Daisuke Kadowaki

p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase

The accumulation of p-cresyl sulfate (PCS), a uremic toxin, is associated with the mortality rate of chronic kidney disease patients; however, the biological functions and the mechanism of its action remain largely unknown. Here we determine whether PCS enhances the production of reactive oxygen species (ROS) in renal tubular cells resulting in cytotoxicity. PCS exhibited pro-oxidant properties in human tubular epithelial cells by enhancing NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity. PCS also upregulated mRNA levels of inflammatory cytokines and active TGF-β1 protein secretion associated with renal fibrosis. Knockdown of p22(phox) or Nox4 expression suppressed the effect of PCS, underlining the importance of NADPH oxidase activation on its mechanism of action. PCS also reduced cell viability by increasing ROS production. The toxicity of PCS was largely suppressed in the presence of probenecid, an organic acid transport inhibitor. Administration of PCS for 4 weeks caused significant renal tubular damage in 5/6-nephrectomized rats by enhancing oxidative stress. Thus, the renal toxicity of PCS is attributed to its intracellular accumulation, leading to both increased NADPH oxidase activity and ROS production, which, in turn, triggers induction of inflammatory cytokines involved in renal fibrosis. This mechanism is similar to that for the renal toxicity of indoxyl sulfate.

Cys34-Cysteinylated Human Serum Albumin Is a Sensitive Plasma Marker in Oxidative Stress-Related Chronic Diseases

The degree of oxidized cysteine (Cys) 34 in human serum albumin (HSA), as determined by high performance liquid chromatography (HPLC), is correlated with oxidative stress related pathological conditions. In order to further characterize the oxidation of Cys34-HSA at the molecular level and to develop a suitable analytical method for a rapid and sensitive clinical laboratory analysis, the use of electrospray ionization time-of-flight mass spectrometer (ESI-TOFMS) was evaluated. A marked increase in the cysteinylation of Cys34 occurs in chronic liver and kidney diseases and diabetes mellitus. A significant positive correlation was observed between the Cys-Cys34-HSA fraction of plasma samples obtained from 229 patients, as determined by ESI-TOFMS, and the degree of oxidized Cys34-HSA determined by HPLC. The Cys-Cys34-HSA fraction was significantly increased with the progression of liver cirrhosis, and was reduced by branched chain amino acids (BCAA) treatment. The changes in the Cys-Cys34-HSA fraction were significantly correlated with the alternations of the plasma levels of advanced oxidized protein products, an oxidative stress marker for proteins. The binding ability of endogenous substances (bilirubin and tryptophan) and drugs (warfarin and diazepam) to HSA purified from chronic liver disease patients were significantly suppressed but significantly improved by BCAA supplementation. Interestingly, the changes in this physiological function of HSA in chronic liver disease were correlated with the Cys-Cys34-HSA fraction. In conclusion, ESI-TOFMS is a suitable high throughput method for the rapid and sensitive quantification of Cys-Cys34-HSA in a large number of samples for evaluating oxidative stress related chronic disease progression or in response to a treatment.

Organic anion transporters play an important role in the uptake of p -cresyl sulfate, a uremic toxin, in the kidney

BACKGROUND p-Cresyl sulfate (PCS), a recently identified anionic uremic toxin, is the main circulating metabolite of p-cresol. In cases of chronic kidney disease (CKD), it might be associated with cardiovascular outcomes and the progression of CKD. However, the renal excretion pathway of PCS is currently unknown. The objective of the present study was to determine whether organic anion transporters (OATs), which are renal tubular basolateral membrane transporters, play an important role in this process. METHODS The uptake of PCS was investigated using rat renal cortical slices and human proximal tubular cells (HK-2). The active uptake velocity was calculated by subtracting the uptake velocity at 4°C (nonspecific uptake) from that at 37°C. RESULTS As evidenced by renal cortical slice experiments, the uptake of PCS was saturable with a mean K(m) of 231.6 μM, indicating that the active transport is involved in the basolateral uptake of PCS. Similar results were also observed in HK-2 cells. The active transport of PCS was significantly suppressed by inhibitors of OATs, such as probenecid, benzylpenicillin, p-aminohippuric acid and estrone sulfate. Similar inhibitions were observed in the presence of indoxyl sulfate and 3-carboxy-4-methyl-5-propyl-2-furanpropionate, OATs substrates among uremic toxins. In contrast, digoxin and tetraethylammonium that did not interact with OATs had little inhibitory effect. CONCLUSIONS The findings of the present study strongly suggest that PCS serves as a substrate for OATs, is preferentially recognized by OAT3 and plays a key role in the renal tubular secretion process.

