Katsumi Takemura

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.

Synthesis of creatol, a hydroxyl radical adduct of creatinine and its increase by puromycin aminonucleoside in isolated rat hepatocytes

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.

Creatol, an oxidative product of creatinine in hemodialysis patients

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 μM, 11.1 ± 4.8μM and 5.8±2.9 μM, 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 6h 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.

Decreased Serum Antioxidant Activity of Hemodialysis Patients Demonstrated by Methylguanidine Synthesis and Microsomal Lipid Peroxidation

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.

Biosynthesis of methylguanidine in the hepatic peroxisomes and the effect of the induction of peroxisomal enzymes by clofibrate

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.

Biosynthesis of methylguanidine in the hepatic microsomal fraction

We have investigated various synthetic mechanisms for the production of methylguanidine, a potent uremic toxin, and reported a role for active oxygen in its biosynthesis from creatinine in studies using isolated hepatocytes. In this study, we turn our attention to the hepatic microsomes. Liver homogenates were made from rats, and various organelles were obtained by centrifugation and incubated with creatinine. The results show that methylguanidine synthesis occurs only in the microsomal fraction in the presence of nicotinamide adenine dinucleotide phosphate, reduced form. The microsomal activity is inhibited by the addition of methimazole, metyrapone, superoxide dismutase, catalase or dimethylsulfoxide. These results suggest that methylguanidine is synthesized from creatinine by microsomes, and at least 2 enzymes are involved, an FAD-containing monooxygenase and a P-450-dependent oxidase based on the inhibitory effect of methimazole and metyrapone, respectively. Moreover, the inhibition by various scavengers of active oxygen suggests that active oxygen plays a role in the intermediate steps of the enzymatic reaction.

Temporal Changes in Post-Infectious Glomerulonephritis in Japan (1976-2009)

Background The incidence of post-infectious glomerulonephritis (PIGN) in developed countries has decreased over the last 50 years. Here we identified the trends of the incidence of PIGN in Japan during the past four decades. Methods We explored the frequency, clinicopathological findings, and prognosis of PIGN based on 6,369 cases from the Renal Biopsy Database of our institute in the Kanto region of Japan, diagnosed histologically from 1976 to 2009. Results The numbers of PIGN cases were 131 (2.1%) in total, and 2.4%, 1.1%, 2.6% and 2.1% identified in the 1970s, 1980s, 1990s, and 2000s, respectively. Acute glomerulonephritis (AGN), including post-streptococcal glomerulonephritis (PSGN), accounted for almost all of the PIGN cases in the 1970s, but decreased to approx. 40%–50% since the 1990s. In the 1990s, Staphylococcus aureus infection-related nephritis (SARN) showed a rapid increase in rate, reaching 30%. The incidence of hepatitis C virus infection-associated GN (HCVGN) has increased since the 1990s. The average age at onset rose from 33 to 51 years over the study period. These transitions can be summarized as increases in SARN and HCVGN and decreases in PSGN and other types of AGN, since SARN and HCVGN have older onsets compared to PSGN and other AGN types. The clinicopathological features were marked for each PIGN. Regarding the prognosis, the renal death rates of both the SARN and HCVGN groups were significantly higher than those of other PIGN. Conclusion Based on our analysis of the Renal Biopsy Database, the incidence of PIGN in Japan reached its peak in the 1990s. The temporal changes in the incidence of PIGN reflected the trends in infectious diseases of each decade and the continual aging of the population, with a related higher susceptibility to infections.

Biosynthesis of guanidine in isolated rat hepatocytes, perfused rat liver and intact animals

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.