: To address this, we generated mice with conditional MR overexpression targeted to adipocytes (Adipo-MROE) and studied the effect of fat conditioned medium (Fcm, collected from visceral adipose tissue) on vascular reactivity of resistant mesenteric arteries and associated molecular mechanisms. Endothelial-dependent relaxation to acetylcholine (ACh), in the presence or absence of Fcm, was assessed by wire myography. Levels of reactive oxygen species (ROS) were determined in mitochondria isolated from adipose tissues (mito-AT) by electron paramagnetic spin resonance and Amplex Red kit. Inhibitors of NADPH oxidases and mitochondrial ROS were used in our experiments. Results: We previously showed that 1) ROS were increased in adipose tissues from Adipo-MROE vs control (CTR) mice; b) Fcm from Adipo-MROE (Fcm-MR) induced endothelial dysfunction in control arterie, through redox-sensitive mechanisms. Here, we identified the source of ROS in adipocytes, with the hypothesis that NADPH oxidases and mitochondria may play an important role. Levels of superoxide were 3 fold-increased in mito-AT from Adipo-MROE compared to CTR mice (n = 7 mice per group, p < 0.01). We pre-incubated Fcm-MR with either the inhibitor of NADPH oxidase isoforms 1 and 2 (ML171, 10–6 M), either a specific inhibitor of mitochondrial-derived ROS (mito-TEMPO, 10–7 M), and assessed ACh-induced relaxation (10–6 M) in CTR arteries. Normal relaxation was restored by mito-TEMPO (% relaxation: CTR+Fcm-MR, without vs with mito-TEMPO: 47.5 ± 4.4 vs 87.1 ± 2.2, n = 6 mice per group, p < 0.05), whereas Nox1/2 inhibitor-ML171 did not improve endothelial function (% relaxation: 47.7 ± 4.2, ns). Conclusions: Our data demonstrate that adipocyte MR over-activation leads to increased mitochondrial-derived ROS generation in visceral adipose tissue, which contributes to endothelial dysfunction. We describe a novel mechanism that directly links adipose tissue and vascular function. Our study identifies novel mechanisms linking vascular/adipose tissue biology and aldosterone/MR activation, which may be particularly important in vascular dysfunction associated with metabolic syndrome.

The intimate and controversial relationship between voltage-gated proton channels and the phagocyte NADPH oxidase.

One of the most fascinating and exciting periods in my scientific career entailed dissecting the symbiotic relationship between two membrane transporters, the Nicotinamide adenine dinucleotide phosphate reduced form (NADPH) oxidase complex and voltage-gated proton channels (HV 1). By the time I entered this field, there had already been substantial progress toward understanding NADPH oxidase, but HV 1 were known only to a tiny handful of cognoscenti around the world. Having identified the first proton currents in mammalian cells in 1991, I needed to find a clear function for these molecules if the work was to become fundable. The then-recent discoveries of Henderson, Chappell, and colleagues in 1987-1988 that led them to hypothesize interactions of both molecules during the respiratory burst of phagocytes provided an excellent opportunity. In a nutshell, both transporters function by moving electrical charge across the membrane: NADPH oxidase moves electrons and HV 1 moves protons. The consequences of electrogenic NADPH oxidase activity on both membrane potential and pH strongly self-limit this enzyme. Fortunately, both consequences specifically activate HV 1, and HV 1 activity counteracts both consequences, a kind of yin-yang relationship. Notwithstanding a decade starting in 1995 when many believed the opposite, these are two separate molecules that function independently despite their being functionally interdependent in phagocytes. The relationship between NADPH oxidase and HV 1 has become a paradigm that somewhat surprisingly has now extended well beyond the phagocyte NADPH oxidase - an industrial strength producer of reactive oxygen species (ROS) - to myriad other cells that produce orders of magnitude less ROS for signaling purposes. These cells with their seven NADPH oxidase (NOX) isoforms provide a vast realm of mechanistic obscurity that will occupy future studies for years to come.

