Spherical carbon adsorbent (AST-120) protects deterioration of renal function in chronic kidney disease rats through inhibition of reactive oxygen species production from mitochondria and reduction of serum lipid peroxidation.

BACKGROUND/AIM: An imbalance in renal redox status contributes to progression of renal dysfunction. We investigated the effects of an oral charcoal adsorbent (AST-120) on renal redox status, superoxide production from renal mitochondria, and serum lipid peroxidation using chronic kidney disease (CKD) model rats. METHODS: CKD was induced by 5/6 nephrectomy. CKD rats were divided into 2 groups: controls, and those treated with AST-120 for 20 weeks. We evaluated: (1) renal redox status by in vivo low-frequency electron spin resonance imaging (EPRI); (2) renal superoxide scavenging activity (SSA); (3) superoxide production from renal mitochondria; (4) immunostaining for Cu-Zn superoxide dismutase (SOD), and (5) oxidative stress markers including LDL-negative charge (LDL-CMF), serum lipid peroxide (LPO) and urinary hexanoyl-lysine (HEL). The effect of indoxyl sulfate, a uremic toxin, on mitochondrial superoxide production was also investigated. RESULTS: AST-120 treatment improved renal function, renal SSA, renal mitochondrial superoxide production, renal SOD expression, renal redox status by EPRI, and oxidative stress profiles by LDL-CMF, LPO and urinary HEL. Addition of indoxyl sulfate increased mitochondrial superoxide production and AST-120 also decreased this. CONCLUSIONS: Improvements in the redox status and lipid peroxidation induced by AST-120 may delay the progression of CKD.

In vivo imaging of renal redox status during azelnidipine treatment.

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.

Nitric oxide protection against adriamycin-induced tubulointerstitial injury.

It is well known that oxidative stress is related to the pathogenesis of adriamycin (ADR) nephropathy. However, it is unclear how nitric oxide (NO) is associated with the pathophysiological process after ADR administration. The NO level in a kidney homogenate was assayed by electron paramagnetic resonance (EPR) spectrometry using a direct in vivo NO trapping technique after ADR administration. N-(3-(aminomethyl)benzyl)acetamidine (1400W) was used as a specific, inducible nitric oxide synthase (iNOS) inhibitor. The levels of NO after ADR administration gradually increased for 6 h and then decreased until 24 h after ADR administration. The fractional excretion of Na (FE(Na)) in the urine was elevated in the ADR group on day 1. Pre-treatment of the animals with 1400W attenuated the increase in NO levels despite further elevation of FE(Na). These findings suggest that iNOS-derived NO does not produce a harmful effect but rather protects the ADR-treated kidney against sodium excretion.

In vivo detection of intrinsic reactive oxygen species using acyl-protected hydroxylamine in puromycin nephrosis.

Intrinsic reactive oxygen species (ROS) in a rat model of human minimal change nephropathy were detected directly using an in vivo electron paramagnetic resonance (EPR) method with 1-acetoxy-3-carbamoyl-2,2,5,5-tetramethylpyrrolidine (ACP) in real time. The nephrosis was induced by the intravenous administration of 75 mg/kg of puromycin aminonucleoside (PAN). It was found that ROS in the kidney were increased 1 h after the administration of PAN. This increased oxidative stress declined at 24 h and returned to a normal level 3 days after PAN administration. This is the first non-invasive in vivo detection and quantification of specific ROS in an experimental nephrosis model.

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.

Electron paramagnetic resonance imaging of oxidative stress in renal disease.

The importance of analyzing the kinetics of reactive oxygen species or related substances in vivo is increasing. Electron paramagnetic resonance (EPR) is currently a powerful method for in vivo, non-invasive analysis of oxidative stress. We have applied EPR imaging for murine renal ischemia-reperfusion injury, as a model of acute renal damage, and NF-E2-related factor 2 (Nrf2)-deficient mice, a model for chronic progressive renal disease. In the ischemia-reperfusion model, EPR imaging revealed that the renal radical-reducing activity showed only partial recovery when serum creatinine and BUN have recovered. In the Nrf2-deficient mice, we have revealed that the impaired antioxidant activity is brought by both Nrf2 deficiency and the aging process and may play a key role in the onset of autoimmune nephritis in this model. In addition, EPR imaging is recently being applied to the redox analysis of several nephrosis models, hypertensive rats and streptozotocin-induced diabetic rats. This article summarizes the nephrological application of EPR imaging and in vivo EPR.

