Albuminuria, expression of nicotinamide adenine dinucleotide phosphate oxidase and monocyte chemoattractant protein-1 in the renal tubules of hypertensive Dahl salt-sensitive rats.

In chronic renal diseases, experimental and human data suggest that excess albumin filtered through the glomerular capillary barrier is over-reabsorbed by proximal tubular cells, thereby activating these cells and upregulating the expression of chemokines. On the other hand, a high-salt diet has been shown to induce proteinuria in hypertensive Dahl salt-sensitive (DSS) rats, accompanied with the expression of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in the kidney. In the current study, we therefore examined albuminuria and the expressions of NADPH oxidase and monocyte chemoattractant protein-1 (MCP-1) in the renal tubular cells in hypertensive DSS rats, as well as the effects of the antioxidant N-acetylcysteine (NAC) on each of these parameters. DSS rats were fed a normal-salt diet (0.24% NaCl), a high-salt diet (8% NaCl), or a high-salt diet plus NAC supplementation (15 mg/mL drinking water) for 4 weeks. The high-salt diet provoked an increase in glomerular injuries accompanied with albuminuria and in urinary H2O2 and MCP-1 excretion. Immunohistochemical analysis showed the prominent expression of MCP-1 in the dilated tubular cells, where the NADPH oxidase subunit p47phox was also expressed. The current results suggest that albuminuria caused expression of NADPH oxidase and MCP-1 in the dilated renal tubules, resulting in interstitial inflammation and migration of mononuclear cells in DSS rats, because blockade of albuminuria by NAC counteracted the p47phox and MCP-1 expression.     

Advanced oxidation protein products promote inflammation in diabetic kidney through activation of renal nicotinamide adenine dinucleotide phosphate oxidase

The involvement of inflammatory processes has been recognized in development and/or progression of diabetic nephropathy. However, the mechanisms involved in the pathogenesis of renal inflammation have not been completely understood. In this study, we tested the hypothesis that accumulation of advanced oxidation protein products (AOPPs), which occurs in diabetes, may promote inflammatory responses in diabetic kidney. Streptozotocin-induced diabetic rats were randomized to iv injection of vehicle, native rat serum albumin (RSA), and AOPPs-modified RSA (AOPPs-RSA) in the presence or absence of oral administration of apocynin. A control group was followed concurrently. Compared with RSA- or vehicle-treated diabetic rats, AOPPs-RSA-treated animals displayed significant increase in renal macrophage infiltration and overexpression of monocyte chemoattractant protein-1 and TGF-beta1. This was associated with deteriorated structural and functional abnormalities of diabetic kidney, such as glomerular hypertrophy, fibronectin accumulation, and albuminuria. AOPP challenge significantly increased nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent superoxide generation in renal homogenates and up-regulated membrane expression of renal NADPH oxidase subunits p47(phox) and gp91(phox). All these AOPPs-induced perturbations in diabetic kidney could be prevented by the NADPH oxidase inhibitor apocynin. These data suggest that chronic accumulation of AOPPs may promote renal inflammation in diabetes probably through activation of renal NADPH oxidase.     

Acute suppressive effect of hydrocortisone on p47 subunit of nicotinamide adenine dinucleotide phosphate oxidase

We have recently demonstrated that reactive oxygen species (ROS) generation by polymorphonuclear leukocytes (PMNL) and mononuclear cells (MNC) is inhibited following the intravenous administration of hydrocortisone. This is associated with a parallel decrease in intranuclear NFkappaB, known to modulate inflammatory responses including ROS generation. We have also shown that the plasma levels of interleukin-10 (IL-10), an anti-inflammatory and immunosuppressive cytokine produced by TH2 cells, are also increased after hydrocortisone administration. In this study, we have investigated the effect of hydrocortisone on p47(phox) subunit, a key component of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, in MNC and the pharmacodynamics of this effect with ROS generation and plasma IL-10 levels. p47(phox) subunit protein levels in MNC showed a progressive decrease after hydrocortisone administration. It reached a nadir at 4 hours and increased thereafter to a baseline level at 24 hours. ROS generation also decreased, reached a nadir between 2 and 4 hours, and returned to a baseline level at 24 hours. IL-10 concentrations increased, peaked at 4 hours, and reverted to the baseline levels at 24 hours. In conclusion, p47(phox) subunit suppression may contribute to the inhibition of ROS generation in MNC after hydrocortisone administration. This suppression occurs in parallel with the suppression of NFkappaB and an increase in IL-10 plasma levels. Therefore, it would appear that the decrease in intranuclear NFkappaB and an increase in IL-10 may cause the inhibitory modulation on p47(phox) subunit and ROS generation by MNC following hydrocortisone and other glucocorticoids.     