Age-related alterations in the sarcolemmal environment are attenuated by lifelong caloric restriction and voluntary exercise

Age-related loss of skeletal muscle mass and function, referred to as sarcopenia, is mitigated by lifelong calorie restriction as well as exercise. In aged skeletal muscle fibers there is compromised integrity of the cell membrane that may contribute to sarcopenia. The purpose of this study was to determine if lifelong mild (8%) caloric restriction (CR) and lifelong CR+voluntary wheel running (WR) could ameliorate disruption of membrane scaffolding and signaling proteins during the aging process, thus maintaining a favorable, healthy membrane environment in plantaris muscle fibers. Fischer-344 rats were divided into four groups: 24-month old adults fed ad libitum (OAL); 24-month old on 8% caloric restriction (OCR); 24month old 8% caloric restriction+wheel running (OCRWR); and 6-month old sedentary adults fed ad libitum (YAL) were used to determine age-related changes. Aging resulted in discontinuous membrane expression of dystrophin glycoprotein complex (DGC) proteins: dystrophin and α-syntrophin. Older muscle also displayed decreased content of neuronal nitric oxide synthase (nNOS), a key DGC signaling protein. In contrast, OCR and OCRWR provided significant protection against age-related DGC disruption. In conjunction with the age-related decline in membrane DGC patency, key membrane repair proteins (MG53, dysferlin, annexin A6, and annexin A2) were significantly increased in the OAL plantaris. However, lifelong CR and CRWR interventions were effective at maintaining membrane repair proteins near YAL levels of. OAL fibers also displayed reduced protein content of NADPH oxidase isoform 2 (Nox2) subunits (p67phox and p47phox), consistent with a perturbed sarcolemmal environment. Loss of Nox2 subunits was prevented by lifelong CR and CRWR. Our results are therefore consistent with the hypothesis that lifelong CR and WR are effective countermeasures against age-related alterations in the myofiber membrane environment.

NOX2 Inhibition Impairs Early Muscle Gene Expression Induced by a Single Exercise Bout.

Reactive oxygen species (ROS) participate as signaling molecules in response to exercise in skeletal muscle. However, the source of ROS and the molecular mechanisms involved in these phenomena are still not completely understood. The aim of this work was to study the role of skeletal muscle NADPH oxidase isoform 2 (NOX2) in the molecular response to physical exercise in skeletal muscle. BALB/c mice, pre-treated with a NOX2 inhibitor, apocynin, (3 mg/kg) or vehicle for 3 days, were swim-exercised for 60 min. Phospho–p47phox levels were significantly upregulated by exercise in flexor digitorum brevis (FDB). Moreover, exercise significantly increased NOX2 complex assembly (p47phox–gp91phox interaction) demonstrated by both proximity ligation assay and co-immunoprecipitation. Exercise-induced NOX2 activation was completely inhibited by apocynin treatment. As expected, exercise increased the mRNA levels of manganese superoxide dismutase (MnSOD), glutathione peroxidase (GPx), citrate synthase (CS), mitochondrial transcription factor A (tfam) and interleukin-6 (IL-I6) in FDB muscles. Moreover, the apocynin treatment was associated to a reduced activation of p38 MAP kinase, ERK 1/2, and NF-κB signaling pathways after a single bout of exercise. Additionally, the increase in plasma IL-6 elicited by exercise was decreased in apocynin-treated mice compared with the exercised vehicle-group (p < 0.001). These results were corroborated using gp91-dstat in an in vitro exercise model. In conclusion, NOX2 inhibition by both apocynin and gp91dstat, alters the intracellular signaling to exercise and electrical stimuli in skeletal muscle, suggesting that NOX2 plays a critical role in molecular response to an acute exercise.

Reactive oxygen species derived from NADPH oxidase 1 and mitochondria mediate angiotensin II-induced smooth muscle cell senescence

