Association of NAD(P)H oxidase p22 phox gene variation with advanced carotid atherosclerosis in Japanese type 2 diabetes

OBJECTIVE: To evaluate the association between the C242T polymorphism of the p22 phox gene, an essential component of NAD(P)H oxidase in the vasculature, with intima-media thickness (IMT) of the carotid artery and risk factors for atherosclerosis in type 2 diabetic subjects. RESEARCH DESIGN AND METHODS: C242T polymorphism of the p22 phox gene was detected by polymerase chain reaction-restriction fragment-length polymorphism in 200 Japanese type 2 diabetic subjects and 215 nondiabetic subjects. We examined the association with this mutation and carotid atherosclerosis as well as the patients' clinical characteristics and the level of 8-hydroxy-2'deoxyguanosine (8-OHdG) as an index of oxidative DNA damage. RESULTS: The diabetic subjects with the TC+TT genotypes displayed a significantly lower average IMT (1.13 +/- 0.31 vs. 1.31 +/- 0.34 mm; P = 0.0099) and a not significantly lower serum 8-OHdG level than those with the CC genotype, despite no difference in the risk factors. Stepwise multiple regression analysis showed that the risk factors for increased IMT in the diabetic subjects were systolic blood pressure (P = 0.0042) and p22 phox CC genotype (P = 0.0151). In nondiabetic subjects, the average IMT of the TC+TT group was not different from that of the CC group (0.85 +/- 0.14 vs. 0.94 +/- 0.30 mm, P = 0.417). Fasting plasma insulin concentration (41.4 +/- 15.6 vs. 64.2 +/- 59.4 pmol/l, P = 0.0098) and insulin resistance index of homeostasis model assessment (HOMA-R) (1.58 +/- 0.66 vs. 2.60 +/- 2.56, P = 0.0066) were significantly lower in the TC+TT group than in the CC group. CONCLUSIONS: These results show that the C242T mutation in the p22 phox gene is associated with progression of asymptomatic atherosclerosis in the subjects with type 2 diabetes and is also associated with insulin resistance in nondiabetic subjects.     

NAD(P)H oxidase p22phox Gene C242T polymorphism and lipoprotein oxidation

BACKGROUND: Vascular NAD(P)H oxidase is a key enzyme of superoxide anion production in human vessel walls. The C242T mutation in the CYBA gene coding for p22phox, a component of the enzyme, may change the redox state. The aim of this study was to evaluate the influence of the polymorphism on serum concentrations of oxidative stress markers. METHODS: Serum samples were collected from 134 Type 2 diabetic patients and analyzed for oxidized high-density lipoprotein (HDL) by in-house ELISA, and oxidized low-density lipoprotein (LDL) and thiobarbituric acid reactive substance (TBARS) by commercial kits. For genotyping, the Taqman PCR method was adapted to detect the polymorphism. RESULTS: Circulating concentrations of oxidized HDL were about 1.5-fold lower in those of the CT/TT genotypes than the CC genotype [3.3 +/- 0.3 and 5.0 +/- 0.3 U/dl (mean +/- S.E.M.), respectively; multiple regression analysis, p=0.006], whereas concentrations of oxidized LDL were slightly greater (1.1-fold, p=0.01) in those with the CT/TT genotypes. However, no significant difference was observed in TBARS between the genotypes. CONCLUSIONS: The effect was inconsistent among the markers, but these results suggest that the CYBA C242T polymorphism is involved in NAD(P)H oxidase activity and affects oxidation of lipoproteins by altering the redox state in the vasculature.     

NAD(P)H氧化酶p22phox亚基C242T基因多态性与脑梗死的关系

目的了解上海地区汉族脑梗死患者烟酰胺腺嘌呤二核苷酸磷酸氧化酶[NAD(P)H氧化酶]p22phox亚基C242T基因多态性和等位基因频率的分布情况,探讨其多态性与脑梗死的关系。方法采用聚合酶链反应-限制性片段长度多态性技术(PCR-RFLP),对176例脑梗死患者和131名健康老年人的C242T位点进行基因分型,计算等位基因的频率分布。结果脑梗死患者血清三酰甘油(TG)、高密度脂蛋白胆固醇(HDL-C)、低密度脂蛋白胆固醇(LDL-C)、血糖浓度和血压与老年健康对照组之间差异具有统计学意义(P<0.05)。脑梗死组与老年健康对照组的CC、CT、TT基因型频率分别为92.05%、7.95%、0与93.89%、5.34%、0.76%;C、T等位基因频率分别为96.02%、3.98%与96.56%、3.44%,2组间的差异均无统计学意义(P>0.05)。结论 p22phox亚基C242T位点的单核苷酸多态性与脑梗死的发生可能无关。     