Identification by an EPR technique of decreased mitochondrial reducing activity in puromycin aminonucleoside-induced nephrosis.

The temporal changes in the electron paramagnetic resonance (EPR) signal intensities of a nitroxide radical, 4-hydroxy 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidney in rat puromycin aminonucleoside (PAN) nephrosis were investigated in vivo and in vitro. The rats of the PAN nephrosis group received intraperitoneal injections of PAN at 75 mg/kg body weight while those of control group received saline. The in vivo renal half-lives of TEMPOL were calculated from the decay curve of EPR signal intensities after the intravenous injection of the TEMPOL solution. The mitochondrial half-lives were obtained from the decay curve of the EPR signals after mixing the mitochondrial fraction of the kidney and TEMPOL solution. The in vivo half-lives of TEMPOL of the kidney from 7 to 14 d after PAN administration were significantly longer than those of the controls. The mitochondrial half-lives of TEMPOL on the 9th day after the PAN administration prolonged remarkably compared to the controls (378 +/- 69 vs. 676 +/- 183 s, p <.01). These findings indicate that the in vivo and mitochondrial reducing activity in PAN treated rats decreased markedly, because the half-life of TEMPOL in the kidney reflects the renal reducing activity.

EPR imaging of reducing activity in Nrf2 transcriptional factor-deficient mice.

Mice that lack the Nrf2 (NF-E2-related factor 2) transcription factor develop a lupus-like autoimmune nephritis. The tissue-reducing activity of Nrf2-deficient mice was evaluated using a combination of real-time EPR imaging and spin probe kinetic analysis. Substantial delay in the spin probe 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl (Carbamoyl-PROXYL) disappearance in the liver and kidneys of Nrf2-deficient mice was observed by EPR imaging. The half-life of the spin probe in the upper abdominal area was prolonged in both the Nrf2-deficient mice and in aged mice. The combination of Nrf2 deficiency and aging in female mice resulted in the most prolonged half-life of disappearance, which was four times longer than that of juvenile female mice with a wild-type genotype. These results indicate that the low reducing activity in these organs is brought about by both Nrf2 deficiency and the aging process, and it may play a key role in the onset of autoimmune nephritis. This combination of the EPR imaging and half-life analysis appears to be a very powerful tool in the real-time analysis of reducing activity.

Treatment with the purine synthesis inhibitor mizoribine for ANCA-associated renal vasculitis.

BACKGROUND: The common treatment for antineutrophil cytoplasmic autoantibody (ANCA)-associated renal vasculitis is oral cyclophosphamide (CYC)-corticosteroid combination therapy. However, there are serious complications associated with long-term use of CYC. In this study, we investigate the efficacy of a purine synthesis inhibitor, mizoribine, for patients at high risk for relapse. METHODS: Our study included 5 patients, 4 patients with myeloperoxidase (MPO)-ANCA-associated renal vasculitis and 1 patient with proteinase 3 (PR3)-ANCA-associated renal vasculitis, who had achieved remission through treatment with methylprednisolone pulse therapy, corticosteroids, and CYC. When their ANCA titers were found to be greater than normal range after remission status, mizoribine treatment was initiated. RESULTS: Median time from initial treatment to first administration of mizoribine was 40.0 months (range, 24 to 51 months). Median follow-up was 13.0 months (range, 6 to 16 months). Before initiation of mizoribine treatment, no patient had experienced relapse and ANCA titers were less than the detectable range in all patients at 3 months before mizoribine administration. When mizoribine administration was started, ANCA titers were elevated in all patients (median MPO-ANCA, 101 ELISA units [EU]; range, 65 to 154 EU; PR3-ANCA, 55 EU), but no new symptoms or signs of relapse were noted. After 2 months of mizoribine treatment, only 1 patient had experienced a relapse; however, ANCA titers had decreased in all other patients and normalized in 3 patients. No adverse effects appeared in any patient. CONCLUSION: Considering the balance between suppression of disease activity and adverse effects of treatment, mizoribine may be useful for preemptive treatment for patients with ANCA-associated renal vasculitis at high risk for relapse.

