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Leukocyte activation and the resulting oxidative stress induced by bioincompatible materials during hemodialysis impact the prognosis of patients. Despite multiple advances in hemodialysis dialyzers, the prognosis of hemodialysis patients with complications deeply related to oxidative stress, such as diabetes mellitus, remains poor. Thus, we re-evaluated the effects of hemodialysis on multiple reactive oxygen species using electron spin resonance-based methods for further improvement of biocompatibility in hemodialysis. We enrolled 31 patients in a stable condition undergoing hemodialysis using high-flux polysulfone dialyzers. The effects of hemodialysis on reactive oxygen species were evaluated by two methods: MULTIS, which evaluates serum scavenging activities against multiple hydrophilic reactive oxygen species, and i-STrap, which detects lipophilic carbon-center radicals. Similar to previous studies, we found that serum hydroxyl radical scavenging activity significantly improved after hemodialysis. Unlike previous studies, we discovered that scavenging activity against alkoxyl radical was significantly reduced after hemodialysis. Moreover, patients with diabetes mellitus showed a decrease in serum scavenging activity against alkyl peroxyl radicals and an increase in lipophilic carbon-center radicals after hemodialysis. These results suggest that despite extensive improvements in dialyzer membranes, the forms of reactive oxygen species that can be eliminated during dialysis are limited, and multiple reactive oxygen species still remain at increased levels during hemodialysis.

To clarify the clinical significance of the redox-controlling effects of Kampo, a traditional Japanese herbal medicine, we determined the scavenging activities of various reactive oxygen species in clinically used Kampo formulae using an electron spin resonance-based technique. Formulae containing Rhei Rhizoma (i.e., mashiningan and daiobotanpito) showed high scavenging activity against the alkoxyl radical, and crude extract quantity was significantly correlated with scavenging activity. Hydroxyl radical scavenging activity was positively correlated with the quantity of Zingiberis Rhizoma. Strong hydroxyl radical scavenging activity was also found in formulae containing both Bupleuri Radix and Scutellariae Radix, a widely used anti-inflammatory combination. Formulae containing a clinically common combination of Scutellariae Radix, Coptidis Rhizoma, and Phellodendri Cortex induced high superoxide scavenging activity. Singlet oxygen scavenging activity was high in formulae containing Bupleuri Radix and Glycyrrhizae Radix. In contrast, formulae containing Rehmanniae Radix showed generally low reactive oxygen species scavenging activities, and the quantity of Rehmanniae Radix was negatively correlated with hydroxyl radical and singlet oxygen scavenging activities. These results indicate that the antioxidative effects of Kampo formulae are not uniform but complexly varied against multiple reactive oxygen species. Some formulae have almost no antioxidant effects but may act as pro-oxidants.

Kangen-karyu, a prescription containing six herbs, has been shown to achieve its pharmacological effect through oxidative stress-dependent pathways in animal models. The aim of this study is to investigate the relationship between the antioxidative effect and pharmacological mechanisms of Kangen-karyu, specifically its body temperature elevating effect in humans. Healthy human volunteers, age 35 ± 15 years old, were enrolled in this study. Surface body temperature, serum nitrite, reactive oxygen species (ROS) scavenging activities, and inflammatory cytokines were investigated before and 120 min after Kangen-karyu oral intake. Kangen-karyu significantly increased the surface-body temperature of the entire body; this effect was more remarkable in the upper body and continued for more than 120 min. Accompanying this therapeutic effect, serum nitrite levels were increased 120 min after oral administration. Serum ROS scavenging activities were enhanced against singlet oxygen and were concomitantly decreased against the alkoxyl radical. Serum nitrite levels and superoxide scavenging activities were positively correlated, suggesting that Kangen-karyu affects the O2 (•-)-NO balance in vivo. Kangen-karyu had no effect on IL-6, TNF-α and adiponectin levels. These results indicate that the therapeutic effect of Kangen-karyu is achieved through NO- and ROS-dependent mechanisms. Further, this mechanism is not limited to ROS production, but includes ROS-ROS or ROS-NO interactions.

Oketsu is a characteristic condition that plays an important role in Kampo, Japanese traditional medicine, and includes multiple aspects of hemodynamic disorders. This study aims to clarify the microcirculation of Oketsu and the pharmacological effect of Keishibukuryogan, an anti-Oketsu Kampo prescription, using live imaging techniques. Oral administration of Keishibukuryogan induced significant vasodilation of murine subcutaneous arterioles compared to the preadministration level. This vasodilatation peaked 60 min after administration and persisted for 90 min. The blood velocity in the subcutaneous capillary was also increased by Keishibukuryogan in generally the same manner. In rat mesenteric arterioles, Keishibukuryogan administration improved microhemodynamic parameters, including the resolution of erythrocyte congestion and the cell-free layer, which are representative of Oketsu pathology. Live imaging revealed an increase of diaminofluorescein-2 diacetate fluorescence, a nitric oxide (NO) specific reagent, in the arterial endothelium following Keishibukuryogan administration. This fluorescence was most remarkable at vascular bifurcations but was present throughout the mesenteric arterioles. This study demonstrates the successful imaging of Oketsu pathology with respect to microcirculation and the anti-Oketsu effects of Keishibukuryogan, namely, vasodilation of arterioles, increased blood velocity, and resolution of erythrocyte congestion. The anti-Oketsu effect of Keishibukuryogan is related to endothelial NO production.