Cellular senescence has emerged as an important player in both physiology and pathology. Excessive reactive oxygen species (ROS) is known to mediate cellular senescence. NADPH oxidases are major sources for ROS production in the vascular wall; the roles of different NADPH oxidase isoforms in cellular senescence remain unclear, however. We investigated the roles of two NADPH oxidase isoforms in mitochondrial dysfunction during angiotensin II (Ang II)-induced cellular senescence of human aortic vascular smooth muscle cells (VSMCs). Ang II (10−7M) stimulated ROS generation, exhibiting early increases between 30 and 60min and sustained increases between 24h and 72h, and induced VSMCs senescence after 48h or 72h treatment as assessed with senescence-associated β-galactosidase activity and the expression of two cell cycle inhibitors, p21 and p16. ROS scavengers and membrane-permeable catalase (catalase-PEG) reduced Ang II-stimulated cellular senescence. Furthermore, small interfering RNA (siRNA) of NADPH oxidase catalytic subunit Nox1, but not that of another isoform Nox4, inhibited Ang II-induced cellular senescence. Nox1 siRNA inhibited both early and sustained ROS increases induced by Ang II. In addition, a mitochondrial-specific antioxidant, mitoQ10, effectively inhibited Ang II-induced ROS increases and cellular senescence. Ang II decreased ATP synthesis and induced mitochondrial membrane depolarization, which were attenuated by pre-treating cells with Nox1 siRNA, mitoQ10 or catalase-PEG. The effect of Ang II on the mitochondrial regulator peroxisome-proliferator-activated receptor gamma coactivator-1α (PGC-1α) and its downstream genes was examined. Ang II stimulated S570 phosphorylation of PGC-1α with concomitant decreases in catalase and uncoupling protein-2 (UCP-2) levels between 12h and 72h, which were inhibited by Nox1 siRNA. Knockdown of both catalase and UCP-2 mimicked Ang II-induced VSMC senescence. These results suggested that Ang II-stimulated Nox1 activation mediates mitochondrial dysfunction, probably by decreasing PGC-1α activity and increasing mitochondrial oxidative stress, and leads to cellular senescence of VSMCs.

Podocyte injury: the role of proteinuria, urinary plasminogen, and oxidative stress.

Podocytes are the key target for injury in proteinuric glomerular diseases that result in podocyte loss, progressive focal segmental glomerular sclerosis (FSGS), and renal failure. Current evidence suggests that the initiation of podocyte injury and associated proteinuria can be separated from factors that drive and maintain these pathogenic processes leading to FSGS. In nephrotic urine aberrant glomerular filtration of plasminogen (Plg) is activated to the biologically active serine protease plasmin by urokinase-type plasminogen activator (uPA). In vivo inhibition of uPA mitigates Plg activation and development of FSGS in several proteinuric models of renal disease including 5/6 nephrectomy. Here, we show that Plg is markedly increased in the urine in two murine models of proteinuric kidney disease associated with podocyte injury: Tg26 HIV-associated nephropathy and the Cd2ap-/- model of FSGS. We show that human podocytes express uPA and three Plg receptors: uPAR, tPA, and Plg-RKT. We demonstrate that Plg treatment of podocytes specifically upregulates NADPH oxidase isoforms NOX2/NOX4 and increases production of mitochondrial-dependent superoxide anion (O2-) that promotes endothelin-1 synthesis. Plg via O2- also promotes expression of the B scavenger receptor CD36 and subsequent increased intracellular cholesterol uptake resulting in podocyte apoptosis. Taken together, our findings suggest that following disruption of the glomerular filtration barrier at the onset of proteinuric disease, podocytes are exposed to Plg resulting in further injury mediated by oxidative stress. We suggest that chronic exposure to Plg could serve as a "second hit" in glomerular disease and that Plg is potentially an attractive target for therapeutic intervention.

Effects of dark chocolate on NOX-2-generated oxidative stress in patients with non-alcoholic steatohepatitis.

Activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is considered a pathogenetic mechanism determining fibrosis and disease progression in non-alcoholic steatohepatitis (NASH). Polyphenols exert antioxidant action and inhibit NADPH oxidase in humans. To analyse the effect of cocoa polyphenols on NADPH oxidase isoform 2 (NOX2) activation, oxidative stress and hepatocyte apoptosis in a population affected by NASH. In a cross-sectional study comparing 19 NASH and 19 controls, oxidative stress, as assessed by serum NOX2 activity and F2-isoprostanes, and hepatocyte apoptosis, as assessed by serum cytokeratin-18 (CK-18) levels, were measured. Furthermore, the 19 NASH patients were randomly allocated in a crossover design to 40 g/day of dark chocolate (>85% cocoa) or 40 g/day of milk chocolate (<35% cocoa), for 2 weeks. sNOX2-dp, serum isoprostanes and CK-18 were assessed at baseline and after 2 weeks of chocolate intake. Compared to controls, NASH patients had higher sNOX2-dp, serum isoprostanes and CK-18 levels. A significant difference for treatments was found in subjects with respect to sNOX2-dp, serum isoprostanes and serum CK-18. The pairwise comparisons showed that, compared to baseline, after 14 days of dark chocolate intake, a significant reduction in sNOX2-dp serum isoprostanes and CK-18 M30 was found. No change was observed after milk chocolate ingestion. A simple linear regression analysis showed that ∆ of sNOX2-dp was associated with ∆ of serum isoprostanes. Cocoa polyphenols exert an antioxidant activity via NOX2 down-regulation in NASH patients.