NAD（P）H氧化酶p22phox亚基C242T基因多态性与脑梗死的关系

目的了解上海地区汉族脑梗死患者烟酰胺腺嘌呤二核苷酸磷酸氧化酶[NAD（P）H氧化酶]p22phox亚基C242T基因多态性和等位基因频率的分布情况,探讨其多态性与脑梗死的关系。方法采用聚合酶链反应-限制性片段长度多态性技术（PCR-RFLP）,对176例脑梗死患者和131名健康老年人的C242T位点进行基因分型,计算等位基因的频率分布。结果脑梗死患者血清三酰甘油（TG）、高密度脂蛋白胆固醇（HDL-C）、低密度脂蛋白胆固醇（LDL-C）、血糖浓度和血压与老年健康对照组之间差异具有统计学意义（P〈0.05）。脑梗死组与老年健康对照组的CC、CT、TT基因型频率分别为92.05%、7.95%、0与93.89%、5.34%、0.76%;C、T等位基因频率分别为96.02%、3.98%与96.56%、3.44%,2组间的差异均无统计学意义（P〉0.05）。结论 p22phox亚基C242T位点的单核苷酸多态性与脑梗死的发生可能无关。     

Investigation of the C242T polymorphism of NAD(P)H oxidase p22 phox gene and ischaemic heart disease using family-based association methods

Ischaemic heart disease is a complex phenotype arising from the interaction of genetic and environmental factors. Excessive production of reactive oxygen species leading to endothelial dysfunction is believed to be important in the pathogenesis of ischaemic heart disease. The NAD(P)H oxidase system generates superoxide anions in vascular cells; however, the role of the C242T polymorphism of the NAD(P)H oxidase p22 phox gene in ischaemic heart disease is unclear due to contradictory results from case-control studies. Consequently, we applied family-based association tests to investigate the role of this polymorphism in ischaemic heart disease in a well-defined Irish population. A total of 1023 individuals from 388 families (discordant sibships and parent/child trios) were recruited. Linkage disequilibrium between the polymorphism and ischaemic heart disease was tested using the combined transmission disequilibrium test (TDT)/sib-TDT (cTDT) and pedigree disequilibrium test (PDT). Both cTDT and PDT analyses found no statistically significant excess transmission of either allele to affected individuals (P =0.30 and P =0.28, respectively). Using robust family-based association tests specifically designed for the study of complex diseases, we found no evidence that the C242T polymorphism of the p22 phox gene has a significant role in the development of ischaemic heart disease in our population.     

老年2型糖尿病患者血糖变异性与糖尿病肾病的关系

目的:研究老年2型糖尿病患者血糖变异性与糖尿病肾病的关系.方法:对114例老年2型糖尿病患者按照24 h尿微量白蛋白量分为:单纯糖尿病组(DM组)36例,早期糖尿病肾病组(EDN组)44例,临床糖尿病肾病组(DN组)34例.检测收缩压(SBP)、舒张压(DBP)、空腹血糖(FBG)、糖化血红蛋白(HbA1C)、血肌酐(sCr)、半胱氨酸蛋白酶抑制剂C(Cys C),并采用动态血糖监测系统(CGMS)计算日内平均血糖波动幅度(MAGE)、最大血糖波动幅度(LAGE)、全天血糖值标准差(SDBG)、曲线下面积(AUC)和日间血糖平均绝对差(MODD)来评价血糖变异性.结果:DN组SBP、DBP、HbA1C、sCr和Cys C显著高于EDN组和DM组(P＜0.05).EDN组Cys C显著高于DM组(P＜0.05).DN组SDBG、MAGE、LAGE、AUC、MODD显著高于EDN组和DM组(P＜0.05),日内及日间血糖波动幅度显著增加,EDN组的CGMS各项指标均显著高于DM组(P＜0.05).结论:老年2型糖尿病患者血糖变异性与DN的发生、发展及严重程度有着密切关系.     