Exercise program for patients with chronic kidney disease currently recognized as a useful method to improve multiple factors of patients’ condition, however there still remains systemic risks including cardiovascular events. We examined the risk of oxidative stress brought by the intra-dialysis exercise program using electron spin resonance-based multiple radical scavenging (MULTIS) method. Thirty-nine stable hemodialysis (HD) patients of multiple dialysis centers were examined. The renal rehabilitation program was aerobic training using the lower limb ergo-meter twice a week during HD, with Borg scale 11 to 13 and the work time was 15 to 60 min. Eleven patients of them continued the program up to 9 mo. Oxidative stress was evaluated by MULTIS method measuring serum scavenging activities against five reactive oxygen species. Inflammatory cytokines were also measured. The patients received the renal rehabilitation showed significant increase of daily activity, exercise tolerance and muscular strength, accompanied with decreases of systolic blood pressure, LDL and HDL, and an improvement of HD efficiency (Kt/V). The serum alkylperoxyl radical (ROO•) and hydroxyl radical (•OH) scavenging activities were significantly increased by the exercise program within the 3 and 9 mo., respectively. To the contrary, superoxide (O2•‒) and alkoxyl radical (RO•) scavenging activities was decreased. An increase of TNF-a was detected in the patients with the exercise program. No severe unfavorable effects were confirmed during the program. These results indicate that the effects of renal rehabilitation are mostly recognized as favorable, while decreases of O2•‒ and RO• scavenging activities accompanied with an increase of TNF-a should be concerned. The oxidative stress related responses of HD patients are different from healthy subjects.

Patients with advanced-stage chronic renal failure (CKD) are exposed to strong oxidative stress, and hemodialysis (HD) is considered to improve this. Multiple reports have been published on this topic, including our previous reports (J Am Soc Nephrol1997). However, recent advances have revealed that the oxidative stress in CKD does not simply increase unidirectionally. By using the newly developed electron spin resonance (ESR)-based analytical methods, we reevaluated the effects of HD on the oxidative status of patients with CKD. Two methods, namely multiple free radical scavenging assay (MULTIS), which evaluates scavenging activities against multiple reactive oxygen species (ROS) using CYPMPO as a spin trap, and iStrap, which is a sensitive method to detect lipid-derived carbon-center radicals using a spin trapDPhPMPO, were used in this study. Thirty-three patients undergoing maintenance HDwere enrolled. Informed consent was obtained before the trial. Thirteen cases were complicated with type 2 diabetes. Confirming our previous result, serum hydroxyl radical scavenging activity was significantly improved after HD. By contrast, scavenging activities against alkoxyl and alkylperoxyl radicals were significantly reduced after HD, which suggests a deterioration of the radical scavenging system downstream of the radical chain reaction. These reductions in scavenging activities were more obvious in patients with diabetes. The changes in the signal intensity of DPhPMPO in the whole tested group varied, whereas that of the diabetic patients was increased after HD, consistent with the alkylperoxyl radical findings. The superoxide scavenging activity was higher in the CKD subjects than that in the non-CKD subjects before HD, which then decreased after HD. These results indicate that the HD process does not simply decrease oxidative stress. Although intensive improvements have been made on the materials used in the HD session, such as the dialyzer membranes, ROS downstream of radical chain reactions are still not adequately controlled.

The effect of the calcium channel blocker azelnidipine on the redox status of a murine hypertension model was analyzed and imaged using in vivo low frequency electron paramagnetic resonance (EPR). A murine two kidney-one clip (2K1C) hypertension model was produced by a clipping of the right renal artery. The resulting hypertensive mice were treated with low-dose azelnidipine (1 mg/kg/d), with high-dose azelnidipine (3 mg/kg/d) or without azelnidipine (HT group). An EPR system equipped with a loop-gap resonator and an imaging system was employed. Redox status was evaluated as organ reducing activity measured by means of the decay rate (half-lives) of the spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL). Four weeks after clipping the mice demonstrated hypertension as expected. After the additional 2 weeks of azelnidipine treatments, the Carbamoyl-PROXYL half-lives of the Low and High azelnidipine groups measured in the upper abdominal area were significantly shorter than those of the HT group, suggesting improvements in the reducing activity. The blood pressures of the three groups showed no significant differences at this time, and there was no correlation between the renal reducing activity and either blood pressure or serum creatinine values. EPR imaging studies revealed that the improvement in abdominal reducing activity was mainly recognized in the kidney but not in the liver. These results indicate that azelnidipine ameliorates renal redox status through an improvement in reducing activity independent of blood pressure control.

Creatol (CTL) is a hydroxyl radical adduct of creatinine (Cr). The serum methylguanidine (MG) level and the MG/Cr molar ratio are reported to be biomarkers for oxidative stress. The aim of this study was to examine whether urinary excretion of CTL, another oxidative stress-related marker, is increased in patients with chronic renal failure (CRF). One hundred twenty-four non-dialyzed patients with chronic renal failure (serum Cr level, 1.3-10.0 mg/dL) were recruited from our hospitals. Urine and serum levels of CTL and MG were determined by high-performance liquid chromatography with the use of 9, 10- phenanthrenequinone as a fluorogenic reagent. The CTL/Cr and (CTL+MG)/Cr molar ratios in spot urine samples were also compared with those in 24-h urine samples. The urinary CTL/Cr and (CTL+MG)/Cr molar ratios increased with decreases in Cr clearance in patients with CRF. Correlations between serum and spot urine (CTL+MG)/Cr and between serum and spot urine CTL/Cr were quite similar to those in 24-h urine samples. CTL/Cr and (CTL+MG)/Cr molar ratios in both 24-h urine and spot urine samples appear to be useful indices of the severity of CRF.

We measured the hydroxyl radical (.OH) generation in fourteen patients undergoing coronary artery bypass grafting (CABG), of whom seven patients underwent on-pump CABG with cardiopulmonary bypass (CPB) and seven patients underwent off-pump CABG without CPB. To detect .OH generation, we measured the urinary excretion of .OH products of creatinine (Cr), creatol (CTL; 5-hydroxycreatinine) and methylguanidine (MG) with HPLC using the one point sampling and collected urine during and after the operation. The urinary CTL value corrected urinary Cr value of on-pump CABG significantly increased about 3-5 times from the beginning of CPB to 4 h after operation compared to the baseline value before CPB in both the collected urine and the one point sampling urine. The urinary MG/Cr value in both groups did not change significantly. Significantly increased .OH generation was found during and soon after on-pump CABG.