Stable nitroxide Tempol ameliorates brain injury by inhibiting lipid peroxidation in a rat model of transient focal cerebral ischemia.

Oxygen free radicals have been implicated in the pathogenesis of cerebral ischemia and reperfusion injury. 4-Hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (Tempol) has been reported as a stable nitroxide and a membrane-permeable free radical scavenger. This study was performed to investigate the mechanism of Tempol in attenuating ischemia-reperfusion injury in a rat model of transient focal cerebral ischemia. We measured the cerebral 2,3-dihydroxybenzoic acid (DHBA) level as the amount of hydroxyl radical production using a microdialysis technique with salicylic acids trapping during ischemia and reperfusion. The concentration of cerebral thiobarbituric acid reactive substances (TBARS), representing the extent of lipid peroxidation by free radicals, and the area of cerebral infarction were also measured. The level of cerebral 2,3-DHBA was increased during ischemia and reperfusion, especially during the early reperfusion stage at the periphery of the infarct area (nearly 500-fold). Intravenous administration of Tempol at the time of reperfusion reduced 2,3-DHBA production (Vehicle group: 472.2+/-196.2, Tempol group: 238.3+/-77.2) and the cerebral TBARS level (Vehicle group: 541.7+/-84.7, Tempol group: 339.0+/-147.2 nmol/g), and decreased the size of the cerebral infarction (Vehicle group: 202.2+/-98.4, Tempol group: 98.5+/-13.7 mm(3)). In contrast, Tempol administered 15 min prior to reperfusion reduced neither the TBARS level nor the size of the infarction. These results indicate that Tempol administration at the time of reperfusion reduced lipid peroxidation by scavenging free radicals, resulting in a reduction of the infarct size.

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.

Increased levels of lipid peroxides as predictive of symptomatic vasospasm and poor outcome after aneurysmal subarachnoid hemorrhage.

OBJECT: Cerebral vasospasm remains a devastating medical complication of aneurysmal subarachnoid hemorrhage (SAH). Reactive oxygen species and subsequent lipid peroxidation are reported to participate in the causes of cerebral vasospasm. This clinical study was performed to investigate the relationships between levels of lipid peroxides in cerebrospinal fluid (CSF) and both delayed cerebral vasospasm and clinical outcome after SAH. METHODS: Levels of phosphatidylcholine hydroperoxide (PCOOH) and cholesteryl ester hydroperoxide (CEOOH) in the CSF were measured in 20 patients with aneurysmal SAH. The patients' CSF was collected within 48 hours of hemorrhage onset and on Day 6 or 7 post-SAH. On Day 7, angiography was performed to verify the degree and extent of the vasospasm. The relationship between the patients' clinical profiles and the levels of lipid peroxides in the CSF were investigated. Both PCOOH and CEOOH were detectable in CSF, and their levels decreased within 7 days after onset of SAH. The levels of CEOOH within 48 hours after onset of hemorrhage were significantly higher in patients in whom symptomatic vasospasm later developed than in patients in whom symptomatic vasospasm did not develop (p = 0.002). Levels of PCOOH measured within 48 hours after onset of hemorrhage were significantly higher in patients with poor outcomes than in patients with good outcomes (p = 0.043). CONCLUSIONS: Increased levels of lipid peroxides measured in the CSF during the acute stage of SAH were predictive of both symptomatic vasospasm and poor outcome. Measurements of lipid peroxides in the CSF may be useful prognostically for patient outcomes as well as for predicting symptomatic vasospasm.

In vivo temporal EPR imaging for estimating the kinetics of a nitroxide radical in the renal parenchyma and pelvis in rats.

The kinetics of a nitroxide radical in the renal parenchyma and pelvis in rats were investigated by employing an in vivo EPR imaging system equipped with a surface-coil-type resonator (SCR). The exposed kidney of a living rat was inserted into the single-turn coil of the SCR, with the renal major axis aligned with the direction of alternative magnetic field (B(1)). After the injection of nitroxide radical via the tail vein, EPR measurements were repeated. From the temporal EPR images of the kidney on the 2-D projection to the plane which is perpendicular to the direction of B(1,) the decay rate of nitroxide radical in the renal parenchyma and pelvis was estimated. The parenchymal decay rate was found to be significantly shorter than that for the pelvis.