Composition and functions of vascular nicotinamide adenine dinucleotide phosphate oxidases

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases are important sources of reactive oxygen species (ROS) and are expressed in at least three different homologues in the vasculature. The enzymes consist of a membrane complex of one of the large catalytically active Nox proteins and p22phox and different cytosolic subunits. Reactive oxygen species formation by the nicotinamide adenine dinucleotide phosphate oxidases Nox1 and Nox2 in arteries is a consequence of an activation of the enzymes by different stimuli such as growth factors, cytokines, and cardiovascular risk factors (cigarette smoke, high blood pressure, oxidized lipids). Nox4, in contrast, is constitutively active, and therefore, ROS formation by this enzyme is controlled on the expression level of the protein. The negative vascular effects of ROS, such as endothelial dysfunction, vascular hypertrophy, aneurysm formation, and inflammatory activation, appear to be the consequence of an activation of Nox1 and Nox2. Nox4, in contrast, potentially elicits positive effects because it promotes differentiation and reduces proliferation of cells. Consequently, selective pharmacologic inhibition of Nox proteins has a potential to interfere with cardiovascular disease initiation and progression.     

Tea polyphenols regulate nicotinamide adenine dinucleotide phosphate oxidase subunit expression and ameliorate angiotensin II-induced hyperpermeability in endothelial cells.

Out-of-control reactive oxygen species (ROS) signaling is one of the key events in the pathogenesis of endothelial dysfunction and essential hypertension. We observed that tea polyphenols decreased the production of ROS via regulation of the protein expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in bovine carotid artery endothelial cells (BCAECs). Both green tea polyphenols (GTP) and black tea polyphenols (BTP) down-regulated the expression of NADPH oxidase subunits p22phox and p67phox while up-regulating catalase expression (p < 0.05, respectively). Pre-treatment with GTP or BTP for 24 h significantly decreased the superoxide anion level (p < 0.05) and permeable fluorescence intensities in Ang II-stimulated BCAECs. A decrease in cell permeability was also observed by pre-treatment with diphenylene iodonium chloride (DPI) or vitamin E (p < 0.05, respectively). The result demonstrates that tea polyphenols alleviate angiotensin (Ang) II-induced hyperpermeability mainly by decreasing ROS production. Our results suggest that tea polyphenols regulate ROS-related protein expression and may be beneficial in preventing endothelial cell dysfunction and development of cardiovascular diseases, including hypertension.     

Induction of ischemic tolerance in rat liver via reduced nicotinamide adenine dinucleotide phosphate oxidase in Kupffer cells

AIM To elucidate the mechanisms of hepatocyte preconditioning by H2O2 to better understand the pathophysiology of ischemic preconditioning.METHODS The in vitro effect of H2O2 pretreatment was investigated in rat isolated hepatocytes subjected to anoxia/reoxygenation. Cell viability was assessed with propidium iodide fluorometry. In other experiments, rat livers were excised and subjected to warm ischemia/reperfusion in an isolated perfused liver system to determine leakage of liver enzymes. Preconditioning was performed by H2O2 perfusion, or by stopping the perfusion for 10 min followed by 10 min of reperfusion.To inhibit Kupffer cell function or reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase,gadolinium chloride was injected prior to liver excision, or diphenyleneiodonium, an inhibitor of NADPH oxidase, was added to the perfusate, respectively. Histological detection of o~gen radical formation in Kupffer cells was performed by perfusion with nitro blue tetrazolium.RESULTS Anoxia/reoxygenation decreased hepatocyte viability compared to the controls. Pretreatment with H2O2 did not improve such hepatocyte injury. In liver perfusion experiments, however, H2O2 preconditioning reduced warm ischemia/reperfusion injury, which was reversed by inhibition of Kupffer cell function or NADPH oxidase. Histological examination revealed that H2O2 preconditioning induced oxygen radical formation in Kupffer cells. NADPH oxidase inhibition also reversed hepatoprotection by ischemic preconditioning.CONCLUSION H2O2 preconditioning protects hepatocytes against warm ischemia/reperfusion injury via NADPH oxidase in Kupffer cells, and not directly. NADPH oxidase also mediates hepatoprotection by ischemic preconditioning.     

Association of atrial nicotinamide adenine dinucleotide phosphate oxidase activity with the development of atrial fibrillation after cardiac surgery.

