Heterogeneity of prognosis in adult IgA nephropathy, especially with mild proteinuria or mild histological features.

OBJECTIVE: The present study was undertaken to clarify the clinical course and prognosis of adult patients with primary IgA nephropathy (IgAN), especially with mild proteinuria or mild histological alternations. PATIENTS AND METHODS: A population of 735 IgAN patients whom we were able to observe for more than two years was examined. RESULTS: A total of 115 patients (15.6%) was on dialysis during the observation period. The overall 5-year renal survival rate was 92.0%. On the other hand, 166 patients (22.6%) were in clinical remission. A group with mild proteinuria included 197 patients (26.8%). Forty-seven patients of this group showed minor glomerular abnormalities, whereas 12 patients with mild proteinuria showed severe mesangial involvement. Three patients with mild proteinuria were on dialysis during the observation period, whose proteinuria was increased during the clinical course. A group with minor glomerular abnormalities included 82 patients (11.2%). Forty-seven patients of this group showed mild proteinuria, of whom 12 patients showed moderate proteinuria. However, three patients with minor glomerular abnormalities who were not on dialysis showed loss of renal function. CONCLUSION: These results indicated the heterogeneity of the course and prognosis in IgAN. Even if a patient's initial clinical or histological findings are comparatively mild, strict follow-up management is needed.

Role of nitric oxide in the synthesis of guanidinosuccinic acid, an activator of the N-methyl-D-aspartate receptor.

BACKGROUND: We propose that reactive oxygen and argininosuccinic 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 of NO, and dimethyl sulfoxide, a hydroxyl radical 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 inhibited by ornithine, arginine, or citrulline with ammonia, but not by ornithine without ammonia, where arginine production is limited. CONCLUSION: GSA is formed from 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 scavenges 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.

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.

Prevalence of Japanese dialysis patients with an A-to-G mutation at nucleotide 3243 of the mitochondrial tRNA(Leu(UUR)) gene.

BACKGROUND: A high prevalence of an A-to-G mutation at nucleotide 3243 of the mitochondrial genome in patients with diabetes mellitus (DM) and/or deafness has been reported previously. We investigated the prevalence of this mutation in Japanese dialysis patients with associated DM and/or deafness. METHODS: We studied 106 dialysis patients with DM, 26 with DM and deafness, and 26 with deafness alone, using peripheral leucocytes to detect an A-to-G transition at nucleotide 3243 of the mitochondrial gene. RESULTS: We identified this transition in 1 of 26 patients with DM and deafness. None of the 106 DM or 26 dialysis patients with deafness but no DM was positive for this mutation. A 42-year-old male patient on continuous ambulatory peritoneal dialysis (CAPD) who carried this mutation had a 20-year history of sensory hearing loss as well as hypertrophic cardiomyopathy. CONCLUSION: We found that a mitochondrial gene mutation at nucleotide 3243 was present in one dialysis patient with NIDDM and deafness. The prevalence of this mutation was found to be below 1% in diabetic end-stage renal disease patients in Japan.

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 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.

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.

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.

Intraperitoneal hyaluronan production in stable continuous ambulatory peritoneal dialysis patients.

