Advanced glycosylated end products-mediated activation of polymorphonuclear neutrophils in diabetes mellitus and associated oxidative stress

Two important consequences of hyperglycemia in diabetes are development of oxidative stress and formation of advanced glycation end products (AGE) which are known to be associated with diabetic complications. Relationship between AGE formation and development of oxidative stress (OS) is yet to be established. In the present study, the involvement of AGE in PMN-mediated ROS generation and the associated OS were investigated in type 2 diabetic mellitus (DM) patients. We assessed OS parameters (serum MDA, FRAP and GSH), PMN oxidative functions (respiratory burst and superoxide production) and total serum AGE in 90 subjects divided equally in three groups--control group, Group I consisting of type 2 diabetic patients without microvascular complications and Group II consisting of type 2 diabetic patients with microvascular complications. PMNs isolated from both groups (I and II) exhibited higher level of respiratory burst (RB) and produced increased amount of superoxide anion as compared to the controls. The increase was more pronounced in diabetes with complications, as compared to those without. Serum malondialdehyde (MDA) level was elevated, whereas glutathione (GSH) and ferric reducing ability of plasma (FRAP) levels were significantly reduced in diabetes as compared to the controls, suggesting the presence of oxidative stress in DM. A positive correlation between PMN oxidative function and OS parameters suggested the involvement of PMN in the development of OS in DM. Serum AGE level was also elevated in diabetic groups as compared to the controls. Further, the positive correlation between serum AGE level and PMN oxidative function suggested the involvement of AGE in increased RB and generation of reactive oxygen species (ROS) by resting diabetic PMN. The results of the study indicate that AGE-PMN interaction possibly upregulates NADPH oxidase, leading to enhanced ROS generation and thus contributes to the pathogenesis in diabetes.     

Dissociation between aggregation and superoxide production in human granulocytes

Aggregation and the activation of the granulocyte (PMN) superoxide (O2-) generating system occur when certain stimuli are added to resting cells. It had previously been postulated that PMN aggregation is essential for maximal O2- production. This study was undertaken to test the hypothesis that PMN aggregation is required for full expression of PMN O2- production. We examined aggregation and O2- production induced by four stimuli; concanavalin A (Con A), phorbol myristate acetate (PMA), N-formylmethionyl-leucyl-phenylalanine (FMLP), and ionophore A23187. Cytochalasin B enhanced aggregation by all four stimuli but only enhanced the rate of O2- production by Con A; 2-deoxyglucose inhibited aggregation by all stimuli. Dissociation of PMN aggregation and O2- production was achieved by using NEM, TPCK, and divalent cations. NEM and TPCK prevent Con A-induced O2- production but have no effect on Con A-induced aggregation. PMA-stimulated PMN generate O2- in the presence or absence of Ca++ and Mg++. In contrast, PMA stimulated maximum PMN aggregation only in the presence of both Ca++ and Mg++. Thus PMN can generate O2- without aggregating, and PMN can aggregate without producing O2-. PMN from patients with chronic granulomatous disease do not generate O2- or undergo membrane potential depolarization in response to PMA. These PMN aggregated when stimulated with PMA, providing evidence that depolarization is not required for PMN aggregation. We conclude that aggregation and the activation of the O2- generating system, though temporally related, are not necessarily causally related.     

Production of superoxide and nitric oxide by granulocytes in non-insulin-dependent diabetic patients with and without diabetic nephropathy

Production of the superoxide radical anion O2-. and the nitric oxide radical NO-. by granulocytes was studied in 14 patients with type 2 diabetes without nephropathy, 21 patients with type 2 diabetes and diabetic nephropathy, and 19 healthy subjects, both without and after stimulation with opsonized zymosan. O2-. production by both resting and stimulated granulocytes was increased in type 2 diabetes patients without nephropathy but decreased in type 2 diabetes patients with nephropathy, compared with healthy subjects. NO. generation was highly augmented in type 2 diabetes patients without nephropathy by both resting and stimulated cells; values for type 2 diabetes patients with nephropathy were intermediate between the type 2 diabetes patients without nephropathy and the healthy subjects. These data point to granulocytes as one of possible sources of oxidative stress in type 2 diabetes.     

