Urate, a naturally occurring product of purine metabolism, is a scavenger of biological oxidants implicated in numerous disease processes, as demonstrated by its capacity of neuroprotection. It is present at higher levels in human blood (200 500 microM) than in other mammals, because humans have an effective renal urate reabsorption system, despite their evolutionary loss of hepatic uricase by mutational silencing. The molecular basis for urate handling in the human kidney remains unclear because of difficulties in understanding diverse urate transport systems and species differences. Here we identify the long-hypothesized urate transporter in the human kidney (URAT1, encoded by SLC22A12), a urate anion exchanger regulating blood urate levels and targeted by uricosuric and antiuricosuric agents (which affect excretion of uric acid). Moreover, we provide evidence that patients with idiopathic renal hypouricaemia (lack of blood uric acid) have defects in SLC22A12. Identification of URAT1 should provide insights into the nature of urate homeostasis, as well as lead to the development of better agents against hyperuricaemia, a disadvantage concomitant with human evolution.

Cardiovascular disease (CVD) is prevalent in patients with chronic kidney disease (CKD). In hemodialysis (HD) patients, some protein-bound uremic toxins are considered to be associated with CVD. However, it is not yet known which uremic toxins are important in terms of endothelial toxicity. Serum samples were obtained from 45 HD patients before and after HD. Total and free serum concentrations of indoxyl sulfate, indoxyl glucuronide, indoleacetic acid, p-cresyl sulfate, p-cresyl glucuronide, phenyl sulfate, phenyl glucuronide, phenylacetic acid, phenylacetyl glutamine, hippuric acid, 4-ethylphenyl sulfate, and 3-carboxy-4-methyl-5-propyl-2-furanpropionic acid (CMPF) were simultaneously measured by liquid chromatography/electrospray ionization-mass spectrometry/mass spectrometry (LC/ESI-MS/MS). The effects of these solutes at their pre-HD mean and maximum serum concentrations on reactive oxygen species (ROS) production in human umbilical vein endothelial cells (HUVEC) were measured with a ROS probe. Serum levels of 11 of the solutes (all except 4-ethylphenyl sulfate) were significantly increased in HD patients compared to healthy subjects. All 12 solutes showed changes in their protein-binding ratios. In particular, indoxyl sulfate, p-cresyl sulfate, CMPF, and 4-ethylphenyl sulfate showed high protein-binding ratios (>95 %) and low reduction rates by HD (<35 %). Indoxyl sulfate at its mean and maximum pre-HD serum concentrations-even with 4 % albumin-stimulated ROS production in HUVEC most intensely, followed by CMPF. In conclusion, the serum levels of 11 protein-bound uremic toxins were increased in HD patients. Indoxyl sulfate, p-cresyl sulfate, and CMPF could not be removed efficiently by HD due to their high protein-binding ratios. Indoxyl sulfate most intensely induced endothelial ROS production, followed by CMPF.

Abstract. In uremic patients, various uremic toxins are accumulated and exert various biologic effects on uremia. Indoxyl sulfate (IS) is one of uremic toxins that is derived from dietary protein, and serum levels of IS are markedly increased in both uremic rats and patients. It has been previously reported that the accumulation of IS promotes the progression of chronic renal failure (CRF). This study demonstrates the role of rat organic anion transporters (rOATs) in the transport of IS and the induction of its nephrotoxicity. The administration of IS to 5/6-nephrectomized rats caused a faster progression of CRF, and immunohistochemistry revealed that IS was detected in the proximal and distal tubules where rOAT1 (proximal tubules) and/or rOAT3 (proximal and distal tubules) were also shown to be localized. In in vitro study, the proximal tubular cells derived from mouse that stably express rOAT1 (S2 rOAT1) and rOAT3 (S2 rOAT3) were established. IS inhibited organic anion uptake by S2 rOAT1 and S2 rOAT3, and the Ki values were 34.2 and 74.4 M, respectively. Compared with mock, S2 rOAT1 and S2 rOAT3 exhibited higher levels of IS uptake, which was inhibited by probenecid and cilastatin, organic anion transport inhibitors. The addition of IS induced a decrease in the viability of S2 rOAT1 and S2 rOAT3 as compared with the mock, which was rescued by probenecid. These results suggest that rOAT1 and rOAT3 play an important role in the transcellular transport of IS and the induction of its nephrotoxicity.

Summary: Recent studies suggest that advanced glycation endproducts play an important role in cardiovascular complications of ageing, diabetes and end-stage renal failure. Since highly elevated levels of advanced glycation endproducts are present in serum of patients on maintenance haemodialysis, an accurate and rapid assay for their determination would be useful. This would be particularly valuable for monitoring the removal of advanced glycation endproducts by novel dialysis membranes, as well as the effect of new drugs for the inhibition of their formation.Measurement of advanced glycation endproducts in serum was performed by two competitive ELISAs, using a monoclonal antibody directed against imidazolone, an advanced glycation endproduct formed by the reaction of arginine with 3-deoxyglucosone, and a polyclonal antibody directed against keyhole limpet haemocyanin-advanced glycation endproduct, as well as by quantitative fluorescence spectroscopy.Each of the assays showed significant differences between the controls and the maintenance haemodialysis patients. Advanced glycation endproduct levels determined by each of the ELISAs correlated with total and protein-bound fluorescence, but not with each other, suggesting a variable distribution of advanced glycation endproducts on serum proteins among the maintenance haemodialysis patients.

Background/Aim: Indoxyl sulfate, a uremic toxin, is considered a risk factor for cardiovascular disease (CVD) in chronic kidney disease (CKD). The present study aimed to determine whether indoxyl sulfate increases the expression of intercellular adhesion molecule-1 (ICAM-1) and monocyte chemotactic protein-1 (MCP-1) by reactive oxygen species (ROS)-induced activation of nuclear factor-ĸB (NF-ĸB) in vascular endothelial cells. Methods: Human umbilical vein endothelial cells (HUVEC) were incubated with indoxyl sulfate. The expression of ICAM-1 and MCP-1 in HUVEC was analyzed by quantitative reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. Phospho-NF-ĸB p65 (Ser 536), an active form of the NF-ĸB subunit, was determined by Western blotting. Results: Indoxyl sulfate significantly increased the mRNA expression of ICAM-1 and MCP-1 in HUVEC in a time- and concentration-dependent manner. Inhibitors of NF-ĸB (ammonium pyrrolidinedithiocarbamate and isohelenin) and an antioxidant (N-acetyl-L-cysteine) suppressed the indoxyl sulfate-induced expression of ICAM-1 and MCP-1 in HUVEC. Indoxyl sulfate increased phospho- NF-ĸB p65 in HUVEC, and N-acetyl-L-cysteine suppressed it. Conclusions: Indoxyl sulfate upregulates the expression of ICAM-1 and MCP-1 by ROS-induced activation of NF-ĸB in vascular endothelial cells. Thus, indoxyl sulfate may play an important role in the development of CVD in CKD by increasing the endothelial expression of ICAM-1 and MCP-1.