Antioxidant capacity in rat skeletal muscle tissues determined by electron spin resonance.

The amount of radical scavenging activity in muscle is unknown. The present study examines whether electron spin resonance (ESR) could measure and distinguish antioxidant capacity in muscle with different contractile and metabolic characteristics. Specimens of the soleus, plantaris, gastrocnemius (deep/surface portions), heart and diaphragm were obtained from female Wistar rats (n=7; 12 weeks old). Scavenging activity against superoxide anions in these specimens were determined by ESR using a spin-trapping chemical (5,5-dimethyl-1-pyrroline-N-oxide). The ESR signal intensity of reaction mixtures containing muscle tissues was significantly lower in the heart, soleus, diaphragm and deep portion of the gastrocnemius than in the plataris and surface portion of the gastrocnemius. Thus, the amount of scavenging activity converted into superoxide dismutase activity was the highest in the heart, and higher in the soleus, diaphragm and deep portion of the gastrocnemius than in other muscles (ANOVA, P<0.01). In addition, scavenging activity significantly correlated with citrate synthase activity (r=0.72, P<0.01, n=42) and myoglobin content (r=0.63, P<0.01, n=42). These findings suggested that ESR and spin-trapping can be detect differences in free radical scavenging activity among muscle tissues with different metabolic characteristics.

Management of unruptured cerebral aneurysms in patients with polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (PKD) is a hereditary disorder characterized by bilateral multiple renal cysts and early onset chronic renal failure. PKD patients tend to suffer their subarachnoid hemorrhage at a younger age. Unruptured aneurysms in PKD patients are not always innocuous, and proactive treatment has been indicated for these lesions. However, the management of PKD patients undergoing unruptured cerebral aneurysm surgery has been documented on only a few occasions. The purpose of this study was to better define the management of unruptured cerebral aneurysms in patients with PKD. METHODS: We present a retrospective review of the management of unruptured cerebral aneurysms in 16 patients with PKD. Eight patients were maintained through chronic hemodialysis whereas the remaining 8 patients did not require hemodialysis, at the time of treatment of their cerebral aneurysms. The mean follow-up period was 24 months. RESULTS: In the nonhemodialysis patients prophylactic hemodialysis was routinely performed after cerebral angiography to prevent deterioration of the pre-existing renal dysfunction. Microsurgical clipping of the aneurysm was performed in 15 patients (7 nonhemodialysis and 8 hemodialysis patients) and intravascular coil embolization was performed in 1 nonhemodialysis patient. One nonhemodialysis patient who underwent microsurgical clipping required a temporary hemodialysis after surgery, but the patient was not shifted to chronic hemodialysis. No patients developed postprocedural complications, and each showed an excellent recovery. CONCLUSION: PKD patients with unruptured cerebral aneurysms can be safely treated with an appropriate treatment strategy including the use of prophylactic hemodialysis.

Effect of CV159-Ca(2+)/calmodulin blockade on redox status hepatic ischemia-reperfusion injury in mice evaluated by a newly developed in vivo EPR imaging technique.

1,4-Dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridinedicarboxylic acid methyl 6-(5-phenyl-3-pyrazolyloxy)hexyl ester (CV159) exhibits selective blocking of Ca(2+)/calmodulin and inhibits Ca(2+) overloading in living organisms. The effects of this antagonist in mice with hepatic ischemia-reperfusion injury were investigated using electron paramagnetic resonance imaging (EPRI) and ex vivo EPR (x-band EPR) techniques. The EPRI determined that the 3-carbamoyl-2,2,5,5-tetramethylpyrrolidine-1-oxyl half-life in CV159-treated mice was significantly shorter than that in untreated mice and was almost equal to that in the sham group. Both the cytosolic and the mitochondrial superoxide scavenging activities in CV-treated mice were significantly higher than that in untreated mice. Faint staining of the anti-superoxide dismutase antibody and strong staining of anti-inducible nitric oxide synthase antibody were observed in the liver of control group. In contrast to these findings, immunostaining of these antibodies in the liver of CV159-treated mice were reversed compared to control group. Western blotting showed that CV159 contributed to the high superoxide dismutase expression and low expression of inducible nitric oxide synthase. The alanine aminotransferase level in CV159-treated mice significantly decreased in comparison to that observed in the untreated mice. We conclude that CV159 retains its organ-reducing activity against radicals in hepatic reperfusion injury, which is mediated by the inhibition of Ca(2+) overloading.