We investigated the protective effects of Glycyrrhizae Radix extract against peroxynitrite (ONOO-)-induced oxidative stress under in vivo as well as in vitro conditions. The extract showed strong ONOO- and nitric oxide (NO) scavenging effects under in vitro system, in particular higher activity against ONOO-. Furthermore, elevations of plasma 3-nitrotyrosine levels, indicative of in vivo ONOO- generation and NO production, were shown using a rat in vivo ONOO(-)-generation model of lipopolysaccharide injection plus ischemia-reperfusion. The administration of Glycyrrhizae Radix extract at doses of 30 and 60 mg/kg body weight/day for 30 days significantly reduced the concentrations of 3-nitrotyrosine and NO and decreased inducible NO synthase activity. In addition, the nitrated tyrosine protein level and myeloperoxidase activity in the kidney were significantly lower in rats given Glycyrrhizae Radix extract than in control rats. However, the administration of Glycyrrhizae Radix extract did not result in either significant elevation of glutathione levels or reduction of lipid peroxidation in renal mitochondria. Moreover, the in vivo ONOO- generation system resulted in renal functional impairment, reflected by increased plasma levels of urea nitrogen and creatinine, whereas the administration of Glycyrrhizae Radix extract reduced these levels significantly, implying that the renal dysfunction induced by ONOO- was ameliorated. The present study suggests that Glycyrrhizae Radix extract could protect the kidneys against ONOO- through scavenging ONOO- and/or its precursor NO, inhibiting protein nitration and improving renal dysfunction caused by ONOO-.

Relation between anemia resistant to recombinant human erythropoietin (rHuEPO) therapy and the oxidative stress in hemodialysis (HD) patients was investigated. Stable HD patients who had consistent hemoglobin concentrations on a constant dose of rHuEPO were studied. Patients were excluded if there were factors that might affect hemopoiesis or administration of antioxidant supplements. Patients were classified into three groups: High (9000 U/week), Low (1500-4500 U/week) and No rHuEPO group. Thiobarbituric acid reactive substances (TBARS) of sera and erythrocyte were examined. Serum superoxide and hydroxyl radical scavenging activities were measured using electron spin resonance. TBARS in the erythrocyte was higher in High rHuEPO group compared with No rHuEPO group, though the serum TBARS were similar. A diminution of serum hydroxyl radical scavenging activity was observed in High rHuEPO group. Hydroxyl radical signal intensity showed a strong correlation with the serum ferritin in High rHuEPO group, although ferritin concentrations were not different among the 3 groups. Superoxide scavenging activity showed no differences. These results indicate that increased lipid peroxidation in erythrocyte, raised by decreased serum hydroxyl radical scavenging activity, is one cause of rHuEPO resistant anemia. Serum ferritin may be involved in this hydroxyl radical production.

Synthesis of guanidinosuccinic acid (GSA) and methylguanidine (MG) is markedly increased in end-stage renal disease (ESRD), and these substances have been known as uremic toxins.

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.

Puromycin aminonucleoside (PAN) has been known to induce proteinuria. The increased generation of reactive oxygen species (ROS) has been implicated in this toxicity of PAN. We have reported that PAN increases the synthesis of methylguanidine (MG) and creatol which are the products of the reaction of creatinine and the hydroxyl radical in isolated rat hepatocytes. However, the mechanism for the increased ROS induced by PAN is still unclear. In this paper, we investigate the role of protein kinase C (PKC) on the PAN induced reactive oxygen generation in isolated rat hepatocytes. Isolated hepatocytes were incubated in Krebs-Henseleit bicarbonate buffer containing 3% BSA, 16.6 mM creatinine and tested reagents. MG and creatol were determined by high-performance liquid chromatography using 9,10-phenanthrenequinone for the post-labeling. PAN increased MG and creatol synthesis in isolated rat hepatocytes by 60%. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7), a PKC inhibitor, at 10 and 100 microM significantly inhibited MG and creatol synthesis with or without PAN. The inhibition rate is dose dependent from 10 to 100 microM. H1004, a reagent used as control for H-7, did not affect (at 10 microM) or increased little (at 100 microM) the synthesis of MG and creatol. Ro31-8425, a potent PKC inhibitor, significantly inhibited (at 10 microM) MG synthesis in the presence of PAN. PKC in the membrane fraction, a marker of PKC activation, increased over the initial concentration by a factor of 1.65-fold at 60 min incubation and 2.16-fold at 120 min with PAN, while it changed little without PAN. These results indicate that PAN activates PKC resulting in increased hydroxyl radical generation in isolated rat hepatocytes.

Creatol (CTL) is a product resulting from the reaction of creatinine (Cr) with the hydroxyl radical and is identified as a precursor of methylguanidine (MG), a uremic toxin. In this study, we investigated serum CTL levels together with those of Cr and MG in 66 patients who were on maintenance hemodialysis (HD). Prior to dialysis, the mean serum levels of Cr, CTL and MG were 967 (= 11.1 mg/dl)+/-267 microM, 11.1+/-4.8 microM and 5.8+/-2.9 microM, respectively. The mean CTL level was about 1.1% that of Cr, and the CTL plus MG level was about 1.4% that of the Cr level. The reduction rates of Cr, CTL and MG by a single HD were 62.6 6.1%, 71.0+/-10.3% and 51.9+/-11.6%, respectively. The CTL level at 0.5, 1 and 6 h after HD increased rapidly by 20.7+/-8.7%, 31.7+/-14.7% and 80.1+/-27.3%, respectively. There was a significant correlation between CTL or CTL/Cr and parathyroid hormone in patients who had just undergone parathyroidectomy. No significant correlation was found between CTL or CTL/Cr and those factors which seems to be related to the predialysis levels of reactive oxygen. Therefore, because of the good clearance of CTL and its rapid conversion to MG, its usefulness for the estimation of hydroxyl radical generation in HD patients is limited.

