Sohji Nagase

Association of ecNOS gene polymorphisms with end stage renal diseases

Nitric oxide (NO) is a very potent regulator of intrarenal hemodynamics and is thought to be an important factor in the deterioration of renal function. Several polymorphisms of the endothelial NO synthase (eNOS) gene have been reported. For instance, tandem 27-bp repeats in intron 4 of the eNOS gene are polymorphic, i.e. eNOS4a allele has 4 and eNOS4b has 5 tandem repeats, and the association between eNOS4a and myocardial infarction has been reported. In addition, a missense Glu298Asp mutation in exon 7 of the eNOS gene is reported to be a risk factor for hypertension or myocardial infarction. In this study, we investigated the frequencies of these 2 polymorphisms of eNOS gene in patients with end-stage renal diseases (ESRD), and compared them with those of healthy subjects.Genomic DNA was obtained from regularly hemodialyzed patients and healthy volunteers. The allele frequencies of eNOS4a and eNOS4b in intron 4 were analyzed by PCR and the missense Glu298Asp mutation in exon 7 were determined by PCR FMLP analysis.The allele frequency of eNOS4a (eNOS4a/b and eNOS4a/a) in non-diabetic group is significantly higher than that in healthy controls (27.3% vs. 19.0%, p = 0.01) though there is no significant difference between diabetic group and healthy controls. On the other hand, the frequencies of missense Glu298Asp mutation in both non-diabetic and diabetic groups are significantly higher than that in healthy controls (22.5% in non-diabetic, 20.8% in diabetic and 7.4% in control group, p = 0.002: non-diabetic vs. control, p = 0.01: diabetic vs. control).This study clarified that the polymorphisms in intron 4 and exon 7 of eNOS gene are the genetic risk factors for ESRD. The polymorphisms in intron may change the transcriptional activity and those in exon may alter the 3 dimensional structure of the enzyme, and may affect the progression of renal diseases via decreased NO synthesis. Further study is required to clarify the detailed mechanisms.

Stable nitroxide Tempol ameliorates brain injury by inhibiting lipid peroxidation in a rat model of transient focal cerebral ischemia.

Oxygen free radicals have been implicated in the pathogenesis of cerebral ischemia and reperfusion injury. 4-Hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (Tempol) has been reported as a stable nitroxide and a membrane-permeable free radical scavenger. This study was performed to investigate the mechanism of Tempol in attenuating ischemia-reperfusion injury in a rat model of transient focal cerebral ischemia. We measured the cerebral 2,3-dihydroxybenzoic acid (DHBA) level as the amount of hydroxyl radical production using a microdialysis technique with salicylic acids trapping during ischemia and reperfusion. The concentration of cerebral thiobarbituric acid reactive substances (TBARS), representing the extent of lipid peroxidation by free radicals, and the area of cerebral infarction were also measured. The level of cerebral 2,3-DHBA was increased during ischemia and reperfusion, especially during the early reperfusion stage at the periphery of the infarct area (nearly 500-fold). Intravenous administration of Tempol at the time of reperfusion reduced 2,3-DHBA production (Vehicle group: 472.2+/-196.2, Tempol group: 238.3+/-77.2) and the cerebral TBARS level (Vehicle group: 541.7+/-84.7, Tempol group: 339.0+/-147.2 nmol/g), and decreased the size of the cerebral infarction (Vehicle group: 202.2+/-98.4, Tempol group: 98.5+/-13.7 mm(3)). In contrast, Tempol administered 15 min prior to reperfusion reduced neither the TBARS level nor the size of the infarction. These results indicate that Tempol administration at the time of reperfusion reduced lipid peroxidation by scavenging free radicals, resulting in a reduction of the infarct size.

Biosynthesis of Methylguanidine in Isolated Rat Hepatocytes and in vivo

To clarify the organ in which methylguanidine is synthesized, high doses of creatinine, which is known to stimulate the synthesis of methylguanidine, were administered to male Wistar rats intraperitoneally. Various tissues of the rats were frozen by a freeze clamp method before and 1, 2 and 3 h after injection, and methylguanidine was determined by high-pressure liquid chromatography using 9,10-phenanthrenequinone for fluorometric determination. We found evidence that the liver, kidney, lung, muscle, red blood cells and gut flora synthesize methylguanidine. In addition, we measured the synthesis of methylguanidine in isolated hepatocytes prepared from normal rats following the addition of creatinine, arginine and guanidinoacetic acid to the incubation medium. Synthesis of methylguanidine was observed only in those incubations which contained creatinine, and was dependent on the concentration of creatinine in the media and on the incubation period. Isolated rat hepatocytes also synthesized guanidine in the presence of guanidinoacetic acid. These results indicate that the liver is one of the organs which synthesize methylguanidine and also that creatinine is the precursor.