Superoxide production from human polymorphonuclear leukocytes by human mannan-binding protein (MBP)

Mannan-binding protein (MBP) is a Ca(2+)-dependent mammalian lectin that plays an important role in innate immunity. In this study, we found that ligand-bound MBP stimulates polymorphonuclear leukocytes (PMN) to induce cell aggregation and superoxide production. The biological response of PMN to ligand-bound MBP was dose- and time-dependent. The PMN aggregation and superoxide production induced by ligand-bound MBP was blocked completely by pertussis toxin, and partially blocked by a platelet activation factor receptor antagonist, TCV-309. These findings suggest that the ligand-bound MBP stimulates PMN through a putative MBP receptor(s) on PMN.     

Phagocytosis, bactericidal capacity, and superoxide anion (O2-) production by polymorphonuclear neutrophils from patients with diabetes mellitus

Phagocytosis, bactericidal capacity and superoxide anion production of polymorphonuclear neutrophils (PMN) were estimated in 30 patients with well-controlled insulin-dependent diabetes (IDD) and in 50 patients with non insulin-dependent diabetes (NIDD). The estimations were additionally done in 20 elderly patients without glucose intolerance. The estimations of bactericidal capacity were performed in autologous-, zymosan activated-, inactivated- and control plasma. The phagocytosis of viable staphylococci was unchanged in all evaluated groups. The bactericidal capacity in all diabetic patients was significantly reduced. It was fully correctable in patients with IDD by suspension of cells in control or zymosan activated plasma. The improvement of PMN bactericidal capacity in patients with NIDD in similar conditions was less distinct. The superoxide anion production in patients with IDD was similar to values noticed in healthy persons. Whereas, the O2- production in patients with NIDD as well as in elderly patients were significantly reduced and correlated significantly with bactericidal capacity impairment. The possible mechanism of noticed disturbances were discussed.     

Association between dietary fat and antioxidant status of Tunisian type 2 diabetic patients

BACKGROUND: The antioxidant enzymes: superoxide dismutase (Cu/Zn SOD) and glutathione peroxidase (GSH-Px) provide a defense against the damage of cells by reactive oxygen species, which increased in diabetic state. It was demonstrated that dietary treatment could improve the antioxidant status in patients with type 2 diabetes mellitus. This study was undertaken to determine if erythrocyte Cu/Zn SOD and GSH-Px activities correlate with dietary nutrients in 35 selected type 2 diabetic patients (21 women and 14 men) without diabetic complications. RESULTS: We found that erythrocyte Cu/Zn SOD was diminished in patients with poor controlled diabetes and GSH-Px activity was significantly decreased in obese compared with non-obese type 2 diabetic patients (1.07+/-0.87 and 2.36+/-1.99 U/ml, respectively; P=0.024). Both erythrocyte Cu/Zn SOD and GSH-Px activities were positively correlated to erythrocyte omega3-polyunsaturated fatty acids (PUFA). In non-obese diabetic patients, only GSH-Px activity was correlated negatively to the fraction of linoleic acid (18:2omega6) and arachidonic acid (20:4omega6) in erythrocytes phospholipids. CONCLUSIONS: The data of this study reveal that activities of erythrocyte antioxidant enzymes were altered in type 2 diabetic patients. Further studies are needed to determine if diet supplemented with omega3-PUFA is required to improve antioxidant defense system in diabetic state.     

Increased nonenzymatically glycosylated proteins in the vitreous humor of diabetic animals

One of the earliest pathologic changes of diabetes mellitus is increased nonenzymatic glycosylation (i.e., glycation) of proteins, which results in abnormal aggregation of collagen fibrils and production of superoxide radicals. These abnormalities may be responsible for the precocious senescence of connective tissue associated with the disease. We sought to determine whether glycation is increased in the vitreous humor of short-term diabetic cats (6 months' duration) and rabbits (2 months' duration), using a nitroblue tetrazolium colorimetric assay for fructosamine. Vitreous protein fructosamine concentration was significantly higher in diabetic cats and rabbits, compared with that in control (nondiabetic) animals. These results indicate that glycation is increased in the vitreous humor of short-term diabetic animals, and therefore may be one of the initial triggers for clinically apparent diabetic retinopathy.     