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.

A state of peroxidation is one of the factors contributing to uremia. For example, we have reported that certain species of reactive oxygen, particularly the hydroxyl radical, play an important role in the biosynthesis of methylguanidine which contributes to toxicity in patients with uremia. However, it is uncertain which enzymes are involved in the synthesis of methylguanidine from creatinine. In this study, we attempt to show methylguanidine synthesis in the presence of peroxisomal enzymes that catalyze the β-oxidation of fatty acids. In addition, we investigate the effect of clofibrate, which induces peroxisomal enzymes or glutathione peroxidase activity, on methylguanidine synthesis in the peroxisomal fraction. Male Wistar rats were fed with the chow containing 0.5% clofibrate to induce peroxisomal enzymes and control rats were fed with ordinary laboratory chow. Peroxisomal fractions were obtained from liver homogenates by centrifugation, and incubated with creatinine in 0.1 M potassium phosphate buffer pH 7.4 at 37°C. Results show that methylguanidine is synthesized from creatinine concomitant with the synthesis of hydrogen peroxide from endogenous substrates in the peroxisomal fraction. This methylguanidine synthesis is inhibited by the addition of dimethylsulfoxide, glutathione, or sodium azide (p < 0.01). The rate of methylguanidine synthesis in clofibrate-treated rats was significantly less than that in control rats (p < 0.02). These results suggest that methylguanidine is synthesized in the peroxisomal fraction, and reactive oxygen species which are generated through this enzymatic reaction, participate in methylguanidine synthesis. Moreover, the induction of a scavenger system, especially glutathione peroxidase takes precedent over the generation of reactive oxygen species in peroxisomes treated with clofibrate.

Synthesis of guanidinosuccinic acid (GSA), a uremic toxin, has been suggested to relate to the urea concentration and synthetic rate. Among the urea cycle enzymes, inhibition of argininosuccinate (ASA) lyase by urea has been reported. Argininosuccinate which contains a GSA structure is a candidate of a GSA precursor. We found that another uremic toxin, methylguanidine, is formed from creatinine with reactive oxygen species. Therefore, we investigated in vitro whether GSA is formed from ASA with reactive oxygen species. GSA was measured by HPLC by a post-column-labeling method using 9,10-phenathrequinone. When 1 mmol/l ASA was reacted with the hydroxyl radical-generating system for 5 min at pH 7.4, 9 mumol/l GSA was formed. Dimethylsulfoxide, a hydroxyl radical scavenger, markedly inhibited GSA synthesis. The superoxide radical generated by xanthine and xanthine oxidase reaction also formed 1 mumol/l GSA from 1 mumol/l ASA and the GSA formation was inhibited by superoxide dismutase or catalase almost completely. Addition of FeCl2 to the xanthine/xanthine oxidase reaction further increased GSA synthesis. These results indicate that GSA is formed from ASA by reaction with the hydroxyl radical and the superoxide radical.

This study aims to raise the possibility of methylguanidine, a peroxidative product of creatinine, as a measure of the peroxidative state. As a known standard, we measured the inhibitory effect of uremic serum on the NADPH-dependent microsomal lipid peroxidation. This is an established method for evaluating the peroxidative state and is compared to the effect of uremic serum on methylguanidine synthesis. The study shows decreased serum antioxidant activity in hemodialysis patients by both methods, though there is no correlation between them. These results support the use of methylguanidine as a peroxidative marker and suggest a difference in the reactive oxygen species involved in the reactions of methylguanidine synthesis and microsomal lipid peroxidation.

維持血液透析患者においてmorphine製剤投与により意識障害を呈した2例を経験したので報告する. 症例1: 46歳女性. 血液透析歴6か月. 肺癌の多発骨転移による疼痛に対して硫酸morphine徐放錠を20mg/日で投与したところ, 投与3日目で強い眠気を訴え, 4日目に半昏睡状態となった. morphine製剤の投与中止後, 徐々に意識状態は改善したが, 会話が可能となるまで約10日を要した. 症例2: 71歳男性. 血液透析歴5年. 前立腺癌の多発骨転移による疼痛に対して硫酸morphine徐放錠20mg/日を2日間, 10mg/日を25日間投与したところ傾眠傾向が出現した. 同剤の投与を中止後, 傾眠傾向は消失した.これら2症例は血液透析を受けていないmorphine被投与例 (7例) と比較して, 血清morphine濃度は比較的低値であったが, その主要代謝物であるmorphine-6-glucuronide (M6G) とmorphine-3-glucuronide (M3G) の濃度は高値であった. 腎不全患者において, M6G, M3Gが蓄積性を持つことやM6Gがmorphineの活性代謝産物であることが知られており, M6Gの蓄積が両症例での意識障害の原因と推定された. 血液透析患者に対してmorphineを使用する際には, M6Gの蓄積による意識障害が容易に起こるため, 投与量の決定に慎重な配慮が必要と考えられた.

Biochemical assay and immunohistochemical staining of neutral endopeptidase were performed on bladder cancer cells. In superficial bladder cancer the enzyme activity and immunohistochemical intensity of staining were high, while invasive bladder cancer showed only a low level of activity. This finding suggests that neutral endopeptidase is expressed at a certain stage of cell differentiation, during the neoplastic process in the bladder. Gene expression is assumed to be closely correlated with this mechanism. From the results of this study neutral endopeptidase will serve as a new tumor marker for bladder cancer as well as acute lymphatic leukemia.