OBJECTIVES: Our goal was to evaluate the role of myocardial nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and plasma markers of oxidative stress in the pathogenesis of post-operative atrial fibrillation (AF). BACKGROUND: Atrial fibrillation is a common complication of cardiac surgery, leading to increased morbidity and prolonged hospitalization. Experimental evidence suggests that oxidative stress may be involved in the pathogenesis of AF; however, the relevance of this putative mechanism in patients undergoing cardiac surgery is unclear. METHODS: We measured basal and NADPH-stimulated superoxide production in right atrial appendage samples from 170 consecutive patients undergoing conventional coronary artery bypass surgery. Plasma markers of lipid and protein oxidation (thiorbabituric acid-reactive substances, 8-isoprostane, and protein carbonyls) were also measured in blood samples drawn from a central line before surgery and after reperfusion. RESULTS: Patients who developed AF after surgery (42%) were older and had a significantly increased atrial NADPH oxidase activity than patients who remained in sinus rhythm (SR) (in relative light units/s/mug protein: 4.78 +/- 1.44 vs. 3.53 +/- 1.04 in SR patients, p < 0.0001). Plasma markers of lipid and protein oxidation increased significantly after reperfusion; however, neither pre-operative nor post-operative measurements differed between patients who developed AF and those who remained in SR after surgery. Multivariate analysis identified atrial NADPH oxidase activity as the strongest independent predictor of post-operative AF (odds ratio 2.41; 95% confidence interval 1.71 to 3.40, p < 0.0001). CONCLUSIONS: Atrial NADPH oxidase activity is independently associated with an increased risk of post-operative AF, suggesting that this oxidase system may be a key mediator of atrial oxidative stress leading to the development of AF after cardiac surgery.     

Nox/Duox family of nicotinamide adenine dinucleotide (phosphate) oxidases.

Reactive oxygen species are classically described as occurring as an accidental byproduct of respiration, and are generally thought to be deleterious to biologic systems. The phagocyte nicotinamide adenine dinucleotide phosphate oxidase provides an example of deliberate reactive oxygen species generation, but the function of this enzyme is to oxidatively modify bacteria as part of bactericidal mechanisms. The discovery of a family of nicotinamide adenine dinucleotide (phosphate) oxidases related to the phagocyte oxidase, the Nox/Duox family, provides additional examples of deliberate generation of reactive oxygen species. This article describes this new family of enzymes and considers hypotheses for their function. Potential roles of Nox/Duox in generation of reactive oxygen species that function in cell signaling (related to growth and angiogenesis), immune function, hypoxic response, and oxidative modification of extracellular matrix proteins are discussed.     

尼克酰胺腺嘌呤二核苷酸磷酸氧化酶在血管性认知障碍中的作用及其机制的研究进展

氧化应激(OS)是导致血管性认知障碍(VCl)的因素之一.尼克酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶(NOX)产生的活性氧(ROS)是VCI重要的致病分子.VCI的危险因素如高血糖及缺血/再灌注等在上游增加NOX的活化;其他危险因素如高龄、高血压及卒中是NOX依赖ROS产生的下游损伤.VCI的成因包括与NOX相关的...     

还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶在脂多糖诱导的小鼠急性心肌损伤组织中的表达及意义

目的探讨还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的非吞噬细胞氧化酶(Nox)家族在脂多糖(LPS)诱导的小鼠急性心肌损伤组织中的表达及意义。方法选取8~10周龄无特定病原体C57/BL6雄性小鼠30只,随机分为对照组和LPS组,每组15只。LPS组小鼠通过腹腔注射大剂量LPS(10 mg/kg)诱导急性心肌损伤模型,对照组腹腔注射等量生理盐水,观察6 h后取材,逆转录-聚合酶链反应(RT-PCR)检测Nox、B细胞淋巴瘤/白血病蛋白-2(Bcl-2)、Bcl-2相关X蛋白(Bax)、半胱氨酸天门冬氨酸特异性蛋白酶3(Caspase 3)基因表达,Western blotting检测Nox 2、Nox 4、Bax、Bcl-2和Caspase 3蛋白表达,免疫组织化学法检测4羟基壬烯醛(4-HNE)表达,末端脱氧核糖核酸转移酶(Td T)介导的脱氧尿苷三磷酸(dUTP)切口末端标记技术(TUNEL)法检测心肌组织细胞凋亡,比较两组结果差异。使用SPSS 13.0统计软件对数据进行分析,两组比较采用t检验。结果相比对照组,LPS组小鼠心肌Nox 2、Nox 4、Bax基因和蛋白以及Caspase 3蛋白表达水平明显升高,Bcl-2基因和蛋白表达水平明显降低,差异具有统计学意义(P0.05)。免疫组织化学检测结果表明相比对照组,LPS组心肌组织4-HNE表达明显增加,差异具有统计学意义(P0.05)。TUNEL检测结果表明相比对照组,LPS组心肌组织心肌凋亡细胞明显增加,差异具有统计学意义(P0.05)。结论 NADPH氧化酶通过调控氧化应激和细胞凋亡参与急性心肌损伤的发生发展。     