NAD(P)H oxidase-stimulating activity of serum from type 2 diabetic patients with retinopathy mediates enhanced endothelial expression of E-selectin

Endothelial expression of E-selectin is enhanced in diabetic patients with retinopathy, however, the underlying mechanisms are unclear. Therefore, this study was aimed to determine if endothelial expression of E-selectin is stimulated with serum from type 2 diabetic patients with retinopathy, and whether this process is related to NAD(P)H oxidase-derived oxidative stress. Serum was obtained from type 2 diabetic patients with (T2DR) or without (T2DM) retinopathy, and age-matched non-diabetic healthy person (Control). Serum was added to in vitro-grown human coronary artery endothelial cells (HCAEC), after which E-selectin expression, reactive oxygen species (ROS) production, and NAD(P)H oxidase activity were measured. Serum from T2DR induced a significantly higher expression of E-selectin than serum from T2DM and control in association with an enhanced production of ROS in HCAEC. T2DR serum enhanced E-selectin expression in a ROS-dependent manner since this process was significantly attenuated not only by tiron (1 mM), a superoxide scavenger, but also by DPI (10 micromol/L) and apocynin (100 micromol/L), inhibitors of NAD(P)H oxidase. Furthermore, the activity of NADH oxidase was markedly increased by T2DR serum, and this was accompanied by the enhanced membrane translocation of p47phox, a cytosolic subunit of NAD(P)H oxidase. These findings suggest that serum from T2DR induced up-regulation of E-selectin expression in HCAEC, and this process might be dependent on activation of endothelial NADH oxidase via an enhanced membrane translocation of p47phox.     

Oxidized low-density lipoprotein induces calcium influx in polymorphonuclear leukocytes

Polymorphonuclear leukocytes (PMN) have been suggested to play a role in atherosclerosis, but intracellular signaling after stimulation with oxidized low-density lipoprotein (LDL) is unknown. We investigated mechanistic aspects of oxidized LDL-induced superoxide production by human PMN, with special emphasis on intracellular Ca(2+) concentration ([Ca(2+)](i)). Oxidized LDL, but not native LDL, evoked an early but sustained increase in [Ca(2+)](i) and a delayed production of superoxide. The increase in [Ca(2+)](i) could be reduced by fucoidan and completely prevented by U73122, suggesting involvement of the scavenger receptor and coupling to the phospholipase C signal transduction pathway. Furthermore, we provide evidence that the increase in [Ca(2+)](i) partly results from protein kinase C-dependent Ca(2+) influx. The relevance of this Ca(2+) entry for oxidized LDL-stimulated effects is illustrated by the finding that superoxide production was markedly reduced in the absence of external Ca(2+). Finally, inhibition of phagocytosis by cytochalasin B abolished oxidized LDL-stimulated superoxide production without affecting, however, the Ca(2+) mobilization. These effects of oxidized LDL on [Ca(2+)](i) and on respiratory burst of PMN may underlie the occurrence of elevated levels of [Ca(2+)](i) of resting PMN in hypercholesterolemia and represent a mechanism by which PMN can amplify processes in the early phase of atherosclerosis.     

Polymorphonuclear neutrophil dysfunctions in streptozotocin-induced type 1 diabetic rats

Since conflicting results have been reported on non-specific immune response in type 1 diabetes, this study evaluates polymorphonuclear neutrophil (PMN) functions in the infection free Long Evan diabetic rats (type 1) by using tests that include: polarization assay, phagocytosis of baker\'s yeasts (Saccharomyces cerevisiae) and nitroblue tetrazolium (NBT) dye reduction. Polarization assay showed that neutrophils from diabetic rats were significantly activated at the basal level compared to those from the controls (p < 0.001). After PMN activation with N-formylmethionyl-leucyl-phenylalanine (FMLP), control neutrophils were found to be more polarized than those of the diabetic neutrophils and the highest proportions of polarization were found to be 67 % and 57 % at 10(-7) M FMLP, respectively. In the resting state, neutrophils from the diabetic rats reduced significantly more NBT dye than that of the controls (p < 0.001). The percentages of phagocytosis of opsonized yeast cells by the neutrophils from control and diabetic rats were 87 % and 61 %, respectively and the difference was statistically significant (p < 0.001). Evaluation of the phagocytic efficiency of PMNs revealed that control neutrophils could phagocytose 381 +/- 17 whereas those from the diabetic rats phagocytosed 282 +/- 16 yeast cells, and the efficiency of phagocytosis varied significantly (p < 0.001). Further, both the percentages of phagocytosis and the efficiency of phagocytosis by the diabetic neutrophils were inversely related with the levels of their corresponding plasma glucose (p = 0.02; r = -0.498 and p < 0.05; r = -0.43, respectively), which indicated that increased plasma glucose reduced the phagocytic ability of neutrophils. Such relationship was not observed with the control neutrophils. These data clearly indicate that PMN functions are altered in the streptozotocin (STZ)-induced diabetic rats, and hyperglycemia may be the cause for the impairment of their functions leading to many infectious episodes.     