NADPH氧化酶p22phox亚基C242T基因多态性与糖尿病患者颈总动脉粥样硬化相关性研究

目的：研究2型糖尿病（T2DM）患者NADPH氧化酶p22phox亚基242位点基因多态性分布,探讨该基因多态性与糖尿病患者颈总动脉粥样硬化的相关性。方法：将273例T2DM患者分为颈总动脉内-中膜（IMT）正常、增厚、无斑块与有斑块4组,应用聚合酶链反应-限制性片段长度多态性技术对他们的NADPH氧化酶p22phox亚基242位点进行基因型分析。结果：NADPH氧化酶p22phox亚基242位点基因多态性在IMT正常和IMT增厚的T2DM患者中的分布存在显著性差异（P<0.05）;在无斑块和有斑块的T2DM的患者中,NADPH氧化酶p22phox亚基242位点基因多态性亦存在显著性差异（P<0.05）。结论：NADPH氧化酶p22phox亚基242位点（C-T）基因多态性与T2DM患者颈总动脉IMT的增厚、斑块的发生存在相关性。     

Association between diabetic nephropathy and left ventricular longitudinal myocardial function in type 1 diabetes mellitus patients with preserved ejection fraction

The International Journal of Cardiovascular Imaging - Left ventricular (LV) longitudinal myocardial dysfunction can be observed even in type 2 diabetes mellitus (DM) (T2DM) patients with preserved...     

Smoking and progression of diabetic nephropathy in type 1 diabetes

OBJECTIVE: Cigarette smoking contributes to development of diabetic nephropathy. However, long-term studies on the effect of smoking on decline in kidney function in diabetic nephropathy are lacking. We assessed the impact of smoking on progression of diabetic nephropathy in type 1 diabetic patients enrolled in a prospective observational cohort study started in 1983. RESEARCH DESIGN AND METHODS: We identified all albuminuric type 1 diabetic patients (n = 301) followed for at least 3 years, median (range) 7 years (3-14), who underwent at least yearly measurement of glomerular filtration rate (GFR) by the (51)Cr-EDTA plasma clearance technique (n = 8, range 3-24). In total, 192 men and 109 women were included (age [mean +/- SD] 36 +/- 11 years, duration of diabetes 22 +/- 8 years); 271 patients were treated with antihypertensive drugs, predominantly ACE inhibitors in 179 patients. Patients were classified as smokers if they smoked more than one cigarette per day during a portion of or the entire observation period. Blood pressure, albuminuria, HbA(1c), and serum cholesterol were measured every 3-4 months during the study. RESULTS: In all 301 patients, the mean (SE) rate of decline in GFR (deltaGFR) was 4.0 (0.2) mlx min(-1) x year(-1) during the investigation period. No difference in Delta GFR was demonstrated between nonsmokers (n = 94), deltaGFR 4.5 (0.4), ex-smokers (n = 31), deltaGFR 3.1 (0.7), and smokers (n = 176), deltaGFR 3.9 (0.3) ml x min(-1) x year(-1), respectively (NS). Adjustment for other risk factors for progression of diabetic nephropathy did not alter the results: smoking was not associated with deltaGFR, whereas blood pressure, albuminuria, HbA(1c), and serum cholesterol were demonstrated to be independent progression promoters. CONCLUSIONS: In our study, smoking was not associated with decline in kidney function in type 1 diabetic patients with diabetic nephropathy.     

Pulse pressure predicts incident cardiovascular disease but not diabetic nephropathy in patients with type 1 diabetes (The FinnDiane Study)

OBJECTIVE

Pulse pressure (PP), an estimate of arterial stiffness, has been shown to be associated with incident cardiovascular disease (CVD) in patients with type 1 diabetes (T1D). However, diabetic kidney disease, a strong predictor of CVD, was not previously taken into account. Furthermore, the role of PP as a predictor of diabetic nephropathy is not known. Therefore, we prospectively investigated the associations between PP and these diabetes complications in patients with T1D.

