Michihiro Gotoh

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.

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.

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.

A case of renovascular hypertension associated with neurofibromatosis.

We report a case of renovascular hypertension associated with neurofibromatosis complicated by moderate proteinuria. A 16-year-old female was admitted to Kensei General Hospital with a complaint of headache and a blood pressure of 230/120 mm Hg. She was referred to us for further evaluation of the hypertension. On examination, cafe-au-lait spots were seen over her extremities and flank, and a bruit was heard in the right upper abdomen. The urinary protein excretion was 2.1 g/day. The plasma renin activity (PRA) and plasma aldosterone concentration were high, but the levels of catecholamines were normal. The renogram was asymmetric and on venous sampling, the PRA in the right renal vein was 58.3 ng/ml/h and that in the left was 22.1 ng/ml/h. CT scan detected an approximately 10-mm mass in the proximal right renal artery. Arteriography disclosed severe stenosis in the right renal artery and the superior mesenteric artery. Therefore, we concluded that her hypertension resulted from stenosis of the right renal artery due to neurofibromatosis. Accordingly, she underwent an operation to reconstruct that artery. After the operation, her blood pressure and PRA normalized without administration of any anti-hypertensive drug and urinary protein disappeared.

Role of reactive oxygen and argininosuccinate in guanidinosuccinate synthesis in isolated rat hepatocytes.

The synthesis of guanidinosuccinic acid (GSA) increases in uremics, and GSA is implicated as a uremic toxin. The GSA synthesis increases roughly in proportion to the serum urea level that increases in patients with renal failure. Urea is a specific inhibitor of argininosuccinase, the fourth urea cycle enzyme, and might lead to the increase of argininosuccinate (ASA). We found that GSA is formed from ASA by reactive oxygen species in vitro. In this paper, we investigated GSA synthesis from ASA in isolated rat hepatocytes and the effect of reactive oxygen species on this synthesis. When isolated rat hepatocytes were incubated with 5 mmol/l ASA, GSA was formed linearly with time up to 6 h (16 nmol/g wet liver/6 h). GSA was formed depending on the ASA concentration up to 10 mmol/l. Dimethylsulfoxide, a hydroxyl radical scavenger, inhibited GSA synthesis by 65%. GSA was actively formed when the hepatocytes were incubated with 32 mmol/l urea. The GSA formation in the presence of urea was also inhibited by dimethylsulfoxide, although the inhibition was less marked. FeCl2, that increases the hydroxyl radical generation, increased GSA synthesis. These results indicate that GSA is formed from ASA in isolated hepatocytes. The results also suggest that reactive oxygen species are important for GSA synthesis in the cells.

Renal oncocytomatosis in a long‐term hemodialysis patient treated by laparoscopic surgery

A 50‐year‐old female, who had been on maintenance hemodialysis for 22 years, consulted our clinic because of a left renal mass detected incidentally by ultrasonography. Computed tomography (CT) demonstrated a solid hypervascular mass, suggesting a renal cell carcinoma (RCC), in the left atrophic kidney. Left hand‐assisted laparoscopic nephrectomy (HALN) was performed. The histopathological diagnosis was renal oncocytomatosis. Renal oncocytomatosis in a long‐term hemodialysis patient is extremely rare. We report the first case of renal oncocytomatosis in a long‐term hemodialysis patient treated with hand‐assisted laparoscopic nephrectomy.

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.

Increased Lipid Peroxidation by Rat Liver Microsomes in Experimental Renal Failure

This study investigated the peroxidative state of renal failure by measuring lipid peroxidation. The generation of lipid peroxides by rat hepatic microsomes was compared between experimental renal failure caused by 7/8 nephrectomy and healthy control animals. The concentrations of BUN at the point of sacrifice were 84.2 +/- 18.4 (mean +/- SD) in nephrectomized and 19.8 +/- 4.3 mg/dl in the control group (p < 0.01, n = 5). Microsomal generation of thiobarbituric acid reactive substance was 31.2 +/- 2.8 in the nephrectomized group and 20.9 +/- 4.9 nmol/incubation/min in the control group. There was a significant difference between the groups (p < 0.01, n = 5). In summary, the generation rate of lipid peroxides in the microsomes obtained from the rats with experimental renal failure was significantly higher than the control. This study confirms an increased peroxidative state, and specifies one of the sites of increased lipid peroxide generation in renal failure.

Elimination of Lipid Peroxide during Hemodialysis

Background/Aims: This study is aimed to show the antioxidative effect of hemodialysis (HD) by demonstrating the elimination of toxic lipid peroxides. Methods: Blood samples were obtained from patients on regular maintenance HD before and 15, 30, 60, 120 and 240 min after the start of each HD session. Plasma cholesteryl ester hydroperoxide (CE-OOH), phosphatidylcholine hydroperoxide (PC-OOH), and eliminators of lipid peroxides (LOOH) such as apolipoprotein A-I (apoA-I) and lecithin:cholesterol acyltransferase (LCAT) were investigated. The hydroxyl radical scavenging activity was measured for the evaluation of the pro-oxidative side. Results: CE-OOH and PC-OOH were elevated in patients with chronic kidney disease both on and not on HD, while these values were much higher in HD patients. CE-OOH quickly dropped during the first 30 min of HD, then gradually decreased until 240 min. CE-OOH concentrations were related to those of apoA-I. In contrast, PC-OOH showed an increase 30 min after the start of HD, a change which resembled that of LCAT and was the reverse of the hydroxyl radical scavenging activity. Conclusion: These results demonstrate the antioxidative action through CE-OOH elimination involving apoA-I. The pro- and antioxidative effects of HD on LOOH are not uniform but PC-OOH is mainly influenced prooxidatively.