Alterations of phosphoinositide-specific phospholipase C and protein kinase C in the myocardium of spontaneously hypertensive rats

Summary In order to determine whether phosphoinositide metabolism is altered in hypertensive cardiac hypertrophy, phospholipase C (PLC) and protein kinase C activities were measured in hearts from 4- and 20-week-old spontaneously hypertensive rats (SHR) and age-matched, normotensive Wister-Kyoto rats (WKY). PLC activities were assayed using phosphatidylinositol (PI) and phosphatidylinositol-4,5-bisphosphate (PIP₂) as substrates to assess the substrate specificity. PI-hydrolyzing PLC activity (PI-PLC) was predominantly located in the cytosol, and its activity was similar in both strains. Membrane-bound PIP₂-hydrolyzing PLC activity (PIP₂-PLC) was significantly lower in 20-week-old SHR than in WKY, but there was no significant difference in soluble PIP₂-PLC. Protein kinase C activity was significantly elevated in 20-week-old SHR and Ca²⁺-phospholipid-dependent phosphorylation was observed in the proteins of molecular weight 26, 32, 43, and 95 KDa. In 4-week-old prehypertensive SHR, there were no significant differences in PI-PLC, PIP₂-PLC, or protein kinase C activities as compared with age-matched WKY. These data demonstrated that protein kinase C and membrane-bound PIP₂-PLC are altered during the period of hypertension development. These alterations may have important roles in the development or maintenance of hypertensive cardiac hypertrophy in SHR.Until April 31, 1990 N. Makita, Department of Cardiovascular Medicine, Hokkaido University School of Medicine, Kita 15, Nishi 7, Kita-Ku Sapporo 060, Japan After May 1, 1990 N. Makita, Division of Nephrology, S-3223, Medical Center North, Vanderbilt University, Nashville. Tennessee 37232, USA     

胰高血糖素样肽1对1型糖尿病大鼠心肌烟酰胺腺嘌呤二核苷酸氧化酶亚单位表达的影响

目的 探讨胰高血糖素样肽1 （GLP-1）对1型糖尿病大鼠心肌组织烟酰胺腺嘌呤二核苷酸（NADPH）氧化酶亚单位p22phox和Nox4表达的影响.方法 将42只雄性SD大鼠用随机数字表法分为正常对照组（NC组,n=7）和糖尿病造模组（n=35）.采用链脲佐菌素（STZ）对糖尿病造模组制备1型糖尿病大鼠模型.将造模成功的29只1型糖尿病大鼠用随机数字表法分为糖尿病组（DM组,n=10）,糖尿病GLP-1低剂量治疗组（DL组,n=10）和糖尿病GLP-1高剂量治疗组（DH组,n=9）.DL组予以艾塞那肽1μg/kg,2次/d皮下注射,DH组予以艾塞那肽5μg/kg,2次/d皮下注射.艾塞那肽治疗8周后处死动物.用实时荧光定量聚合酶链反应测定4组大鼠心肌p22phox和Nox4 mRNA的表达,免疫组化法检测心肌铜-锌-超氧化物歧化酶（Cu-Zn-SOD）蛋白表达.多组比较采用单因素方差分析.结果 与NC组比较,DM组大鼠心肌p22phox和Nox4 mRNA表达显著升高（t=5.77、5.36,均P＜0.05）,心肌Cu-Zn-SOD蛋白表达显著升高（t=59.91,P＜0.05）.艾塞那肽治疗8周后,与DM组比较,DL组和DH组大鼠心肌p22phox和Nox4 mRNA表达均显著降低（t=16.86、7.66和16.11、7.59,均P＜0.05）,心肌Cu-Zn-SOD蛋白表达均显著降低（t=56.00、47.05,均P＜0.05）.与DL组比较,DH组大鼠心肌p22phox和Nox4 mRNA表达显著降低（t=10.14、8.67,均P ＜0.05）,而心肌Cu-Zn-SOD表达无明显变化（=81.91,P＞0.05）.结论 GLP-1可剂量依赖性地下调1型糖尿病大鼠心肌p22phox和Nox4的表达,非剂量依赖性地上调心肌Cu-Zn-SOD表达,减轻氧化应激对心肌的损害,对糖尿病大鼠心肌产生保护作用.     