Plasma levels of guanidine (G) are reported to be increased in uremic patients and are synthesized from various guanidino compounds via a chemical reaction involving the hydroxyl radical in vitro. To identify both the metabolic precursor and the synthesizing organ of G, we investigated the concentrations of G in various organs of rats administered several guanidino compounds and we attempted to synthesize G biologically using isolated rat hepatocytes or perfused rat liver. In addition, we investigated the effect of the peroxidative state on the G synthesis in isolated hepatocytes using various reagents which alter this condition. Results show that the concentration of G increased in the kidney, liver and muscle following the administration of L-canavanine. In addition, G increased in the kidney at 90 min after the administration of guanidinoacetic acid (GAA). Moreover, G is synthesized from L-canavanine in isolated rat hepatocytes and perfused rat liver, and G synthesis in hepatocytes is partially inhibited by the addition of superoxide dismutase, catalase, glutathione or ethylenediaminetetraacetic acid. These results suggest that L-canavanine is possibly a biological precursor and GAA is an endogenous precursor of G. Furthermore, it is suggested that these reactions are closely related to the peroxidative state.

Paraquat,有機溶媒,殺虫剤の中毒は腎臓の尿細管障害を起すが,その毒性は活性酸素の産生,チトクロームP-450monooxigenaseやglutathione-S-transferaseにより代謝され親電子性代謝産物となり標的物質への結合,代謝産物による障害(蓚酸やフッ素)などによる. puromycin aminonucleosideはアデノシンに類似の低分子有機化合物でネフローゼを起すが,著者らはこれが活性酸素の産生を増加させることを発見し,糸球体の活性酸素の画像化を供覧した.

各種の疾患において活性酸素が組織障害の直接的な原因物質であることが判明してきた.原子や分子が不対電子を有するとフリーラジカルと呼ばれ,一般に高い反応性を持つ.酸素がその分子の場合,活性酸素と呼ぶ.高い反応性を持つ活性酸素は生体に重要な物質の合成反応に用いられるが,制御されない物質の酸化は物質の変性を起こし,さらなる傷害を起こす.腎疾患を例にとると尿毒症の発症に伴い生成が著しく増加するメチルグアニジンは活性酸素とクレアチニンの反応生成物である.また,ネフローゼを起こすピューロマイシンアミノヌクレオシドは細胞における活性酸素の産生を増加させ,その産生増加はアラキドン酸代謝やPAFと関連していることが示唆された.外因物質による生体内の活性酸素の増加は,発癌や老化との関連があり,また,細胞における活性酸素の産生調節は疾病の治療薬の開発にとって重要である.

Imaging of hydroperoxides in a rat glomerulus stimulated by puromycin aminonucleoside Background To determine the locus of the increased oxidation induced by puromycin aminonucleoside (PAN), we imaged hydroperoxides in glomeruli stimulated by PAN in vivo and in vitro. Methods Dichlorofluorescein diacetate (DCFH-DA) in cells makes dichlorofluorescein, a substance that fluoresces when reacted with hydroperoxides. Fluorescence was detected using a photon detection video camera connected to a microscope. Two kinds of isolated glomeruli of Wistar rats were examined. One was the glomerulus obtained from rats on the seventh day following the injection of PAN. In this case, glomeruli were incubated in a buffer containing 5 mM DCFH-DA. Another was the glomerulus collected at 30 minutes after a large amount of DCFH-DA was intravenously injected. These glomeruli were incubated with either PAN or phorbol myristate acetate (PMA) in Krebs-Henseleite bicarbonate buffer. Results The images from the glomeruli treated by PAN in vivo resemble pictures of a galaxy by telescope. When the glomeruli were treated by PAN in vitro , two localized points appeared in each glomerulus after 15 minutes of incubation with PAN, and after 75 minutes of incubation, the fluorescence spread throughout the glomerulus. When glomeruli were incubated with PMA, two points that gave a very strong fluorescence were observed in each glomerulus, but they did not spread throughout the glomeruli. In both experiments, glomeruli without stimulants did not fluoresce. Conclusion Increases in hydroperoxides were observed in the glomeruli from rats made nephrotic by exposure to PAN, and were also observed in glomeruli following 15 minutes of incubation with PAN in vitro.

While it is well known that hemodialysis (HD) patients with end stage renal failure are exposed to high oxidative stress, there is not a general opinion regarding whether antioxidant activity is high or low in these patients. We evaluated the variation of plasma hydroxyl radical scavenging activity (p-HRSA) by a single-session of HD in 69 patients by using a new system, reactive flow-injection electron spin resonance. And then comparing p-HRSA with their biochemical parameters, we tried to elucidate what components affected p-HRSA in the HD patients. The average of p-HRSA significantly increased after HD and the variation of p-HRSA by HD was correlated with that of plasma total protein (TP). In 5 patients however, their p-HRSA decreased after HD, in spite of increasing TP. In pre-HD, the p-HRSA values and hydroxyl radical scavenging activity of low-molecular weight fraction of plasma were significantly higher in these 5 patients than in patients whose p-HRSA increased after HD. These 5 patients were in an inflammatory state. These findings suggest that p-HRSA is mainly affected by TP, but caution should be exercised in patients who have high p-HRSA before HD and whose p-HRSA does not increase after HD.

The protective effect of (-)-epicatechin 3-O-galate (ECg) against peroxynitrite (ONOO-)-mediated damage was examined using an animal model and a cell culture system. In rats subjected to lipopolysaccharide (LPS) administration plus ischemia-reperfusion, the plasma 3-nitrotyrosine level an indicator of ONOO- production in vivo, was elevated, whereas it declined significantly and dose-dependently after the oral administration of ECg at doses of 10 and 20 micromoles/kg body weight/day for 20 days prior to the process. Moreover, oral administration of ECg significantly enhanced the activities of the antioxidant enzymes, superoxide dismutase, catalase and glutathione peroxidase, and the antioxidant glutathione, showing enhancement of the biological defense system against the damage induced by ONOO-. In addition, the significant increase in the renal mitochondrial thiobarbituric acid-reactive substance level of LPS and ischemic-reperfused control rats was attenuated in rats given ECg. Furthermore, the elevations in the plasma urea nitrogen and creatinine (Cr) levels and the urinary methylguanidine/Cr ratio induced by the procedure were attenuated markedly after oral administration of ECg, implying amelioration of renal impairment. The addition of ECg (25 or 125 microM) prior to 3-morpholinosydnonimine (SIN-1, 800 microM) exposure reduced ONOO- formation and increased the viability of cultured renal epithelial (LLC-PK1) cells in a dose-dependent manner. In particular, ECg inhibited ONOO(-)-mediated apoptotic cell death, which was confirmed by decreases in the DNA fragmentation rate and the presence of apoptotic morphological changes, i.e. small nuclei and nuclear fragmentation. Furthermore, adding ECg before SIN-1 treatment regulated the cell cycle by enhancing G2/M phase arrest. This study provides evidence that ECg has protective activity against the renal damage induced by excessive ONOO- in cellular and in vivo systems.