Complement inhibition reduces injury in the type 2 diabetic heart following ischemia and reperfusion

Chronic inflammation exacerbates the cardiovascular complications of diabetes. Complement activation plays an important role in the inflammatory response and is known to be involved in ischemia-reperfusion (I/R) injury in the nondiabetic heart. The purpose of this study was to determine if increased complement deposition explains, in part, the increased severity of neutrophil-mediated I/R injury in the type 2 diabetic heart. Nondiabetic Zucker lean control (ZLC) and Zucker diabetic fatty (ZDF) rats underwent 30 min of coronary artery occlusion followed by 120 min of reperfusion. Another group of ZDF rats was treated with the complement inhibitor FUT-175 before reperfusion. Left ventricular (LV) tissue samples were stained for complement deposition and neutrophil accumulation following reperfusion. We found significantly more complement deposition in the ZDF LV compared with the ZLC (P < 0.05), and complement deposition was associated with significantly greater neutrophil accumulation. In whole blood samples taken preischemia and at 120 min reperfusion, neutrophils exhibited significantly more CD11b expression in the ZDF group compared with the ZLC group (P < 0.05). Furthermore, intracellular adhesion molecule (ICAM)-1 expression following I/R was increased significantly in ZDF hearts compared with ZLC hearts (P < 0.001). These results indicate that, in the ZDF heart, increased ICAM-1 and polymorphonuclear neutrophil (PMN) CD11b expression play a role in increasing PMN accumulation following I/R. The infarct size of the ZDF was significantly greater than ZLC (P < 0.05), and treatment with FUT-175 significantly decreased infarct size, complement deposition, and PMN accumulation in the diabetic heart. These findings indicate an exacerbated inflammatory response in the type 2 diabetic heart that contributes to the increased tissue injury observed following ischemia and reperfusion.     

Association of the SOD2 Ala(-9)Val and SOD3 Arg213Gly polymorphisms with diabetic polyneuropathy in patients with diabetes mellitus type 1

Single-nucleotide polymorphisms of the genes for mitochondrial (SOD2) and extracellular (SOD3) superoxide dismutases were tested for association with diabetic polyneuropathy (DPN) in diabetes mellitus (DM) type 1. Patients (n = 180) were divided into two groups with nonoverlapping (polar) phenotypes. Group DPN+ included 86 individuals with DPN and DM type 1 record of no more than 5 years. Group DPN-included 94 patients with DM type 1 record of more than 10 years but without clinical signs of DPN. Fisher's exact test revealed significant differences in allele and genotype frequencies for the two groups. Higher frequencies of SOD2 allele Val and genotype Val/Val and of SOD3 allele Arg and genotype Arg/Arg were established for group DPN+. On this evidence, SOD2 and SOD3 were associated with DPN in DM type 1.     

Relation of CuZnSOD activity with renal insufficiency in hypertensive diabetic patients

Diabetes and renal insufficiency are interrelated metabolic disorders closely associated with redox homeostasis disturbances. The aim of this study was to compare the activity of copper zinc superoxide dismutase (CuZnSOD) in the erythrocytes of hypertensive diabetic patients with or without renal insufficiency with normal healthy control subjects. In both groups of diabetic patients, blood glucose level and the content of glycosylated hemoglobin (HbA1c) were higher than in the control group. However, CuZnSOD activity was significantly higher than control only in hypertensive diabetic patients with renal insufficiency. Our results suggest that disturbances in superoxide homeostasis do correlate with long-term complication in diabetes, i.e. diabetic renal insufficiency and hypertension.     

Hydrogen peroxide and peroxynitrite enhance Ca2+ mobilization and aggregation in platelets from type 2 diabetic patients

Cytosolic Ca²⁺ mobilization, especially Ca²⁺ entry, is enhanced in platelets from type 2 diabetic individuals, which might result in platelet hyperaggregability. In the present study, we report an increased oxidant production in resting and stimulated platelets from diabetic donors. Pretreatment of platelets with catalase or trolox, an analog of vitamin E, reversed the enhanced Ca²⁺ entry, evoked by thapsigargin plus ionomycin or thrombin, observed in platelets from diabetic subjects, so that in the presence of these scavengers Ca²⁺ entry was similar in platelets from healthy and diabetic subjects. In contrast, mannitol was without effect on Ca²⁺ mobilization. Catalase and trolox reduced thrombin-induced aggregation in platelets from type 2 diabetic subjects, while mannitol did not modify thrombin-induced platelet hyperaggregability. We conclude that H₂O₂ and ONOO⁻ are likely involved in the enhanced Ca²⁺ mobilization observed in platelets from type 2 diabetic patients, which might lead to platelet hyperactivity and hyperaggregability.     