Protein kinase C and platelet reactivity in spontaneously hypertensive rats

Since protein kinase C (PKC) plays an important role in the control of platelet biological responses, we investigated whether it can be involved in the enhanced platelet reactivity to thrombin which is observed in spontaneously hypertensive rats (SHR) in comparison to that observed in controls (WKY). PKC activity was determined by measuring the phosphorylation of P47 protein (the endogenous substrate of PKC in the platelet). Mean effective concentration (EC50) values for phorbol ester and synthetic diacylglycerol (direct activators of PKC) were similar in SHR and WKY, thus revealing similar intrinsic activity of PKC in both rat strains. EC50 values for thrombin were approximately 30% lower in SHR v WKY. Enhanced PKC activity in SHR is likely the result of an increased diacylglycerol formation and release of Ca2+ from intracellular pools, consequent to an increased thrombin-induced phospholipase C activity.     

尼克酰胺腺嘌呤二核苷酸对阿霉素所致大鼠心肌损伤的保护作用

目的观察尼克酰胺腺嘌呤二核苷酸(NADH)对阿霉素(ADR)损伤的大鼠心肌保护作用。方法将60只雄性健康Wistar大鼠随机分为ADR组、NADH组、ADR NADH组及对照组。在实验第8周处死大鼠.光镜下观察心肌形态结构及病理改变;用生化方法测定大鼠心肌组织、心肌细胞线粒体中谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活性及丙二醛(MDA)水平。结果光镜下ADR NADH组大鼠心肌组织损伤程度较ADR组明显减轻;从心肌组织GSH-Px、SOD活性可以看出,与ADR组[(3.97±1.11.15.63±3.79)kU/g Pr]比较,ADR NADH组[(5.88±0.97,49.27±4.22)kU/g Pr]明显增高,二者比较差异有统计学意义(q=7.22、32.18,P<0.01);心肌组织MDA水平,ADR组[(4.55±1.51)μmoI/g Pr]明显高于ADR NADH组[(3.64±0.75)μmoL/g Pr],二者比较差异有统计学意义(q=8.48,P<0.01)。结论NADH对阿霉素性心肌损伤有一定的保护作用,这种保护作用可能主要与NADH提高受损心肌细胞抗氧化能力有关。     

Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase (ThoX, LNOX, Duox) genes and proteins in human thyroid tissues.

The large homolog of NADPH oxidase flavoprotein LNOX2, and probably LNOX1, are flavoproteins involved in the thyroid H(2)O(2) generator. Western blot analysis of membrane proteins from normal human thyroid, using antipeptide antibodies, indicated that LNOX1,2 are 164-kDa glycoproteins and that N-glycosylated motifs account for at least 10-20 kDa of their total apparent molecular mass. Northern blot analysis of 23 different human tissues demonstrated that LNOX2 messenger RNA (mRNA) is strongly expressed only in the thyroid gland, although blast analysis of expressed sequence tags databases indicated that LNOX genes are also expressed in some nonthyroid cells. We investigated LNOX1,2 gene and protein expressions in normal and pathological human thyroid tissues using real-time kinetic quantitative PCR and antipeptide antibodies, respectively. In normal tissue, LNOX1,2 are localized at the apical pole of thyrocytes. Immunostaining for LNOX1,2 was heterogeneous, inside a given follicle, with 40-60% of positive follicular cells. Among normal and pathological tissues, variations of LNOX1 and LNOX2 mRNA levels were parallel, suggesting a similar regulation of both gene expressions. Whereas LNOX mRNAs seemed slightly affected in benign disease, the expression of protein was highly variable. In multinodular goiters, 40-60% of cells were stained. In hypofunctioning adenomas, LNOX immunostaining was highly variable among follicles, whereas sodium/iodide (Na+/I-) symporter immunostaining was decreased. In hyperfunctioning thyroid tissues, only few cells (0-10%) were weakly stained, whereas sodium/iodide symporter staining was found in the majority of follicular cells. In conclusion, LNOX proteins are new apical glycoproteins with a regulation of expression that differs from other thyroid markers.