Epidermal growth factor-induced hydrogen peroxide production is mediated by dual oxidase 1

Stimulation of mammalian cells by epidermal growth factor (EGF) elicits complex signaling events, including an increase in hydrogen peroxide (H2O2) production. Understanding the significance of this response is limited by the fact that the source of EGF-induced H2O2 production is unknown. Here we show that EGF-induced H2O2 production in epidermal cell lines is dependent on the agonist-induced calcium signal. We analyzed the expression of NADPH oxidase isoforms and found both A431 and HaCaT cells to express the calcium-sensitive NADPH oxidase, Dual oxidase 1 (Duox1) and its protein partner Duox activator 1 (DuoxA1). Inhibition of Duox1 expression by small interfering RNAs eliminated EGF-induced H2O2 production in both cell lines. We also demonstrate that H2O2 production by Duox1 leads to the oxidation of thioredoxin-1 and the cytosolic peroxiredoxins. Our observations provide evidence for a new signaling paradigm in which changes of intracellular calcium concentration are transformed into redox signals through the calcium-dependent activation of Duox1.

Evaluation of NADPH oxidases as drug targets in a mouse model of familial amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by progressive loss of motor neurons, gliosis, neuroinflammation and oxidative stress. The aim of this study was to evaluate the involvement of NADPH oxidases (NOX) in the oxidative damage and progression of ALS neuropathology. We examined the pattern of NOX expression in spinal cords of patients and mouse models of ALS and analyzed the impact of genetic deletion of the NOX1 and 2 isoforms as well as pharmacological NOX inhibition in the SOD1G93A ALS mouse model.A substantial (10–60 times) increase of NOX2 expression was detected in three etiologically different ALS mouse models while up-regulation of some other NOX isoforms was model-specific. In human spinal cord samples, high NOX2 expression was detected in microglia. In contrast to previous publications, survival of SOD1G93A mice was not modified upon breeding with constitutive NOX1 and NOX2 deficient mice. As genetic deficiency of a single NOX isoform is not necessarily predictive of a pharmacological intervention, we treated SOD1G93A mice with broad-spectrum NOX inhibitors perphenazine and thioridazine. Both compounds reached in vivo CNS concentrations compatible with NOX inhibition and thioridazine significantly decreased superoxide levels in the spinal cord of SOD1G93A mice in vivo. Yet, neither perphenazine nor thioridazine prolonged survival. Thioridazine, but not perphenazine, dampened the increase of microglia markers in SOD1G93A mice. Thioridazine induced an immediate and temporary enhancement of motor performance (rotarod) but its precise mode of action needs further investigation. Additional studies using specific NOX inhibitors will provide further evidence on the relevance of NOX as drug targets for ALS and other neurodegenerative disorders.

Modulation of NADPH-oxidase gene expression in rolB-transformed calli of Arabidopsis thaliana and Rubia cordifolia

Expression of rol genes from Agrobacterium rhizogenes induces reprogramming of transformed plant cells and provokes pleiotropic effects on primary and secondary metabolism. We have previously established that the rolB and rolC genes impair reactive oxygen species (ROS) generation in transformed cells of Rubia cordifolia and Arabidopsis thaliana. In the present investigation, we tested whether this effect is associated with changes in the expression levels of NADPH oxidases, which are considered to be the primary source of ROS during plant–microbe interactions. We identified two full-length NADPH oxidase genes from R. cordifolia and examined their expression in non-transformed and rolB-transformed calli. In addition, we examined the expression of their homologous genes from A. thaliana in non-transformed and rolB-expressing cells. The expression of Rboh isoforms was 3- to 7-fold higher in both R. cordifolia and A. thaliana rolB-transformed cells compared with non-transformed cells. Our results for the first time show that Agrobacterium rolB gene regulates particular NADPH oxidase isoforms.

Nobiletin attenuates cardiac dysfunction, oxidative stress, and inflammatory in streptozotocin: induced diabetic cardiomyopathy.