Proanthocyanidin promotes free radical-scavenging activity in muscle tissues and plasma.

The present study was carried out to clarify the effect of oral administration of proanthocyanidin (PA) on radical-scavenging activity in muscle and plasma using electron spin resonance (ESR). Eight-week-old male Wistar rats were orally administered with 3 doses per day of 1 mL of 0.05% (PA0.05), 0.5% (PA0.5) or 5% (PA5) PA for 1 week. Control animals received the same volume of distilled water. We also examined the effect of a single dose of 0.5% PA. Blood and muscle were collected from rats 1 h after the final administration. Scavenging activity against superoxide anions in the plasma and m. soleus (Sol), m. plantaris (Pla), deep and surface areas of the m. gastrocnemius (GasD and GasS, respectively) and myocardium (Hrt) was determined using ESR with the spin trap, 5,5-dimethyl-1-pyrroline-N-oxide The scavenging activity in plasma for all groups given PA was 34%-44% higher than the control (p<0.05). The scavenging activity in Hrt, Sol and GasD increased by up to 50% compared with the control and tended to increase depending on the dose of PA (p<0.05). The impact of a single dose of PA was undetectable in all tissues. These results suggested that 1 week of oral PA improves the radical-scavenging activity in both plasma and muscle, especially in highly oxidative muscle. A single dose of PA was insufficient to improve the antioxidative capacity of muscle tissues.

Evaluation of adriamycin nephropathy by an in vivo electron paramagnetic resonance.

A rat model for human minimal change nephropathy was obtained by the intravenous injection of adriamycin (ADR) at 5 mg/kg. By using an in vivo electron paramagnetic resonance (EPR) spectrometer operating at 700 MHz, the temporal changes in signal intensities of a nitroxide radical, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidneys of rats with ADR nephropathy were investigated. The decay rate of the EPR signal intensity obtained in the kidney is indicative of the renal reducing ability. It was found that the reducing ability in the kidney declined on the 7th day after ADR administration and recovered after the 14th day. Impairment of the reducing ability occurred before the appearance of continuous urinary protein. The in vitro EPR study showed that this impairment of in vivo renal reducing ability is related to impairment of the reducing ability in the mitochondria.

Normalizing renal reducing ability prevents adriamycin-induced proteinuria.

Reactive oxygen species play an important role in adriamycin (ADR) nephropathy. We showed by in vivo electron paramagnetic resonance (EPR) that renal reducing ability (RRA) declined on the 7th day after ADR administration. Proteinuria appeared after the decline in RRA. The aim of this study was to prove by in vivo EPR whether the decline in RRA is altered by scavengers such as dimethyl sulfoxide (DMSO) and dimethylthiourea (DMTU) and that it is this change which is responsible for the proteinuria in ADR nephropathy. By showing that DMSO and DMTU ameliorate the RRA, we demonstrate that the decline in RRA is related to ADR-induced proteinuria.

Erythropoietin-resistant anaemia in a predialysis patient with Klinefelter syndrome.

A diabetic predialysis patient who had significantly reduced sensitivity to erythropoietin therapy was admitted to Tsukuba University Hospital. Many factors that might have been the cause of the erythropoietin resistance were examined, and a diagnosis of refractory anaemia was made based on a bone marrow aspiration biopsy. A cytogenetic abnormality (47, XXY) was also detected in the bone marrow biopsy specimen, and hence the patient was also diagnosed with Klinefelter syndrome. It was suspected that the sex chromosome abnormality influenced glucose intolerance, renal insufficiency, and erythropoietin resistance due to myelodysplastic changes in the bone marrow.