RESEARCH DESIGN AND METHODS

A total of 4,509 patients from the FinnDiane Study participated. Follow-up data on incident CVD events and renal status (median 5.3 years) were available in 69 and 76% of the patients, respectively. Altogether, 269 patients (8.6%) had an incident CVD event and 370 patients (10.8%) progressed to a higher level of albuminuria or to end-stage renal disease.

RESULTS

PP was higher at baseline in patients who experienced a CVD event (66 ± 18 vs. 52 ± 14 mmHg; P < 0.001) or progressed in their renal status (58 ± 18 vs. 54 ± 15 mmHg; P < 0.01) during follow-up. In a Cox regression model, PP was independently associated with a first ever CVD event (hazard ratio per 10 mmHg 1.22 [95% CI 1.10–1.34]) but not progression of renal disease (1.00 [0.89–1.12]) after adjustments for traditional risk factors.

CONCLUSIONS

PP, a marker of arterial stiffness, is a risk factor for cardiovascular complications but not for diabetic nephropathy in patients with T1D.Whereas systolic blood pressure (SBP) increases with age in the western population, diastolic blood pressure (DBP) generally increases during adulthood, peaks at 55–60 years of age, and thereafter starts to decrease as the result of arterial stiffening (1). This results in an increased pulse pressure (PP) that is associated with cardiovascular disease (CVD) (2). We have shown a premature increase in PP in patients with type 1 diabetes (T1D) compared with nondiabetic subjects (3). The results indicated an accelerated arterial aging in patients with T1D that may contribute to a higher risk of CVD.PP has been shown to be a strong predictor of CVD in the general population, especially in elderly subjects aged >65 years (4,5). The EURODIAB Prospective Complications Study extended this finding to patients with T1D because PP was shown to be associated with incident CVD (6). However, diabetic nephropathy, a strong CVD risk factor (7), was not taken into account. Furthermore, Soedamah-Muthu et al. (8) reported that PP predicted all-cause but not cardiovascular mortality in the same cohort.Although PP was shown to be associated with diabetic nephropathy in a cross-sectional study (3), it is not known whether PP could be used as a predictor of progression of diabetic nephropathy. However, blood pressure per se has been shown to predict incipient and overt diabetic nephropathy, although the results have been conflicting (9–11).Therefore, the aim was to explore the role of PP in the development of CVD and progression of diabetic nephropathy in a large, homogeneous and nationwide cohort of patients with T1D.     

Metabolic syndrome in type 1 diabetes: association with diabetic nephropathy and glycemic control (the FinnDiane study)

OBJECTIVE: The aim of this study was to estimate the prevalence of the metabolic syndrome in Finnish type 1 diabetic patients and to assess whether it is associated with diabetic nephropathy or poor glycemic control. RESEARCH DESIGN AND METHODS: In all, 2,415 type 1 diabetic patients (51% men, mean age 37 years, duration of diabetes 22 years) participating in the nationwide, multicenter Finnish Diabetic Nephropathy (FinnDiane) study were included. Metabolic syndrome was defined according to the National Cholesterol Education Program diagnostic criteria. Patients were classified as having normal albumin excretion rate (AER) (n = 1,261), microalbuminuria (n = 326), macroalbuminuria (n = 383), or end-stage renal disease (ESRD) (n = 164). Glycemic control was classified as good (HbA1c <7.5%), intermediate (7.5-9.0%), or poor (>9.0%). Creatinine clearance was estimated with the Cockcroft-Gault formula. RESULTS: The overall prevalence of metabolic syndrome was 38% in men and 40% in women. The prevalence was 28% in those with normal AER, 44% in microalbuminuric patients, 62% in macroalbuminuric patients, and 68% in patients with ESRD (P < 0.001). Patients with metabolic syndrome had a 3.75-fold odds ratio for diabetic nephropathy (95% CI 2.89-4.85), and all of the separate components of the syndrome were independently associated with diabetic nephropathy. The prevalence of metabolic syndrome was 31% in patients with good glycemic control, 36% in patients with intermediate glycemic control, and 51% in patients with poor glycemic control (P < 0.001). Similarly, metabolic syndrome increased with worsening creatinine clearance. CONCLUSIONS: The metabolic syndrome is a frequent finding in type 1 diabetes and increases with advanced diabetic nephropathy and worse glycemic control.     