Diabetic cardiomyopathy, characterized by the presence of diastolic and/or systolic myocardial dysfunction, is one of the major causes of heart failure. Nobiletin, which is extracted from the fruit peel of citrus, is reported to possess anti-inflammatory, anti-oxidative, and hypolipidemic properties. The purpose of this study was to investigate whether nobiletin exerts the therapeutic effect on streptozotocin-induced diabetic cardiomyopathy (DCM) in mice. 80 experimental male C57BL mice were randomly assigned into four groups: sham+vehicle (VEH/SH), sham+nobiletin (NOB/SH), DCM+vehicle (VEH/DM), and DCM+nobiletin (NOB/DM). Nobiletin treatment ameliorated cardiac dysfunction in the DCM group, as shown by the result of echocardiography and hemodynamic measurements. Nobiletin treatment also blunted the mRNA expression of NADPH oxidase isoforms p67(phox), p22(phox), and p91(phox), and abated oxidative stress. Although administration of diabetic mice with nobiletin did not significantly effect the level of blood glucose, it decreased the TGF-β1, CTGF, fibronectin, and collagen Iα expressions and blunted cardiac fibrosis. In addition, nobiletin inhibited the activation of c-Jun NH2-terminal kinase (JNK), P38, and NF-κB in the cardiac tissue of diabetic mice. Collectively, our study indicates that treatment with nobiletin mitigates cardiac dysfunction and interstitial fibrosis, and these beneficial of nobiletin may belong to the suppression of JNK, P38, and NF-κB signaling pathways.

NOX1/2 activation in human gingival fibroblasts by Fusobacterium nucleatum facilitates attachment of Porphyromonas gingivalis.

Periodontal diseases are infectious polymicrobial inflammatory diseases that lead to destruction of the periodontal ligament, gingiva, and alveolar bone. Sequential colonization of a broad range of bacteria, including Fusobacterium nucleatum and Porphyromonas gingivalis, is an important phenomenon in this disease model. F. nucleatum is a facultative anaerobic species thought to be a key mediator of dental plaque maturation due to its extensive coaggregation with other oral bacteria, while P. gingivalis is an obligate anaerobic species that induces gingival inflammation by secreting various virulence factors. The formation of a bacterial complex by these two species is central to the pathogenesis of periodontal disease. Reactive oxygen species (ROS) are produced during bacterial infections and are involved in intracellular signaling. However, the impact of oral bacteria-induced ROS on the ecology of F. nucleatum and P. gingivalis has yet to be clarified. In the present study, we investigated ROS production induced in primary human oral cells by F. nucleatum and P. gingivalis and its effect on the formation of their bacterial complexes and further host cell apoptosis. We found that in primary human gingival fibroblasts (GFs), two NADPH oxidase isoforms, NOX1 and NOX2, were activated in response to F. nucleatum infection but not P. gingivalis infection. Accordingly, increased NADPH oxidase activity and production of superoxide anion were observed in GFs after F. nucleatum infection, but not after P. gingivalis infection. Interestingly, in NOX1, NOX2, or NOX1/NOX2 knockdown cells, the number of P. gingivalis decreased when the cells were coinfected with F. nucleatum. A similar pattern of host cell apoptosis was observed. This implies that F. nucleatum contributes to attachment of P. gingivalis by triggering activation of NADPH oxidase in host cells, which may provide an environment more favorable to strict anaerobic bacteria and have a subsequent effect on apoptosis of host cells.

CHRONIC ADMINISTRATION OF ALBUMIN MODIFIED BY ADVANCED GLYCATION (AGE) INDUCES EXPRESSION OF PRO‐FIBROTIC, PRO‐APOPTOTIC AND RENIN‐ANGIOTENSIN SYSTEM GENES ON RENAL TISSUE