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.

Heparin has been reported to have many actions similar to calcium-dependent protein kinase (PKC) inhibitors. We have found that puromycin aminonucleoside (PAN) increases hydroxyl radical generation and this was prevented by H-7, a PKC inhibitor in isolated rat hepatocytes. In this study, we investigate the effect of heparin on the increased hydroxyl radical generation as well as PKC activation by PAN in isolated rat hepatocytes. To estimate the amount of hydroxyl radical generation, we measured methylguanidine (MG) and creatol which are the products from the reaction of creatinine and hydroxyl radical. Synthetic rate of MG plus creatol in isolated rat hepatocytes incubated in Krebs-Henseleit bicarbonate buffer containing creatinine and tested reagents were recorded. This rate with or without PAN was 231 +/- 11 or 112 +/- 5.6 nmol/g wet cells/4 h (mean +/- S.E., n = 5), respectively. Heparin concentrations of 3.3, 6.6 and 10 U/ml inhibited MG plus creatol synthesis in the presence of PAN by 30, 38 and 39%, and without PAN by 8.4, 27 and 34%, respectively. Statistical significance was observed except for 3.3 U/ml without PAN. The ratio of PKC in membrane/cytoplasmic fraction, an indicator of PKC activation, increased 2.8- and 3-fold that of the 0 time after 60 and 120 min incubation with PAN while heparin at 10 U/ml almost completely suppressed this increase in the ratio of PKC. The PKC ratio of the membrane/cytoplasmic fraction obtained from hepatocytes with heparin alone or without PAN and heparin was almost unchanged during the tested period. Variation of PKC levels in membrane fraction is similar to that of PKC ratio of the membrane/cytoplasmic fraction. Increased creatol synthesis by PAN and its inhibition by heparin were observed in the same samples as those used for the PKC study. These results indicate that heparin inhibits the increase in hydroxyl radical generation induced by PAN through inhibition of PKC activation in isolated rat hepatocytes.

We have reported that (1) the synthesis of GSA, a uremic toxin, increases depending on the urea concentration and (2) GSA is formed from argininosuccinic acid (ASA) and the hydroxyl radical or SIN-1 which generates superoxide and NO simultaneously. However, an excess of NO, which also serves as a scavenger of the hydroxyl radical, inhibited GSA synthesis. We also reported that arginine, citrulline or ammonia plus ornithine, all of which increase arginine, inhibit GSA synthesis even in the presence of urea. To elucidate the mechanism for increased GSA synthesis by urea, we investigated the effect of urea on ASA and arginine, the immediate precursor of NO. Isolated rat hepatocytes were incubated in 6 ml of Krebs-Henseleit bicarbonate buffer containing 3% bovine serum albumin, 10 mM sodium lactate, 10 mM ammonium chloride and with or without 36 mM of urea and 0.5 or 5 mM ornithine at 37 degrees C for 20 min. In vivo experiments, 4 ml/100 g body weight of 1.7 M urea or 1.7 M NaCl were injected intra-peritoneally into 5 male Wistar rats. Two hours after the intra-peritoneal injection of urea or 1.7 M NaCl, blood, liver and kidney were obtained by the freeze cramp method and amino acids were determined by an amino acid analyzer (JEOL:JCL-300). ASA in isolated hepatocytes was not detected with or without 36 mM (200 mgN/dl) urea, but the arginine level decreased from 36 to 33 nmol/g wet cells with urea. Ornithine which inhibits GSA synthesis, increased ASA markedly in a dose dependent manner and increased arginine. At 2 h after the urea injection the rat serum arginine level decreased by 42% (n = 5), and ornithine and citrulline levels increased significantly. Urea injection increased the ASA level in liver from 36-51 nmol/g liver but this was not statistically significant. We propose that urea inhibits arginine synthesis in hepatocytes, where the arginine level is extremely low to begin with, which decreases NO production which, in turn, increases hydroxyl radical generation from superoxide and NO. This may, also, be an explanation for the reported increase in oxygen stress in renal failure.

The recent development of electron paramagnetic resonance (EPR) permits its application for in vivo studies of nitric oxide (NO). In this study, we tried to obtain 3D EPR images of endogenous NO in the abdominal organs of lipopolysuccaride (LPS) treated mice. Male ICR mice, each weighing about 30 g, received 10 mg/kg of LPS intraperitoneally. Six hours later, a spin trapping reagent comprised of iron and an N-dithiocarboxy sarcosine complex (Fe(DTCS)2, Fe 200 mM, DTCS/Fe = 3) were injected subcutaneously. Two hours after this treatment, the mice were fixed in a plastic holder and set in the EPR system, equipped with a loop-gap resonator and a 1 GHz microwave. NO was detected as an NO-Fe(DTCS)2 complex, which had a characteristic 3-line EPR spectrum. NO-Fe(DTCS)2 complexes in organ homogenates were also measured using a conventional X-band EPR system. NO-Fe(DTCS)2 spectra were obtained in the upper abdominal area of LPS treated mice at 8 h after the LPS injection. 3D EPR tiled and stereoscopic images of the NO distribution in the hepatic and renal areas were obtained at the same time. The NO-Fe(DTCS)2 distribution in abdominal organs was confirmed in each organ homogenate using conventional X-band EPR. This is the first known EPR image of NO in live mice kidneys.