Diabetic nephropathy in patients with type 1 diabetes mellitus.

Proteinuria has been recognized in association with diabetes mellitus as early as the 18th century. This form of renal disease is known as diabetic nephropathy. It is now clear that diabetic nephropathy is the principal cause of end stage renal disease (ESRD) in the western world. According to reports by the United States Renal Data System (USRDS), in the past two decades there has been a continual increase in the incidence of ESRD among patients with diabetes. Many patients have diabetes that progresses to diabetic nephropathy, which is often not discovered until overt nephropathy is present. Many of the complications of diabetes could be minimized if patients received a comprehensive health maintenance program that includes vigorous cardiac risk reduction, routine eye examinations; routine foot examinations; screening and treatment for microalbuminuria, optimal hypertension management; and improved glycemic control. Hence, the key is not only prudent screening of these patients, but referral as well. Using a case study approach, this article illustrates the care of patients with diabetic nephropathy in type 1 diabetes mellitus.     

Relation between development of nephropathy and the p22phox C242T and receptor for advanced glycation end product G1704T gene polymorphisms in type 2 diabetic patients

OBJECTIVE: The development of diabetic nephropathy is considered to be associated with oxidative stress. NADPH oxidase and the receptor for advanced glycation end products (RAGE) have attracted attention as mechanisms of generating oxidative stress. We studied the relation between the genotypes of the NADPH p22phox C242T and RAGE G1704T polymorphisms and the development of diabetic nephropathy in type 2 diabetic patients. RESEARCH DESIGN AND METHODS: Using a retrospective review of clinical data, we allocated 181 Japanese type 2 diabetic patients to one of two groups: patients without diabetic nephropathy (group N; n = 108) and patients developing diabetic nephropathy (group D; n = 73) for 10 years or more. The p22phox C242T and RAGE G1704T polymorphisms were examined by Taqman PCR methods. RESULTS: The frequency of the p22phox CC genotype was significantly higher in group D than in group N (90 vs. 79%; P = 0.0427). The frequency of the RAGE GT + TT genotype was significantly higher in group D than in group N (26 vs. 13%; P = 0.0313). The frequency of the combination of p22phox CC and RAGE GT + TT genotypes was significantly higher in group D than in group N (22 vs. 8%; P = 0.0057). In multiple logistic regression analysis, systolic blood pressure, HbA(1c), triglycerides, and the combination of polymorphisms were shown to be independent variables. CONCLUSIONS: These results suggest that assessment of the combination of NADPH p22phox C242T and RAGE G1704T polymorphisms may be useful in identifying the risk for developing diabetic nephropathy in type 2 diabetic patients.     

Serum adiponectin is increased in type 1 diabetic patients with nephropathy

OBJECTIVE: To elucidate whether serum adiponectin is associated with renal function, low-grade inflammatory markers, metabolic control, and insulin resistance in type 1 diabetic patients with and without nephropathy. RESEARCH DESIGN AND METHODS: A total of 189 type 1 diabetic patients from the Finnish Diabetic Nephropathy Study were divided into three groups based on their urinary albumin excretion rate (AER): patients with normal AER (n = 66) had no antihypertensive medication, while patients with microalbuminuria (n = 63) or macroalbuminuria (n = 60) were all treated with an ACE inhibitor. Renal function was estimated with the Cockcroft-Gault formula. Adiponectin was measured by an in-house time-resolved immunofluorometric assay. RESULTS: Adiponectin concentrations were higher in women than in men, but since there was no significant difference in sex distribution between the groups, data were pooled. Adiponectin concentrations were higher in patients with macroalbuminuria (19.8 +/- 12.0 mg/l) than in patients with microalbuminuria (13.1 +/- 4.8 mg/l) or normoalbuminuria (11.8 +/- 4.2 mg/l). In a univariate analysis, adiponectin was positively associated with creatinine (r = 0.41; P < 0.0001), AER (r = 0.33; P < 0.0001), interleukin-6 (r = 0.22; P = 0.002), systolic blood pressure (r = 0.22; P = 0.004), HbA(1c) (r = 0.17; P = 0.02), total cholesterol (r = 0.16; P = 0.03), and HDL cholesterol (r = 0.16; P = 0.03) and negatively with estimated glomerular filtration rate (GFR; r = -0.52; P < 0.0001) and waist-to-hip ratio (WHR; r = -0.16; P = 0.03). In a multiple linear regression analysis including the above variables, estimated GFR, AER, and WHR were independently associated with adiponectin levels (r(2) = 0.32). CONCLUSIONS: Serum adiponectin concentrations are increased in type 1 diabetic patients with nephropathy, and levels are further associated with renal insufficiency.     