IntroductionDiabetes mellitus (DM) is the major cause of chronic kidney disease in the world. Hyperglycemia participates in the development of diabetic nephropathy (DN), among other pathways, through the increased production of advanced glycation end products (AGEs). AGEs not excreted by the kidneys are distributed into the tissues, remain biologically active and can cause local inflammation, oxidative stress and stimulate other systems, such as the renin‐angiotensin system (RAS). The main hypothesis of this study is that AGEs contribute to DN regardless of hyperglycemia, by changing the expression of pro‐fibrotic, pro‐inflammatory, and anti‐oxidant genes and stimulating the RAS. The second hypothesis is that treatment with the antioxidant N‐acetylcysteine (NAC) could prevent the effects of AGEs.MethodsMale Wistar rats were divided into four groups (n = 9–11 per group) and treated for 90 days: Control albumin (Control Alb, 20 mg/kg/day, i.p.), NAC (600 mg/L of water,ad libitum), Glycated albumin (AGE Alb, 20 mg/kg/day, i.p.), AGE Alb plus NAC. At the end of the treatment, the animals were allocated in metabolic cages for 24 hours for urine sample collection. Urinary variables were evaluated. One kidney of each animal was submitted to mRNA expression of the following genes by RT‐qPCR: transforming growth factor β (Tgfb1), tumor necrosis factor α (Tnf), collagen type III (Col3a1) and type IV (Col4a1), connective tissue growth factor (Ctgf), angiotensinogen (Agt), renin (Ren), angiotensin converting enzyme 1 (Ace), NADPH oxidase isoform 4 (Nox4), thiorredoxin interacting protein (Txnip) and apoptosis regulators Bax and Bcl2 and β‐actin (Actb; reference gene). The other kidney was perfused and submitted to morphological analyses.ResultsRenal variables analyses showed no differences in the body weight/kidney weight ratio, urine flow rate and Na+ and K+ excretion. There was a decrease in the protein/creatinin ratio in animals treated by AGE Alb plus NAC (vs AGE Alb; p=0.0047). There was an increase in the expression of pro‐fibrotic Tgfb1 (AGE Alb vs Control Alb, p= 0.0105) and Col4a1 (AGE Alb vs Control Alb; p= 0.0070) genes. Col4a1 upregulation was corrected by NAC (AGE plus NAC vs AGE Alb; p= 0.0152). Regarding RAS genes, there was an increase in the expression of Agt (AGE Alb vs Control Alb; p= 0.0335) and Ren, (AGE Alb vs Control Alb; p= 0.0122) genes, without changes in Ace mRNA expression. Ren upregulation was corrected by NAC treatment (AGE plus NAC vs AGE Alb; p= 0.0343). Nox4 expression was not changed among groups, however AGE‐treated rats exhibited an increase in Txnip expression (AGE Alb vs Control Alb; p= 0.0120). Bax/Bcl2 expression ratio also increased in AGE Alb‐treated rats (AGE Alb vs Control Alb, p= 0.0356) and was corrected by NAC treatment (AGE plus NAC vs AGE Alb; p= 0.0377). AGE Alb‐treated rats showed an increase in total glomerular area, which was not corrected by NAC treatment (AGE Alb vs Control Alb; p= 0.0436).ConclusionsChronic administration of albumin modified by glycation activates the expression of pro‐fibrotic, pro‐apoptotic and RAS genes and induces glomerular hypertrophy in normoglycemic animals. NAC administration only partially reverted these changes. These results suggest that AGEs exert renal deleterious effects per se.Support or Funding InformationFapesp (2014/17251‐9; 2012/04831‐1).

Effects of NOX‐1 on Real‐Time Blood Nitric Oxide and Hydrogen Peroxide in Acute Hyperglycemia

Hyperglycemia has been associated with vascular endothelial dysfunction in part by a reduction in nitric oxide (NO) production and increased oxidative stress (e.g., increased superoxide (SO) and hydrogen peroxide (H2O2). Endothelial‐derived NO can be significantly reduced by increased SO/H2O2 in part by the activation of NADPH oxidase during hyperglycemia. Of the 7 NADPH oxidase isoforms, NOX1 is mainly expressed in the vasculature and may play a major role in hyperglycemia induced oxidative stress and vascular endothelial dysfunction. To test this hypothesis, we measured blood NO and H2O2 levels in real time via NO and H2O2 microsensors inserted into femoral veins of rats. Hyperglycemia (e.g., 200 mg/dl) was maintained by infusion i.v. of 30% glucose solution for 3 hours with or without a selective NOX1 inhibitor, ML171. We found that hyperglycemia for 3 hours significantly increased blood H2O2 levels by 1.88±0.4 μM (n=6) compared to the saline infused control (P&lt;0.05, n=2). By contrast, ML171 (1 and 5 μM) reduced hyperglycemia‐induced H2O2 levels by 1.36±0.61 μM (n=8) and 4.35±1.02 μM (n=5), respectively, at the end of experiment. Meanwhile, hyperglycemia significantly reduced blood NO levels by 82.48±38.12 nM (n=3) compared to the saline control (P&lt;0.05, n=2). By contrast, ML171 (1 μM) attenuated the hyperglycemia induced decrease in blood NO levels and increased blood NO levels by 71.15±24.00 nM (n=5) at the end of experiment. Our preliminary results indicate that NOX1 activation may contribute to hyperglycemia‐induced oxidative stress and NO reduction. Furthermore, inhibition of NOX1 may mitigate the deleterious effects of hyperglycemia.Support or Funding InformationThis study was supported by Division of Research, the Center for Chronic Disorders of Aging, and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