Antioxidant effects of a dietary supplement: Reduction of indices of oxidative stress in normal subjects by water-soluble chitosan

The effect of water-soluble chitosan, a natural polymer derived from chitin, on indices of oxidative stress was investigated in normal volunteers. Treatment with chitosan for 4 weeks produced a significant decrease in levels of plasma glucose, atherogenic index and led to increase in high density lipoprotein cholesterol (HDL). Chitosan treatment also lowered the ratio of oxidized to reduced albumin and increased total plasma antioxidant activity (TPA). There was good correlation between TPA and oxidized albumin ratio. The results indicate that oxidized albumin ratio represents a potentially useful marker of oxidative stress. In in vitro studies, albumin carbonyls and hydroperoxides were significantly decreased in a time-dependent manner in the presence of chitosan, compared with controls (p<0.05). Chitosan also reduced two stable radicals in a dose- and time-dependent manner. The results suggest that chitosan has a direct antioxidant activity in systemic circulation by lowering the indices of oxidative stress in both in vitro and in vivo studies. This may confer benefits additional to the reduction in plasma carbohydrate and increase in HDL levels. It may also inhibit oxidation of serum albumin commonly observed in patients undergoing hemodialysis, resulting in reduction of oxidative stress associated with uremia.

Effect of olmesartan on oxidative stress in hemodialysis patients

The effect of olmesartan, an inverse angiotensin II type 1 receptor blocker (ARB), on oxidative stress in hemodialysis (HD) patients is not fully understood, and has not been widely investigated in vitro or in vivo. We determined the amount of oxidized albumin and albumin hydroperoxides formed during incubation in the absence and presence of olmesartan by high-performance liquid chromatography (HPLC) and by a ferrous oxidation xylenol assay in an in vitro study. Six hypertensive HD patients were treated with 40 mg of olmesartan once daily, and blood pressure monitoring (BPM) was performed after 0, 4, and 8 weeks of treatment. The ratio of oxidized to unoxidized albumin was also determined. The oxidized albumin ratios and levels of albumin hydroperoxides were significantly decreased in a concentration-dependent manner in the presence of olmesartan, compared with the absence of olmesartan (p<0.05) in in vitro studies. In HD patients, olmesartan also significantly reduced systolic and diastolic blood pressure after 4 weeks, with a further significant decrease after 8 weeks. The ratio of oxidized to unoxidized albumin was markedly decreased after 4 weeks and these lower levels were maintained at 8 weeks. Olmesartan effectively lowered the extent of oxidation of albumin in both in vitro and in vivo studies, and this effect might confer benefits beyond a reduction in blood pressure.

Interaction between Two Sulfate-Conjugated Uremic Toxins, p-Cresyl Sulfate and Indoxyl Sulfate, during Binding with Human Serum Albumin

Recently, p-cresyl sulfate (PCS) has been identified as a protein-bound uremic toxin. Moreover, the serum-free concentration of PCS, which is associated with its efficacy of hemodialysis, appears to be a good predictor of survival in chronic kidney disease (CKD). We previously found that PCS interacts with indoxyl sulfate (IS), another sulfate-conjugated uremic toxin, during renal excretion via a common transporter. The purpose of this study was to further investigate the interaction between PCS and IS on the binding to human serum albumin (HSA). Here, we used ultrafiltration to show that there is only one high-affinity binding site for PCS, with a binding constant on the order of 105 M−1 (i.e., comparable to that of IS). However, a binding constant of the low-affinity binding site for PCS is 2.5-fold greater than that for IS. Displacement of a fluorescence probe showed that PCS mainly binds to site II, which is the high-affinity site for PCS, on HSA. This finding was further supported by experiments using mutant HSA (R410A/Y411A) that displayed reduced site II ligand binding. A Klotz analysis showed that there could be competitive inhibition between PCS and IS on HSA binding. A similar interaction between PCS and IS on HSA was also observed under the conditions mimicking CKD stage 4 to 5. The present study suggests that competitive interactions between PCS and IS in both HSA binding and the renal excretion process could contribute to fluctuations in their free serum concentrations in patients with CKD.

Hemoglobin vesicles, polyethylene glycol (PEG)ylated liposomes developed as a red blood cell substitute, do not induce the accelerated blood clearance phenomenon in mice.