OBJECTIVE: Several cytokines and proteins are excreted intraperitoneally during the course of peritonitis and stable states in continuous ambulatory peritoneal dialysis (CAPD) patients. Dialysate hyaluronan (HYA) is also regarded as a marker of peritoneal healing during bacterial peritonitis. We examined here, intraperitoneal HYA production in stable CAPD patients and compared the results to those of the peritoneal equilibration test (PET), the length of time on dialysis, and other marker proteins. DESIGN: We determined the concentration of HYA and other marker proteins in the 4-hour-dwell dialysate at 1-year intervals. SETTING: CAPD unit in Hitachi General Hospital. PATIENTS: The subjects were 46 stable CAPD patients who underwent 104 PETs. RESULTS: A correlation was found between the length of time on dialysis and the amount of HYA excretion in the 4-hr-dwell dialysate (r = 0.403, p < 0.001). A positive but weak correlation was found between the dialysate-to-plasma ratio of the creatinine concentration and dialysate HYA excretion (r = 0.229, p < 0.05). Seven patients were over the 90th percentile in both the concentration of HYA (>349.2 ng/mL) and the amount of HYA (>743.6 microg/4-hr dwell). Five patients exceeded 1000 microg of HYA excretion in the 4-hr-dwell dialysate, 4 of whom showed an abrupt increase of HYA excretion to more than 1000 microg/4-hr dwell, and discontinued CAPD within 6 months due to ultrafiltration failure. Two of these 4 patients were diagnosed with sclerosing encapsulating peritonitis at autopsy. CONCLUSION: Intraperitoneal HYA production increased with both higher permeable membrane and the length of time on CAPD. Monitoring of HYA in the peritoneal dialysate may be useful as a marker to assess functional and morphological changes in the peritoneum in long-term CAPD patients.

[A case of nephrotic syndrome associated with myasthenia gravis and malignant thymoma].

A 26-year-old woman who presented facial and lower leg edema associated with massive proteinuria was admitted to our hospital in February 1992. Nine months before this admission, she exhibited myasthenia gravis and malignant thymoma, and underwent total thymectomy. On admission, there was no symptom of myasthenia gravis. She was diagnosed as having nephrotic syndrome and the first renal biopsy was performed. The histological findings showed membranous nephropathy. Immunofluorescent microscopy revealed that IgG and C3 were stained in a granular pattern in the periphery, and subepithelial deposits were observed in the basement membrane of the glomerulus by electron microscopy. With the administration of prednisolone, proteinuria disappeared and the nephrotic syndrome remitted. She was admitted again in January 1993 due to proteinuria and lower leg edema following cystitis. The findings of the second renal biopsy were unremarkable. She was administered cyclosporin A to improve the nephrotic syndrome and to reduce the side effects of prednisolone. The proteinuria disappeared again and this effect was dependent on the dose of cyclosporin A. Since the first administration, no symptoms of myasthenia gravis or malignant thymoma have been observed. The relationships among myasthenia gravis, malignant thymoma and nephrotic syndrome were examined. Although the first renal biopsy findings showed membranous nephropathy, from the therapeutic responses of both prednisolone and cyclosporin A, the main course of proteinuria in this case may have been due to minimal change nephrotic syndrome. We consider this case of nephrotic syndrome to be important considering its etiology and the relationship between the histological findings and its clinical course.

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 beta-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 degrees 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.

Favorable effect of hemodialysis on decreased serum antioxidant activity in hemodialysis patients demonstrated by electron spin resonance.

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.

Thiobarbituric acid reactive substances are increased in the subcutaneous fat tissue of patients with end-stage renal disease.

BACKGROUND: Patients with end-stage renal disease are thought to be in a highly peroxidative state, based on studies showing decreased serum antioxidant activity and increased peroxidative products. In this study we confirm these findings by examining lipid peroxidation in subcutaneous fat tissue in uraemia. SUBJECTS AND METHODS: Twenty-seven subcutaneous fat samples were taken from patients with end-stage renal disease when they underwent intervention for arteriovenous fistula for haemodialyis or for catheter insertion for continuous ambulatory peritoneal dialysis. The control samples were taken from 11 patients with normal renal function and without any history of renal disease who had surgical interventions. Lipid peroxides were measured as thiobarbituric acid reactive substance. RESULTS: The concentration of thiobarbituric acid reactive substances in subcutaneous fat tissue in the patients with end-stage renal disease is 1.223 +/- 0.636 nmol/mg fat tissue (mean +/- SD) whereas the level in the control group is 0.097 +/- 0.054 nmol/mg fat tissue. A comparison of the two groups by Student's t test revealed a highly significant difference (P < 0.001). CONCLUSIONS: This study supports the finding of a severe peroxidative state in patients with end-stage renal disease.

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.

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.

Synthesis of guanidinosuccinate from argininosuccinate and reactive oxygen in vitro.

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.