TRPML1‐Mediated Ca2+ Signaling in Cerebral Artery Smooth Muscle

Late endosomes and lysosomes (collectively referred to as endolysosomes) are small acidic organelles present in all cells that maintain a steep Ca2+ gradient compared with the cytosol. Therefore, activation of Ca2+ permeable ion channels within the endolysosomal membrane can transiently generate localized microdomains of elevated Ca2+ within the cytosol. The goal of the current study is to elucidate the importance of Ca2+ signals arising from acidic Ca2+ stores in cerebral artery smooth muscle cells (SMC). Members of the mucolipin transient receptor potential (TRPML) subfamily of cation channels are Ca2+ permeable and selectively localize to endolysosomes. Mutations in the human TRPML1 gene (MCOLN1) result in the heritable childhood neurodegenerative disease type IV mucolipidosis (ML‐IV). Quantitative RT‐PCR analysis showed the presence of TRPML1, TRPML2, and TRPML3 in mRNA extracted from cerebral pial arteries from C57Bl/6 mice, with TRPML1 being most abundant. Furthermore, freshly isolated cerebral pial artery SMC showed positive immunofluorescence labeling for TRPML1, which co‐localized with the endolysosomal marker LAMP‐1. Immunolabeling experiments also suggest colocalization of TRPML1 and NADPH oxidase isoform 2 (NOX2) in endolysosomes in pial artery SMC. Ca2+ signals arising from TRPML1‐mediated endolysosomal release were investigated using a TRPML1‐GCaMP3 fusion protein. This genetic construct is targeted to endolysosomes and selectively detects Ca2+ released into the cytosol from these organelles. In HEK293 cells expressing TRPML1‐GCaMP3, an increase in cytosolic fluorescence was detected in response to the selective TRPML1 activator ML‐SA1 (10 μM), signifying that Ca2+ release from acidic stores through TRPML1 can be detected using these methods. We also investigated the functional significance of TRPML1‐mediated intracellular Ca2+ signals. In cerebral SMC, large conductance Ca2+ activated K+ (BK) channels are activated by spatially and temporally limited release of Ca2+ from the sarcoplasmic reticulum via ryanodine receptors (RyR), which generate microdomains of high cytosolic [Ca2+] known as Ca2+ sparks. This pathway promotes smooth muscle cell hyperpolarization and vasodilation. The activity of RyR is elevated by localized increases in cytosolic [Ca2+] to cause Ca2+‐induced Ca2+ release. Therefore, we hypothesized that Ca2+ release from endolysosomes sensitizes RyR to increase Ca2+ spark and BK channel activity. Ca2+ sparks were recorded from cerebral SMC loaded with the fast Ca2+ indicator fluo‐4AM using high‐speed, high‐resolution confocal microscopy. We found that Ca2+ spark sites co‐localized with endolysosomes labeled with LysoTracker Red DND‐99. Furthermore, preliminary experiments show that Ca2+ spark frequency and BK channel activity are increased by ML‐SA1 (3 μM). These data suggest a novel pathway for cerebral blood flow regulation in which local Ca2+ release from endolysosomes through TRPML1 channels sensitizes RyR, stimulating Ca2+ spark and BK channel activity to promote vasodilation. It is possible that disrupted cerebral blood flow control resulting from loss of TRPML1 activity in SMC contributes to neurodegeneration associated with ML‐IV.Support or Funding InformationSupport: R01HL091905 (SE).

The Role of NADPH Oxidase Isoform 1 (NOX1) in L‐NAME‐Induced Leukocyte‐Endothelial Interactions in Rat Mesenteric Postcapillary Venules