Active oxygen in methylguanidine synthesis.

Methylguanidine (MG), a toxin reported in uremia, is thought to be a product of creatinine oxidation. This study is designed to demonstrate the role of active oxygen in the oxidation of creatinine under conditions compatible with those found in uremia. MG synthesis is moderately stimulated by the superoxide radical derived from 3 mM hypoxanthine and 0.015 units/ml xanthine oxidase and inhibited by the addition of superoxide dismutase. This is increased markedly by the addition of 0.05% hydrogen peroxide and augmented to about 56,000 times the control rate in the presence of hydroxyl radicals derived from the reaction of 10 mM FeSO4 and 0.05% hydrogen peroxide. In addition, MG synthesis is inhibited by the addition of sorbitol, lactulose or ethanol, the scavengers of hydroxyl radicals. These results indicate that creatinine can be oxidized to MG by various species of active oxygen and that one of the mechanisms of MG synthesis is such oxidation. MG, therefore, may be a useful indicator of peroxidation in vivo.

Hemodialysis Does Not Influence the Peroxidative State Already Present in Uremia

Hemodialysis (HD) patients are exposed to high oxidative stress, however, the nature of this stress is still unclear. In this study, we employed a specific lipid peroxidative product, phosphatidylcholine hydroperoxide (PCOOH), and evaluated the peroxidative effect of end stage renal disease by measuring thiobarbituric acid reactive substances (TBARS) and PCOOH in both plasma and erythrocyte membrane. We also surveyed plasma TBARS and PCOOH before and after HD sessions thereby assessing oxidative stress by a single HD procedure. The plasma TBARS level of healthy controls was 2.9 ± 0.4 nmol/ml. Those of HD patients before and after HD session were 5.1 ± 1.4 and 3.1 ± 0.5 nmol/ml, respectively, and the pre-HD plasma TBARS levels were significantly higher than those of controls and after HD. The Plasma PCOOH concentration of patients before HD was 119.7 ± 58.4 pmol/ml and was significantly higher than that of controls which was 88.6 ± 14.3 pmol/ml. After HD, the plasma PCOOH level decreased to 103.2 ± 36.0 pmol/ml, which was still significantly higher than that of controls. In erythrocytes, the PCOOH level of patients was 259.3 ± 105.4 nmol/g RBC and was significantly higher than that of controls with 88.6 ± 32.0 nmol/g RBC. Analyzed with respect to the cause of renal disease, the polycystic kidney disease patients showed significantly lower plasma PCOOH levels than the others. These results suggest that there is an increase of lipid peroxidation in both plasma and erythrocytes of HD patients, though this oxidative stress was not brought about by HD.

FORMATION OF GUANIDINOSUCCINIC ACID, A STABLE NITRIC OXIDE MIMIC, FROM ARGININOSUCCINIC ACID AND NITRIC OXIDE-DERIVED FREE RADICALS

Guanidinosuccinic acid (GSA) is noted for its nitric oxide (NO) mimicking actions such as vasodilatation and activation of the N-methyl-D-aspartate (NMDA) receptor. We have reported that GSA is the product of argininosuccinate (ASA) and some reactive oxygen species, mainly the hydroxyl radical. We tested for GSA synthesis in the presence of NO donors. ASA (1 mM) was incubated with NOR-2, NOC-7 or 3-morpholinosydomine hydrochloride (SIN-1) at 37 degrees C. GSA was determined by HPLC using a cationic resin for separation and phenanthrenequinone as an indicator. Neither NOR-2 or NOC-7 formed GSA. SIN-1, on the other hand, generates NO and the superoxide anion which, in turn, generated peroxynitrite which was then converted to the hydroxyl radical. Incubation of ASA with SIN-1 leads, via this route, to GSA. When ASA was incubated with 1 mM SIN-1, the amount of GSA produced depended on the incubation time and the concentration of ASA. Among the tested SIN-1 concentrations, from 0.5 to 5 mM, GSA synthesis was maximum at 0.5 mM and decreased with increasing concentrations of SIN-1. Carboxy-PTIO, a NO scavenger, completely inhibited GSA synthesis. SOD, a superoxide scavenger, decreased GSA synthesis by 20%, and catalase inhibited GSA synthesis only by 12%; DMSO, a hydroxyl radical scavenger completely inhibited GSA synthesis in the presence of SIN-1. These data suggest that the hydroxyl radical derived from a combination of NO and the superoxide anion generates GSA, a stable NO mimic. Meanwhile, synthesis of GSA by NO produces reactive oxygen and activates the NMDA receptor that generates NO from GSA, suggesting a positive feed back mechanism.

