Interactions of human organic anion as well as cation transporters with indoxyl sulfate

Abstract

Various uremic toxicants including indoxyl sulfate exert a number of biological effects on uremic patients. In order to elucidate the molecular mechanisms for the pharmacokinetics of indoxyl sulfate in human, we examined the interactions of human organic anion transporters (human-OATs) and human organic cation transporters (human-OCTs) with indoxyl sulfate using stable transfectants. Indoxyl sulfate inhibited human-OAT1, human-OAT3 and human-OAT4, but not human-OAT2, human-OCT1 and human-OCT2. Kinetic analysis revealed that the K_i values for human-OAT1, human-OAT3 and human-OAT4 were 22.7, 168.7 and 181.3 μM, respectively. Human-OAT1 and human-OAT3 mediated the uptake of indoxyl sulfate and human-OAT4 mediated not only the uptake but also the efflux of indoxyl sulfate. In conclusion, by comparing the K_i values with the plasma concentration of unbound indoxyl sulfate, it was predicted that human-OAT1 and human-OAT3 mediate the transport of indoxyl sulfate in vivo. In addition, it was suggested that human-OAT1 and human-OAT3 are involved in the urinary excretion of indoxyl sulfate, the exacerbation of renal dysfunction and the induction of uremic encephalopathy by indoxyl sulfate.

Introduction

Uremia is characterized by a variety of symptoms including azotemia, overhydration, acidosis, anemia, bleeding tendency, hypertension, cardiac failure and neuropathy (Niwa, 1996). These symptoms are thought to be caused by the accumulation of uremic toxicants in the blood. We have previously identified indoxyl sulfate as an uremic toxicant derived from dietary protein. Indoxyl sulfate is metabolized by the liver from indole, which is produced from tryptophan by intestinal flora including Escherichia coli. Indoxyl sulfate is normally excreted into the urine primarily by proximal tubular secretion and partly by glomerular filtration (Niwa, 2001). Consistent with this, we have found that there is a marked elevation in the serum levels of indoxyl sulfate in 5/6-nephrectomized uremic rats and in uremic patients Niwa and Ise, 1994, Niwa et al., 1997a. Administration of AST-120, an oral absorbent, decreased the intensity of indoxyl sulfate staining in the proximal tubules, as well as the serum and urinary concentrations of indoxyl sulfate, and prevented the progression of renal dysfunction in rats Niwa et al., 1997a, Miyazaki et al., 2000. Thus, we have proposed that the increased serum concentration of uremic toxicants including indoxyl sulfate and the subsequent accumulation of those within the renal tubules in chronic renal failure may exacerbate the deterioration of renal function Niwa and Ise, 1994, Niwa et al., 1997b. In addition, indoxyl sulfate has been reported to cause a number of biological effects on uremic patients including the inhibition of drug binding to serum albumin, erythropoiesis and lymphocyte blast formation Sakai et al., 1995, Sakai et al., 2001, Tsutsumi et al., 2000.

The secretion of numerous organic anions and cations, including endogenous metabolites, drugs and xenobiotics, is an important physiological function of the renal proximal tubule. The process of secreting organic anions and cations through the proximal tubule cells is achieved via unidirectional transcellular transport involving the uptake of organic anions and cations into the cells from the blood across the basolateral membrane, followed by extrusion across the brush-border membrane into the proximal tubule fluid (Pritchard and Miller, 1993). Recently, cDNAs encoding the human organic anion transporter (human-OAT) family have been successively cloned, including human-OAT1 Reid et al., 1998, Hosoyamada et al., 1999, human-OAT2 (Enomoto et al., 2002a), human-OAT3 (Cha et al., 2001) and human-OAT4 (Cha et al., 2000). The human organic cation transporters (human-OCTs) isolated thus far are human-OCT1 (Gorboulev et al., 1997), human-OCT2 (Gorboulev et al., 1997) and human-OCT3 (Wu et al., 2000).

On the basis of its physicochemical properties, indoxyl sulfate possesses an anionic moiety (Fig. 1). Consistent with this, we have previously demonstrated that rat-OAT1 and rat-OAT3 mediate the uptake of indoxyl sulfate and the induction of nephrotoxicity using in vitro and in vivo models (Enomoto et al., 2002b). However, the molecular mechanisms for the pharmacokinetic handling of indoxyl sulfate in human, including renal excretion and tissue distribution, remain unclear. Thus, we examined the interactions of human-OATs and human-OCTs with indoxyl sulfate using proximal tubule cells stably expressing human-OAT1, human-OAT2, human-OAT3, human-OAT4, human-OCT1 and human-OCT2. We also investigated the interaction of indoxyl sulfate with rat-OAT2, a rodent counterpart of human-OAT2, which has been described to differ from human-OAT2 in its localization (Kojima et al., 2002).