Vascular endothelial dysfunction and subsequent inflammation underlie many pathological diseases such as cardiovascular disease, diabetes, and hypertension. Both events are principally initiated by reduced endothelial‐derived nitric oxide (NO) bioavailability and/or activation of vascular/leukocyte NADPH oxidase. NADPH oxidase has 7 isoforms (NOX1‐5 and Duox1‐2). We currently have limited knowledge regarding the roles of individual NOX isoforms in diseases, particularly NOX1, which is a major NADPH oxidase isoform in the vasculature but not in leukocytes. In this study, we evaluated the roles of NOX1 in vascular endothelial dysfunction‐induced inflammation by recording leukocyte rolling, adherence, and transmigration in a postcapillary venule of rat mesenteric tissue in real time via intravital microscopy. We superfused the mesenteric tissue with Krebs’ buffer, and Krebs’ buffer with a non‐selective NO synthase inhibitor, NG‐nitro‐L‐arginine‐methyl‐ester (L‐NAME, 50 μM) with/without a selective NOX1 inhibitor, ML171. We found that at 2 hrs of superfusion, leukocyte rolling (21±8 cells/min), adherence (3±2 cells/100 μm), and transmigration (2±1 cells/200 μm2) was minimal in Krebs’ control (n=8). By contrast, L‐NAME (n=6) significantly increased leukocyte rolling (74±12), adhesion (19±6), and transmigration (18±4) (all P&lt;0.05 compared to Krebs’ control). Whereas, ML171 (1 μM, n=7) prevented the L‐NAME‐induced effect on leukocyte rolling (25±5), adhesion (5±2), and transmigration (5±1). These findings suggest that NOX‐1 activation participates in L‐NAME induced inflammatory responses; whereas inhibition of NOX1 can mitigate inflammation following vascular endothelial dysfunction.Support or Funding InformationThis study was supported by Division of Research, the Center for Chronic Disorders of Aging, and Department of Bio‐Medical Sciences at Philadelphia College of Osteopathic Medicine.

Age‐Related Perturbations in the Sarcolemmal Environment are Attenuated by Lifelong Caloric Restriction and Voluntary Wheel Running

Sarcopenia is the age‐related reduction in skeletal muscle mass and strength. Previously we observed that lifelong mild caloric restriction (CR) and wheel running (WR) attenuated sarcopenia, in part by reducing the age‐induced disruptions of the sarcolemmal dystrophin‐glycoprotein complex (DGC) proteins. The purpose of this study was to determine if mild (8%) CR and lifelong CR + voluntary WR could maintain a phenotypically young sarcolemmal environment in plantaris muscle fibers. Fischer 344 rats were assigned into four groups: 6‐month‐old sedentary ad libitum (YAL), 24‐month‐old sedentary ad libitum group (OAL), 8% caloric restriction from 11 months – 24 months (OCR), and 8% CR plus voluntary wheel running from 11 months – 24 months (OCRWR). We observed significant age‐related increases in key membrane repair proteins (MG53, p &lt; 0.0001; dysferlin, p &lt; 0.0001; annexin A6, p &lt; 0.0001; and annexin A2, p = 0.004) in the OAL plantaris. Lifelong mild CR and CRWR interventions were effective at maintaining membrane repair proteins near YAL levels. Consistent with a perturbed sarcolemmal environment, we also found reduced protein content of NADPH oxidase isoform 2 (Nox2) subunits (p67phox, p = 0.03; p47phox, p = 0.01; and gp91phox, p = 0.096). Lifelong CR and CRWR protected against the age‐related reduction in gp91phox, p47phox, and p67phox proteins. Nox2 subunit abundance in OCR and OCRWR were not significantly different than YAL, while p67phox was significantly higher in OCR than YAL (p = 0.03). Novel data presented here are consistent with the hypothesis that lifelong CR and WR mitigate sarcopenia, in part, by ameliorating age‐induced alterations in the myofiber membrane environment.Support or Funding InformationNIH (AR054084), NASA (NNX12AR62G), the Sydney and J.L. Huffines Institute, and the College of Health and Education at Texas A&amp;M University

Diabetes and Kidney Disease: Role of Oxidative Stress.

Intrarenal oxidative stress plays a critical role in the initiation and progression of diabetic kidney disease (DKD). Enhanced oxidative stress results from overproduction of reactive oxygen species (ROS) in the context of concomitant, insufficient antioxidant pathways. Renal ROS production in diabetes is predominantly mediated by various NADPH oxidases (NOXs), but a defective antioxidant system as well as mitochondrial dysfunction may also contribute. Recent Advances: Effective agents targeting the source of ROS generation hold the promise to rescue the kidney from oxidative damage and prevent subsequent progression of DKD. Critical Issues and Future Directions: In the present review, we summarize and critically analyze molecular and cellular mechanisms that have been demonstrated to be involved in NOX-induced renal injury in diabetes, with particular focus on the role of increased glomerular injury, the development of albuminuria, and tubulointerstitial fibrosis, as well as mitochondrial dysfunction. Furthermore, novel agents targeting NOX isoforms are discussed. Antioxid. Redox Signal. 25, 657-684.