We have reported that(1)the synthesis of GSA, a uremic toxin, increases depending on the urea concentration and (2) GSA is formed from argininosuccinic acid (ASA) and the hydroxyl radical or SIN-1 which generates superoxide and NO simultaneously. However, an excess of NO, which also serves as a scavenger of the hydroxyl radical, inhibited GSA synthesis. We also reported that arginine, citrulline or ammonia plus ornithine, all of which increase arginine, inhibit GSA synthesis even in the presence of urea. To elucidate the mechanism for increased GSA synthesis by urea, we investigated the effect of urea on ASA and arginine, the immediate precursor of NO.

The recent development of electron paramagnetic resonance (EPR) permits its application for in vivo studies of nitric oxide (NO). In this study, we tried to obtain 3D EPR images of endogenous NO in the abdominal organs of lipopolysuccaride (LPS) treated mice. Male ICR mice, each weighing about 30 g, received 10 mg/kg of LPS intraperitoneally. Six hours later, a spin trapping reagent comprised of iron and an N-dithiocarboxy sarcosine complex (Fe(DTCS)2, Fe 200 mM, DTCS/Fe = 3) were injected subcutaneously. Two hours after this treatment, the mice were fixed in a plastic holder and set in the EPR system, equipped with a loop-gap resonator and a 1 GHz microwave. NO was detected as an NO-Fe(DTCS)2 complex, which had a characteristic 3-line EPR spectrum. NO-Fe(DTCS)2 complexes in organ homogenates were also measured using a conventional X-band EPR system. NO-Fe(DTCS)2 spectra were obtained in the upper abdominal area of LPS treated mice at 8 h after the LPS injection. 3D EPR tiled and stereoscopic images of the NO distribution in the hepatic and renal areas were obtained at the same time. The NO-Fe(DTCS)2 distribution in abdominal organs was confirmed in each organ homogenate using conventional X-band EPR. This is the first known EPR image of NO in live mice kidneys.

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.

Urinary free ATP assay by the firefly luciferin-luciferase method is a rapid and simple method for determining renal function, especially uriniferous tubule function. Normal range of urinary free ATP concentration, daily ATP excretion in urine, urinary ATP/creatinine value and ATP decomposition activity in urine is 1.1 x 10(-9)-3.4 x 10(-8) M, 4.0 x 10(-9)-4.1 x 10(-8) mole, 5.0 x 10(-13)-5.9 x 10(-11) mol/mgCr and 100-77% express for the remaining rate of additional ATP, respectively. A significant correlation was found between free ATP concentration and daily ATP excretion in urine with a correlation coefficient of 0.84. In cases of anti-tumor drug(cisplatin = cis-diamminedichloroplatinum II) administration for urinary-track tumor, abnormal urinary free ATP concentration and ATP decomposition activity in urine were clearly demonstrated after a few days of cisplatin administration. The appearance of a tendency toward abnormal relative ATP values were similar to changes in beta 2-MG and NAG values. Diabetic patients often demonstrate unusually high values of urinary free ATP concentration. In asphyxia of the newborn, urinary ATP/creatinine value were significantly higher than those in healthy newborn, but urinary NAG values did not differ.

Decreased availability of arginine and impaired production of NO (nitric oxide) have been implicated in the development of endothelial dysfunction. Citrulline formed by the NOS reaction is recycled to arginine by the citrulline-NO cycle, which is composed of NOS, argininosuccinate synthetase (AS), and argininosuccinate lyase. Therefore, we investigated the alterations of these enzymes in the aorta of streptozotocin (STZ)-induced diabetic rats. eNOS and AS mRNAs were increased by three- to fourfold 1-2 weeks after STZ treatment and decreased at 4 weeks. AL mRNA was weakly induced. Induction of eNOS and AS proteins was also observed. Cationic amino acid transporter (CAT)-1 mRNA remained little changed, and CAT-2 mRNA was not detected. The plasma nitrogen oxide levels were increased 1-2 weeks after STZ treatment and decreased at 4 weeks. Transforming growth factor-beta1 (TGF-beta1) mRNA in the aorta was also induced. TGF-beta1 induced eNOS and AS mRNAs in human umbilical vein endothelial cells but inhibited the proliferation of HUVEC. These results indicate that eNOS and AS are coinduced in the aorta in early stages of STZ-induced diabetic rats and that the induction is mediated by TGF-beta1. The results also suggest that TGF-beta1 works antiatherogenically at early stages of diabetes by increasing NO production, whereas prolonged elevation of TGF-beta1 functions atherogenically by inhibiting endothelial cell growth.

Gallic acid (GA), a food component that is especially abundant in tea, is an antimutagenic, anticarcinogenic and anti-inflammatory agent. We conducted a study using acidum gallicum tablets that contained 10% GA and 90% glucose and a black tea brew that contained 93% of its GA in free form to determine the pharmacokinetics and relative bioavailability of GA in healthy humans. After the administration of a single oral dose of acidum gallicum tablets or tea (each containing 0.3 mmol GA) to 10 volunteers, plasma and urine samples were collected over various time intervals. Concentrations of GA and its metabolite, 4-O-methylgallic acid (4OMGA), were determined, and the pharmacokinetic parameters were calculated. GA from both the tablets and tea was rapidly absorbed and eliminated with mean half-lives of 1.19 +/- 0.07 and 1.06 +/- 0.06 h and mean maximum concentrations of 1.83 +/- 0.16 and 2.09 +/- 0.22 micromol/L (plasma), respectively. After oral administration of the tablets and black tea, 36.4 +/- 4.5 and 39.6 +/- 5.1% of the GA dose were extracted in urine as GA and 4OMGA, respectively. The relative bioavailability of GA from tea compared with that from the tablets was 1.06 +/- 0.26, showing that GA is as available from drinking tea as it is from swallowing tablets of GA.