The role of oxidative stress after retinal laser photocoagulation in nonproliferative diabetic retinopathy

Diabetic retinopathy (DR) represents the most common chronic complication of diabetes, and it is the leading cause of new cases of blindness in patients between 20-74 years old in developed countries. Laser photocoagulation (LF) represents an efficacious approach to the treatment of DR. Oxidative factors, such as free radicals (FR), are continuously generated in aerobic organisms as a result of different metabolic processes. It is well known that oxidative stress plays a role in the development of DR. The aim of this study was to evaluate the thermal effects of the scatter retinal laser photocoagulation technique on the production of FR. A total of 90 patients were enrolled in this study. They were divided in 3 groups: 30 diabetic patients with DR, 30 diabetic patients without DR, and 30 control individuals without diabetes mellitus (DM). Full scatter retinal LF was performed in all patients with DR. We measured the concentrations of superoxide dismutase (SOD), glutathione peroxidase (GPOD), catalase, and total antioxidative status (TAS). Of the 30 DR patients, 13 showed the appearance or worsening of macular edema after LEF, whereas the other 17 patients showed no change. Thirty days after LF, improvement in visual acuity was observed, but this change was not statistically significant. The mean plasma or erythrocyte lysate concentrations of various antioxidants were significantly lower in the diabetic patients without DR compared to the individuals without DM and in the diabetic patients with DR compared to the individuals without DM; the diabetic patients with DR did not show lower concentrations of the antioxidants compared to the diabetic patients without DR. The concentrations of SOD, GPOD, catalase, and TAS were significantly lower in the diabetic patients with DR after retinal scatter LF, which could be the consequence of retinal oxidative stress caused by the LF thermal effect.     

SIRT3 deficiency delays diabetic skin wound healing via oxidative stress and necroptosis enhancement

Sirtuin 3 (SIRT3) plays a vital role in several dermatological diseases. However, the role and detailed mechanism of SIRT3 in diabetic wound healing are unknown well yet. To explore possible involvement of SIRT3 and necroptosis in diabetic skin wound healing, SIRT3 knockout (KO) mice and 129S1/SvImJ wild‐type (WT) mice were injected with streptozotocin (STZ), and mice skin fibroblasts were exposed to high glucose (HG). It was found that SIRT3 expression decreased in the skin of diabetic patients. SIRT3 deficiency delayed healing rate, reduced blood supply and vascular endothelial growth factor expression, promoted superoxide production, increased malondialdehyde (MDA) levels, decreased total antioxidant capacity (T‐AOC), reduced superoxide dismutase (SOD) activity and aggravated ultrastructure disorder in skin wound of diabetic mice. SIRT3 deficiency inhibited mice skin fibroblasts migration with HG stimulation, which was restored by SIRT3 overexpression. SIRT3 deficiency also suppressed α‐smooth muscle actin (α‐SMA) expression, enhanced superoxide production but decreased mitochondrial membrane potential with HG stimulation after scratch. SIRT3 deficiency further elevated receptor‐interacting protein kinase 3 (RIPK3), RIPK1 and caspase 3 expression both in vitro and in vivo. Collectively, SIRT3 deficiency delayed skin wound healing in diabetes, the mechanism might be related to impaired mitochondria function, enhanced oxidative stress and increased necroptosis. This may provide a novel therapeutic target to accelerate diabetic skin wound healing.     

Arterial hypertension exacerbates oxidative stress in early diabetic retinopathy

The present study was undertaken to investigate the redox status in the retina of an experimental model that combines hypertension and diabetes. Spontaneously hypertensive rats (SHR) and their control Wystar Kyoto (WKY) rats were rendered diabetic and, after 20 days, the rats were sacrificed and the retinas collected. The superoxide production was higher in diabetic than in control WKY (p < 0.03) and SHR rats showed elevated superoxide production compared with WKY groups (p < 0.009). The glutathione antioxidant system was diminished only in diabetic SHR (p < 0.04). Tirosyne nitration was higher in diabetic WKY and control SHR compared with control WKY (p < 0.03), and further increment was observed in diabetic SHR (p < 0.02). The DNA damage estimated by immunohystochemistry for 8-OHdG was higher in control SHR than in WKY, mainly in diabetic SHR (p < 0.0001). Hypertension aggravates oxidative-induced cytotoxicity in diabetic retina due to increasing of superoxide production and impairment of antioxidative system.     

