Yohei Miyamoto

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.

Update on the pharmacokinetics and redox properties of protein-bound uremic toxins.

Protein-bound uremic toxins, such as indoxyl sulfate, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid, p-cresyl sulfate, hippuric acid, and indoleacetic acid, have been the subjects of extensive investigations. In this review, we summarized the recent works providing the new insight on the pharmacokinetics and redox properties of these uremic toxins. They have a common characteristic of being difficult to remove by conventional dialysis because they all bind tightly to serum albumin. They are transported via organic anion transporters to various tissues, and accumulate not only in the kidney but also in other tissues including vascular endothelial cells, smooth muscle cells, osteoblasts, and the central nervous system. Accumulated uremic toxins alter nonrenal drug clearance. Intracellular accumulated uremic toxins have been linked to the induction of oxidative stress and the stimulation of proinflammatory cytokines through the production of reactive oxygen species, which play a role in the progression of chronic kidney disease and the development of complications. Unfortunately, despite the massive amount of information on the undesirable effects of uremic toxins, methods for improving the detoxification of these toxins appear to be lacking.

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.

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.

A human serum albumin–thioredoxin fusion protein prevents experimental contrast-induced nephropathy

Contrast-induced nephropathy (CIN), caused by a combination of the direct tubular toxicity of contrast media, a reduction in medullary blood flow, and the generation of reactive oxygen species, is a serious clinical problem. A need exists for effective strategies for its prevention. Thioredoxin-1 (Trx) is a low-molecular-weight endogenous redox-active protein with a short half-life in the blood due to renal excretion. We produced a long-acting form of Trx as a recombinant human albumin-Trx fusion protein (HSA-Trx) and examined its effectiveness in preventing renal injury in a rat model of ioversol-induced CIN. Compared with saline, a mixture of HSA and Trx, or Trx alone, intravenous HSA-Trx pretreatment significantly attenuated elevations in serum creatinine, blood urea nitrogen, and urinary N-acetyl-β-D-glucosaminidase along with the decrease in creatinine clearance. HSA-Trx also caused a substantial reduction in the histological features of renal tubular injuries and in the number of apoptosis-positive tubular cells. Changes in the markers 8-hydroxy deoxyguanosine and malondialdehyde indicated that HSA-Trx significantly suppressed renal oxidative stress. In HK-2 cells, HSA-Trx decreased the level of reactive oxygen species induced by hydrogen peroxide, and subsequently improved cell viability. Thus, our results suggest that due to its long-acting properties, HSA-Trx has the potential to effectively prevent CIN.

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.

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)(·-).

p‐Cresyl sulfate, a uremic toxin, causes vascular endothelial and smooth muscle cell damages by inducing oxidative stress

The major cause of death in patients with chronic kidney disease (CKD) is cardiovascular disease. Here, p‐Cresyl sulfate (PCS), a uremic toxin, is considered to be a risk factor for cardiovascular disease in CKD. However, our understanding of the vascular toxicity induced by PCS and its mechanism is incomplete. The purpose of this study was to determine whether PCS enhances the production of reactive oxygen species (ROS) in vascular endothelial and smooth muscle cells, resulting in cytotoxicity. PCS exhibited pro‐oxidant properties in human umbilical vein endothelial cells (HUVEC) and aortic smooth muscle cells (HASMC) by enhancing NADPH oxidase expression. PCS also up‐regulates the mRNA levels and the protein secretion of monocyte chemotactic protein‐1 (MCP‐1) in HUVEC. In HASMC, PCS increased the mRNA levels of alkaline phosphatase (ALP), osteopontin (OPN), core‐binding factor alpha 1, and ALP activity. The knockdown of Nox4, a subunit of NADPH oxidase, suppressed the cell toxicity induced by PCS. The vascular damage induced by PCS was largely suppressed in the presence of probenecid, an inhibitor of organic anion transporters (OAT). In PCS‐overloaded 5/6‐nephrectomized rats, plasma MCP‐1 levels, OPN expression, and ALP activity of the aortic arch were increased, accompanied by the induction of Nox4 expression. Collectively, the vascular toxicity of PCS can be attributed to its intracellular accumulation via OAT, which results in an enhanced NADPH oxidase expression and increased ROS production. In conclusion, we found for the first time that PCS could play an important role in the development of cardiovascular disease by inducing vascular toxicity in the CKD condition.

New Insight Into the Redox Properties of Uremic Solute Indoxyl Sulfate as a Pro‐ and Anti‐Oxidant

Indoxyl sulfate, an extensively investigated uremic toxin, is involved in the progression of chronic kidney disease (CKD). Recent clinical data indicate that serum levels of indoxyl sulfate are a powerful predictor of overall and cardiovascular mortality. Under CKD conditions, indoxyl sulfate induces oxidative stress, which involves the production of excessive levels of reactive oxygen species in renal tubular cells, mesangial cells, vascular endothelial cells, and osteoblast cells. In contrast, our recent findings explain, at least in part, the role that indoxyl sulfate plays in protecting against oxidative stress under normal‐physiological conditions. Namely, under CKD conditions, the pro‐oxidant properties of indoxyl sulfate exceed its anti‐oxidant properties. These findings provide new insights into the dual role of indoxyl sulfate, which appears to be concentration‐dependent, with respect to its pro‐ or anti‐oxidative properties.

Direct radical scavenging activity of benzbromarone provides beneficial antioxidant properties for hyperuricemia treatment

Uric acid exerts an important antioxidant effect against external oxidative stress under physiological conditions. However, uric acid itself can increase oxidative stress via reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation in adipocytes and vascular cells. Uric acid transporter 1 is involved in the generation of this oxidative stress. Furthermore, uric acid locally activates the renin-angiotensin system, thus producing angiotensin II and subsequently increasing intracellular oxidative stress. Benzbromarone has been reported to suppress uric acid reabsorption via uric acid transporter 1 inhibition in renal tubular cells. In this study we evaluated the in vitro antioxidant effect of benzbromarone from several perspectives. First, the direct radical-trapping capacity of benzbromarone was measured by chemiluminescence assay and electron paramagnetic resonance spectroscopy. Second, the intracellular antioxidant activity of benzbromarone in hyperuricemia was evaluated using endothelial cells. In light of these results, benzbromarone is hypothesized directly to scavenge the superoxide anion radical. In addition, benzbromarone inhibited reactive oxygen species production that was induced by angiotensin II or uric acid in endothelial cells. These findings suggest that benzbromarone possesses the ability directly to scavenge radicals and may act as an antioxidant against uric acid and angiotensin II-induced oxidative stresses in endothelial cells at therapeutically achievable levels in blood.