Kazumasa Aoyagi

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.

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.

A NEW TUMOR MARKER FOR BLADDER CANCER

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.

Glycyrrhizae Radix attenuates peroxynitrite-induced renal oxidative damage through inhibition of protein nitration

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−.

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.

Inhibition of arginine synthesis by urea: A mechanism for arginine deficiency in renal failure which leads to increased hydroxyl radical generation

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°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.

Reduced serum hydroxyl radical scavenging activity in erythropoietin therapy resistant renal anemia

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.

Protective Activity of (−)-Epicatechin 3- O -gallate against Peroxynitrite-mediated Renal Damage

The protective effect of ( m )-epicatechin 3- O -gallate (ECg) against peroxynitrite (ONOO m )-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 m production in vivo, was elevated, whereas it declined significantly and dose-dependently after the oral administration of ECg at doses of 10 and 20 w moles/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 m . 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 w M) prior to 3-morpholinosydnonimine (SIN-1, 800 w M) exposure reduced ONOO m formation and increased the viability of cultured renal epithelial (LLC-PK 1 ) cells in a dose-dependent manner. In particular, ECg inhibited ONOO m -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 G 2 /M phase arrest. This study provides evidence that ECg has protective activity against the renal damage induced by excessive ONOO m in cellular and in vivo systems.

Pharmacokinetics of Tamsulosin Hydrochloride in Patients with Renal Impairment: Effects of α1‐Acid Glycoprotein

The pharmacokinetics of tamsulosin hydrochloride in patients with renal impairment were compared with those in healthy volunteers, and the factors that influenced plasma levels of tamsulosin were elucidated. A single oral dose of 0.2 mg of tamsulosin was given and blood and urine samples were obtained for 36 hours after administration. Unbound plasma concentration of tamsulosin was measured by a combination of equilibrium dialysis and liquid chromatography tandem mass spectrometry methods to examine the effect of protein binding on the pharmacokinetics of tamsulosin. Mean values for maximum concentration (Cmax) and area under the concentration—time curve (AUC) of total drug (Cmax,t and AUC1 in patients with renal impairment were 73% and 211% greater, respectively, than those in healthy volunteers. Mean Cmax and AUC of unbound drug (Cmax,u and AUCu), however, were almost the same in the two groups. A high correlation was found between α1‐acid glycoprotein (α1‐AGP) concentration and AUCt, but no correlation was found between α1‐AGP concentration and AUCu,0–36 or between creatinine clearance (ClCR) and AUCu,0–36. These results show that in patients with renal impairment, the pharmacokinetics of tamsulosin are affected by the change in protein binding that is associated with alteration of plasma α1‐AGP concentration, but are not largely affected by the decrease in the renal excretion. Although total tamsulosin levels increased as plasma protein binding increased, unbound tamsulosin levels (which are directly associated with the pharmacologic effects) remained unchanged in these patients.