Insulin-induced impairment via peroxynitrite production of endothelium-dependent relaxation and sarco/endoplasmic reticulum Ca(2+)-ATPase function in aortas from diabetic rats

We designed this study to determine whether a high insulin level and a diabetic state need to exist together to cause an impairment of endothelium-dependent relaxation. In diabetic rat aortas organ-cultured with insulin [vs both control rat aortas cultured with insulin and diabetic rat aortas cultured in serum-free medium]: (1) the relaxation responses to both acetylcholine (endothelium-dependent relaxation) and Angeli's salt (nitric oxide donor) were significantly weaker, (2) acetylcholine-stimulated nitric oxide production was significantly smaller, (3) superoxide and nitric oxide production into the culture medium was greater, and (4) the levels of both nitrotyrosine and tyrosine-nitrated sarco/endoplasmic reticulum calcium ATPase (SERCA) protein were greater. The insulin-induced effects were prevented by cotreatment with either a superoxide scavenger or a peroxynitrite scavenger. After preincubation with an irreversible SERCA inhibitor, the relaxation induced by the nitric oxide donor was significantly impaired in control aortas cultured with or without insulin and in diabetic aortas cultured without insulin, but not in diabetic aortas cultured with insulin. These results suggest that the coexistence of a high insulin level and an established diabetic state may lead to an excessive generation of peroxynitrite, and that this may in turn trigger an impairment of endothelium-dependent relaxation via a decrease in SERCA function.     

Evaluation of oxidative stress markers in pathogenesis of diabetic neuropathy

Experimental evidences suggest that hyperglycaemia-induced overproduction of reactive oxygen species and subsequent damage to proteins, lipids and DNA may play a key role in the development of distal symmetric polyneuropathy (DSPN)-the most common complication of diabetes mellitus. The study population consisted of 51 individuals aged 52-82 years classified into 3 groups: 16 patients diagnosed with type 2 diabetes mellitus (T2DM) with DSPN, 16 T2DM patients without DSPN and 19 control subjects without diabetes and neuropathy. The study was conducted to determine the activity of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPX) and total antioxidant status (TAS) in the examined groups. An alkaline comet assay was used to determine the extent of DNA damage of oxidized purines as glicosylo-formamidoglicosylase (Fpg) sites, and oxidized pyrimidines as endonuclease III (Nth) sites. A significant decrease of SOD (P < 0.05), GPX (P < 0.05) and nonsignificant decrease of CAT (P > 0.05), and TAS status (P > 0.05) were seen in T2DM patients with neuropathy compared to T2DM patients as well as controls. T2DM patients with or without neuropathy revealed significantly lower (P < 0.05) plasma concentration of nitrous oxide compared to the control subjects. Endogenous level of oxidative DNA damage in T2DM patients with DSPN was significantly higher compared both to the controls and T2DM patients without DSPN (P < 0.001). Moreover, lymphocytes isolated from T2DM patients with DSPN were more susceptible to oxidative DNA lesions induced by hydrogen peroxide than from T2DM patients without DSPN (P < 0.001). Our results confirm hypothesis that oxidative stress may play a substantial role in the development and progression of diabetic distal symmetric polyneuropathy.     

Lack of association between an ecNOS gene polymorphism and diabetic nephropathy in type 2 diabetic patients with proliferative diabetic retinopathy.

The development of diabetic nephropathy shows remarkable variation among individuals. Therefore, not only hyperglycemia but also genetic factors may contribute to the development of diabetic nephropathy. The aim of the present study was to examine the contribution of the 27-bp repeat polymorphism in intron 4 of the endothelial constitutive nitric oxide synthase gene (ecNOS4) to the development of diabetic nephropathy. For this purpose, we analyzed this polymorphism in 167 Japanese type 2 diabetic patients with proliferative diabetic retinopathy consisting of 102 patients with diabetic nephropathy (with macroalbuminuria) and 65 patients without diabetic nephropathy (with normoalbuminuria). The genotype and allele frequencies were not significantly different between patients with diabetic nephropathy and those without diabetic nephropathy (ecNOS4 "b/b" 79.4% vs. 84.6%, ecNOS4 "b/a" 20.6% vs. 15.4%, "b" allele 89.7% vs. 92.3%, "a" allele 10.3% vs. 7.7%). We conclude that the ecNOS4 polymorphism does not contribute to the development of diabetic nephropathy.