Role of nitric oxide in the synthesis of guanidinosuccinic acid, at activator of the N-methyl-d-asparate receptor. Background We propose that reactive oxygn and arginiosuccinic acid (ASA) form guanidinosuccinic acid (GSA). An alternative to this hypothesis is the so-called guanidine cycle, which consists of a series of hydroxyurea derivatives that serve as intermediates in a pathway leading from urea to GSA. We compare the role of the guanidine cycle to that of nitric oxide (NO) in the synthesis of GSA. Methods The members of the guanidine cycle (hydroxyurea, hydroxylamine plus homoserine, L-canaline, and L-canavanine) were incubated with isolated rat hepatocytes. The known NO donors, NOR-2, NOC-7, and SIN-1, were incubated with ASA in vitro. Ornithine, arginine, or citrulline, which increase arginine, a precursor of NO, were incubated with isolated rat hepatocytes. GSA was determined by high-performance liquid chromatography. Results None of guanidine cycle members except for urea formed GSA. SIN-1, which generates superoxide and NO formed GSA, but other simple NO donors, did not. Both carboxy-PTIO, a scavenger, completely inhibited GSA synthesis by SIN-1. GSA formation by SIN-1 reached a maximum at 0.5 mmol/L and decreased at higher concentrations GSA synthesis, stimulated by urea in isolated hepatocytes, was inhibitd by orinithine, arginine, or citrullin with ammonia, but not by ornithine without ammonia, where arginine production is limited. Conclusion GSA is formed ASA and the hydroxyl radical. When arginine increased in hepatocytes, GSA synthesis decreased. These data suggest that increased NO, which results from high concentrations of arginine, or SIN-1, scavanges the hydroxyl radical. This may explain the decreased GSA synthesis in inborn errors of the urea cycle where ASA is decreased, and also the diminished GSA excretion in arginemia.

important role in the pathogenesis of MRSAassociated GN. Background. We have previously reported that 10 patients who developed glomerulonephritis (GN ) in association with methicillin-resistant Staphylococcus Keywords: enterotoxin; glomerulonephritis; MRSA; aureus (MRSA) infection showed a marked increase superantigen in DR+CD4+ and DR+CD8+ subsets of T cells and in T cells expressing several T-cell receptor ( TCR) Vb+cells, perhaps representing Vb-specific T-cell activation by MRSA-derived superantigens (Kidney Int Introduction 1995; 47: 207–216). In this study we examine cytokine levels, T-lymphocyte subsets, natural killer NK cells, memory T cells, and the expression of IL-2 receptors Methicillin-resistant strains of Staphylococcus aureus (MRSA) were initially detected in 1961 in the United in order to better understand the role of bacterial superantigens and cytokines in the pathogenesis of Kingdom [1] shortly after methicillin came into clinical use. Patients with MRSA infection frequently develop MRSA-associated GN. Methods. Twenty-two patients with MRSA infection septicaemia or toxic shock syndrome, both caused by staphylococcal enterotoxins. Staphylococcal enterotoxwho later developed GN caused by staphylococcal enterotoxin were evaluated immunologically in com- ins are described as ‘superantigens’ that stimulate proliferation of resting T cells [2], resulting in massive parison with patients whose MRSA infection was not followed by GN (non-GN group) and normal T-cell activation and release of T-cell-derived cytokines such as interleukin-1 (IL-1), IL-2, IL-4, IL-6, IL-8, individuals. Results. Among peripheral lymphocytes, the frequency IL-10, tumour necrosis factor ( TNF ) as well as interferon-c ( INF-c) [3–7]. of T cells expressing several TCR Vbs, especially Vb5-family TCR, was higher in the GN group than in We have previously reported that patients who developed glomerulonephritis (GN ) after MRSA infecboth the non-GN group and the normal healthy control group. GN patients also showed increased serum tion showed a marked increase in both DR+CD4+ and DR+CD8+ subsets of T cells and in T cells levels of several cytokines, including tumour necrosis factor-a ( TNF-a), interleukin-1b (IL-1b), IL-2, IL-6, expressing several T-cell receptor (TCR) Vb+cells, perhaps representing Vb-specific T-cell activation by

Creatol is a hydroxyl radical adduct of creatinine and the precursor of methylguanidine (MG), a uremic toxin. We investigate the synthesis of creatol and MG from creatinine and the effect of substances that affect the hydroxyl radical in isolated rat hepatocytes. In the presence of increasing concentrations of creatinine, rising level of creatol were found after 2 h incubation in Krebs-Henseleit bicarbonate buffer. However, further increase of creatol was not observed after 4 and 6h incubations. On the other hand, MG after 2 h incubation achieved a level of about 50% that of creatol and increased depending on both the creatinine concentration and the incubation period. DMSO, a hydroxyl radical scavenger decreased the generation of creatol and MG by about 50% at 2.5 mM and the inhibition depended on DMSO concentration. Puromycin amino-nucleoside (PAN) increased both by about 170%. These findings demonstrated that hepatocytes synthesize creatol prior to MG and are inhibited by a hydroxyl] radical scavenger. They also show that PAN increased hydroxyl radical generation in tissue cells.

There is considerable evidence that uremic patients are in a highly peroxidative state. The purpose of this study was to investigate the serum antioxidant activity that may regulate, or represent, the redox state in vivo. Serum from pre- and posthemodialysis patients and from healthy control subjects was added to a system generating the hydroxyl radical, and then the signal intensities of reactive oxygen species were measured by electron spin resonance and spin-trapping technique. The electron spin resonance signals of the reaction mixture containing prehemodialysis sera were significantly stronger than those of the reaction mixture containing healthy sera (P < 0.001, n = 19), and there was no significant difference in the signals between the reaction mixture containing posthemodialysis and healthy sera. These findings demonstrated that serum antioxidant activity in hemodialysis patients is significantly decreased, and this pathological condition is improved by hemodialysis treatment.