Antioxidant capacity in rat skeletal muscle tissues determined by electron spin resonance

The amount of radical scavenging activity in muscle is unknown. The present study examines whether electron spin resonance (ESR) could measure and distinguish antioxidant capacity in muscle with different contractile and metabolic characteristics. Specimens of the soleus, plantaris, gastrocnemius (deep/surface portions), heart and diaphragm were obtained from female Wistar rats (n=7; 12 weeks old). Scavenging activity against superoxide anions in these specimens were determined by ESR using a spin-trapping chemical (5,5-dimethyl-1-pyrroline-N-oxide). The ESR signal intensity of reaction mixtures containing muscle tissues was significantly lower in the heart, soleus, diaphragm and deep portion of the gastrocnemius than in the plataris and surface portion of the gastrocnemius. Thus, the amount of scavenging activity converted into superoxide dismutase activity was the highest in the heart, and higher in the soleus, diaphragm and deep portion of the gastrocnemius than in other muscles (ANOVA, P<0.01). In addition, scavenging activity significantly correlated with citrate synthase activity (r=0.72, P<0.01, n=42) and myoglobin content (r=0.63, P<0.01, n=42). These findings suggested that ESR and spin-trapping can be detect differences in free radical scavenging activity among muscle tissues with different metabolic characteristics.

Spherical Carbon Adsorbent (AST-120) Protects Deterioration of Renal Function in Chronic Kidney Disease Rats through Inhibition of Reactive Oxygen Species Production from Mitochondria and Reduction of Serum Lipid Peroxidation

Background/Aim: An imbalance in renal redox status contributes to progression of renal dysfunction. We investigated the effects of an oral charcoal adsorbent (AST-120) on renal redox status, superoxide production from renal mitochondria, and serum lipid peroxidation using chronic kidney disease (CKD) model rats. Methods: CKD was induced by 5/6 nephrectomy. CKD rats were divided into 2 groups: controls, and those treated with AST-120 for 20 weeks. We evaluated: (1) renal redox status by in vivo low-frequency electron spin resonance imaging (EPRI); (2) renal superoxide scavenging activity (SSA); (3) superoxide production from renal mitochondria; (4) immunostaining for Cu-Zn superoxide dismutase (SOD), and (5) oxidative stress markers including LDL-negative charge (LDL-CMF), serum lipid peroxide (LPO) and urinary hexanoyl-lysine (HEL). The effect of indoxyl sulfate, a uremic toxin, on mitochondrial superoxide production was also investigated. Results: AST-120 treatment improved renal function, renal SSA, renal mitochondrial superoxide production, renal SOD expression, renal redox status by EPRI, and oxidative stress profiles by LDL-CMF, LPO and urinary HEL. Addition of indoxyl sulfate increased mitochondrial superoxide production and AST-120 also decreased this. Conclusions: Improvements in the redox status and lipid peroxidation induced by AST-120 may delay the progression of CKD.

The antioxidant EPC-K1 ameliorates brain injury by inhibiting lipid peroxidation in a rat model of transient focal cerebral ischaemia

Summary¶Background. Cerebral ischaemia-reperfusion injury is associated with the generation of reactive oxygen species during the early phases of reoxygenation. EPC-K1, a phosphate diester of vitamins C and E, has been reported to possess potent hydroxyl radical scavenging activity. This study was performed to investigate the effectiveness of EPC-K1 in attenuating cerebral ischaemia-reperfusion injury in a rat model of transient focal cerebral ischaemia. Method. We evaluated the efficacy of EPC-K1 by measuring the concentration of cerebral thiobarbituric acid reactive substances (TBARS), an indicator of the extent of lipid peroxidation by free radicals, and infarct size in rats subjected to one hour of cerebral ischaemia and 4, 24, or 72 hours of reperfusion. Findings. EPC-K1 significantly reduced both the cerebral TBARS level and the infarct size in a rat model of transient focal cerebral ischaemia. These results indicate that EPC-K1 administration during the early stages of reperfusion ameliorates ischaemic brain injury by inhibiting lipid peroxidation. Interpretation. This report is the first to describe the protective mechanism of EPC-K1 by measuring both the TBARS level and infarct size in a rat model of transient focal cerebral ischaemia, and may suggest a potential clinical approach for the treatment of ischaemic cerebrovascular disease.

Glomerular crescents predominantly express cadherin–catenin complex in pauci-immune-type crescentic glomerulonephritis

Aims:  To investigate the expression of the cadherin complex in human crescentic glomerulonephritis to elucidate the role of intercellular adherens junction molecules in crescent formation.