NAD(P)H oxidase activity in human neutrophils stimulated by phorbol myristate acetate.

Phorbol myristate acetate activated in normal human neutrophils a single enzymatic entity that was dormant in unstimulated cells, optimally active at pH 7.0, and capable of oxidizing either NADH or NADPH, producing NAD(P)+ and superoxide (O27). Comparative fluorometric and spectrophotometric measurements supported the stoichiometry NAD(P)H + 20(2) leads to NAD(P)+ + 20(27) + H+. the seemingly considerable NAD(P)+ production at pH 5.5 and 6.0 was due largely to nonenzymatic oxidation of NAD(P)H by chain reactions initiated by HO27 (perhydroxyl radical), the conjugate acid of O27. This artifact, responsible for earlier erroneous assignments of an acid pH optimum for NAD(P)H oxidase, was prevented by including superoxide dismutase in fluorometric assays. NAD(P)H oxidase was more active towards NADPH (Km = 0.15 +/- 0.03 mM) than NADH (Km = 0.68 +/- 0.2 mM). No suggestion that oxidase activity was allosterically regulated by NAD(P)H was seen. Phorbol myristate acetate-induced O27 production was noted to be modulated by pH in intact neutrophils, suggesting that NAD(P)H oxidase is localized in the plasma membrane where its activity may be subject to (auto) regulation by local H+ concentrations.     

Assessment of vasomotor endothelial function in patients with diabetes mellitus type 1 at different stages of diabetic nephropathy

AIM: To study a vasomotor endothelial function in patients with diabetes mellitus (DM) type 1 at different stages of diabetic nephropathy (DN). MATERIAL AND METHODS: Twenty six patients with DM type 1 (11 males and 15 females, mean age 25.9 +/- 4.3 years, mean history of DM 12.9 +/- 3.4 years) entered the study. They were divided into 4 groups: group 1--without renal affection, group 2--with microalbuminuria (MAU), group 3--with proteinuria (PU), group 4--with chronic renal failure (CRF). The control group consisted of 7 healthy volunteers. Endothelium-dependent vasodilation (EDVD) was studied in the test with reactive hyperemia provoked by 4-5 min occlusion of the brachial artery by pneumocollar and subsequent assessment of arterial diameter changes after decompression using high-resolution ultrasound dopplerography. RESULTS: Reactive hyperemia resulted in dilation of the artery in all the examinees. This dilation was maximal on second 30 after removal of the collar in the controls, group 2, 3 and 4 and reached 9.2 +/- 2.9, 9.63 +/- 3.62, 7.25 +/- 5.23 and 4.42 +/- 4.05%, respectively. Resting blood flow velocity was similar in all the groups and rose maximally by 95-150%. To estimate EDVD of the brachial artery more precisely, the coefficient of endothelial sensitivity to shift tension was calculated. It made up 0.084 +/- 0.04 (control group), 0.0825 +/- 0.08 (group 1), 0.138 +/- 0.07 (group 2), 0.067 +/- 0.05 (group 3) and 0.052 +/- 0.04 (group 4). CONCLUSION: At the earliest stage of DN (stage MAU), EDVD is not affected as maximal vasodilation of the brachial artery and endothelial sensitivity to shift tension do not differ from the control values. This means that the stage of MAU is reversible in early treatment, but PU and CRF are not reversible stages associated with depletion of endothelial cells and loss of sensitivity to changing hemodynamic conditions.