The hemoglobin vesicle (HbV) is an artificial oxygen carrier encapsulating a concentrated hemoglobin solution in a liposome of which the surface is covered with polyethylene glycol (PEG). It was recently reported that repeated injections of PEGylated liposomes induce the accelerated blood clearance (ABC) phenomenon, in which serum anti-PEG IgM plays an essential role. To examine this issue, we investigated whether HbV induces the ABC phenomenon in mice at a dose of 0.1 mg Hb/kg, a dose that is generally known to induce the ABC phenomenon, or at 1400 mg Hb/kg, which is proposed for clinical use. At 7 days after the first injection of nonlabeled HbV (0.1 mg Hb/kg), the mice received HbV in which the Hb had been labeled with 125I. After a second injection, HbV was rapidly cleared from the circulation, and uptake clearances in liver and spleen were significantly increased. In contrast, at a dose of 1400 mg Hb/kg, the pharmacokinetics of HbV was negligibly affected by repeated injection. It is interesting to note that IgM against HbV was produced 7 days postinjection at both of the above doses, and their recognition site was determined to be 1,2-distearoyl-sn-glycero-3-phosphatidylethanolamine-N-PEG in HbV. These results suggest that a clinical dose of HbV does not induce the ABC phenomenon, and that suppression of ABC phenomenon is caused by the saturation of phagocytic processing by the mononuclear phagocyte system. Thus, we conclude that induction of the ABC phenomenon would not be an issue in the dose regimen used in clinical settings.

The uremic solute indoxyl sulfate acts as an antioxidant against superoxide anion radicals under normal-physiological conditions

The effect of the uremic solute indoxyl sulfate (IS) on scavenging superoxide anion radicals ( ) generated from both the xanthine/xanthine oxidase (X/XO) system and activated neutrophils was investigated by electron paramagnetic resonance spectroscopy, combined with 2‐ethoxycarbonyl‐2‐methyl‐3,4‐dihydro‐2H‐pyrrole‐1‐oxide (EMPO). The findings show that the presence of normal‐physiological serum concentrations of IS (0.1–10 μM) resulted in decreased formation of EMPO‐superoxide adduct without affecting XO activity. Furthermore, IS showed scavenging activity against cell‐derived generated from activated neutrophils. In addition, IS also eliminated hydroxyl radicals. These findings suggest that IS acts as a novel endogenous antioxidant under normal‐physiological conditions.

Vitamin D Receptor Activator Reduces Oxidative Stress in Hemodialysis Patients With Secondary Hyperparathyroidism

Treatment with a vitamin D receptor activator (VDRA) has survival benefits probably related to its effects beyond the traditional role in mineral metabolism. We hypothesized that VDRA reduces oxidative stress in hemodialysis (HD) patients. To test this hypothesis, we investigated the effect of VDRA on the oxidative status of albumin in HD patients with secondary hyperparathyroidism. Eleven HD patients with secondary hyperparathyroidism were treated with calcitriol at an intravenous dose of 1.5 µg/week for four weeks. Serum intact parathyroid hormone, calcium and phosphorus were monitored and we measured the amount of oxidized albumin and albumin hydroperoxides form before and after calcitriol treatment. The ratio of oxidized to un‐oxidized albumin was determined as a representative marker of oxidative stress. The radical scavenging activity of albumin was also evaluated. After four weeks of calcitriol therapy, there were no significant changes in serum intact parathyroid hormone, calcium, or phosphorus levels; however, the ratio of oxidized to un‐oxidized albumin was markedly decreased and serum thiol content was significantly increased after calcitriol treatment. Furthermore, the radical scavenging activity of albumin was greater after calcitriol treatment compared with that of untreated albumin. Our data suggest that intravenous calcitriol treatment reduces oxidative stress and strengthens antioxidant defenses by inhibiting albumin oxidation in HD patients with secondary hyperparathyroidism.

A uremic toxin, 3-carboxy-4-methyl-5-propyl-2-furanpropionate induces cell damage to proximal tubular cells via the generation of a radical intermediate.

3-Carboxy-4-methyl-5-propyl-2-furanpropionate (CMPF), a furan fatty acid uremic toxin (UT) and a substrate for organic ion transporters, contributes to the accumulation of CMPF in renal tubular cells. Although oxidative stress induced by UTs has been proposed as a mechanism of its toxicity in chronic kidney disease, little information is available regarding the redox property of CMPF and its relation to renal cell damage. The findings herein show that CMPF enhances the production of reactive oxygen species (ROS) in HK-2 cells in the presence of angiotensin II (A-II), an inducer of O(2)(·-). When iron is also present, CMPF and A-II induce the Fenton reaction, resulting in a further increase in ROS production. Such CMPF-induced oxidative stress increases TGF-β1 secretion in HK-2 cells, and a positive correlation between CMPF-induced ROS production and the secretion of active TGF-β1 was observed. CMPF caused a reduction in cell viability which was negatively correlated with intracellular ROS production. These negative effects of CMPF in HK-2 cells were completely suppressed by probenecid, an inhibitor of organic anion transport. Interestingly, in vitro ROS assays indicate that CMPF directly interacts with superoxide anion radicals (O(2)(·-)) and peroxy radicals (LOO) to produce CMPF radicals. The subsequent interaction of CMPF radicals with dissolved oxygen leads to the overproduction of O(2)(·-). Based on these findings, we conclude that CMPF, which accumulates in the renal cells, appears to play a prominent role as a pro-oxidant which subsequently leads to renal cellular damage via the overproduction of O(2)(·-).