Major role of organic anion transporters in the uptake of phenolsulfonphthalein in the kidney

Abstract

Phenolsulfonphthalein is used for testing renal function. However, its excretion mechanism has not been elucidated. The purpose of this study was therefore to elucidate the transporter-mediated excretion system for phenolsulfonphthalein. p-Aminohippuric acid, a substrate of rat organic anion transporter1 (rOat1), and cimetidine, a substrate of rOat3, reduced the urinary excretion of phenolsulfonphthalein. The uptake of phenolsulfonphthalein by kidney slices was found to consist of two components. The IC₅₀ values of rOat1 substrates were higher than those of rOat3 substrates. In the presence of cimetidine, the Eadie–Hofstee plot gave a single straight line. The profile of the phenolsulfonphthalein uptake component in the presence of cimetidine was similar to that of the low-affinity component in the absence of cimetidine. We conclude that rOat1 and rOat3 are involved in the renal uptake of phenolsulfonphthalein and that phenolsulfonphthalein is a high-affinity substrate for rOat3 but is a relatively low-affinity substrate for rOat1.

Introduction

The kidney plays an important role in the urinary excretion of drugs and their metabolites via glomerular filtration and tubular secretion. The first step in tubular secretion is the uptake from blood through the basolateral membrane of epithelial cells in the proximal tubules. Transporter-mediated systems have been considered to play major roles in the tubular drug uptake. p-Aminohippuric acid is widely used as a model substrate to investigate renal handling of organic anions because of its high renal clearance and susceptibility to metabolism. Rat organic anion transporter 1 (rOat1/Slc22a6) has recently been isolated and identified as a basolateral p-aminohippuric acid transporter (Sweet et al., 1997). Phenolsulfonphthalein is widely used clinically as a drug for testing renal function because of its high renal clearance (Gault et al., 1967). However, it is thought that phenolsulfonphthalein and p-aminohippuric acid do not to share the same transporter system at the basolateral membrane of proximal tubular cells (Pritchard and Miller, 1992). An accumulation step across the basolateral membrane has been demonstrated. However, the transporters responsible for the renal uptake of phenolsulfonphthalein are not fully understood.

To date, another rOats, named rOat2 (Slc22a7) and rOat3 (Slc22a8), have been identified Kusuhara et al., 1999, Sekine et al., 1998. However, rOat2 and rOat1/3 are transcripts from separate genes located on different chromosomes. rOat2 was first cloned as a novel liver transporter NLT (Simonson et al., 1994). rOat1 and rOat3 are the only organic anion transporters that are localized to the basolateral membrane of proximal tubules Sekine et al., 2000, Tojo et al., 1999. rOat1 and rOat3 mediate the transport of many organic anions including endogenous metabolites, drugs and xenobiotics. It is thought to be the most important physiological process involved in the basolateral uptake of organic anions in renal epithelial cells (Sekine et al., 2000). It has been demonstrated that rOat1 mediates the transport of relatively small hydrophilic organic anions such as p-aminohippuric acid (Uwai et al., 1998). In contrast, the preferred substrates for rOat3 are larger and more hydrophobic compounds (Cha et al., 2001). There is a little information about the contribution of rOat1 and rOat3 to the total renal uptake of organic anions. The aim of the present study was to determine the validity of the hypothesis that phenolsulfonphthalein is cleared from peritubular blood by rat kidney organic anion transporters, particularly by rOat1 and rOat3, and to determine the contribution of rOat1 and rOat3 to the renal uptake of phenolsulfonphthalein. The results demonstrated that phenolsulfonphthalein is a high-affinity substrate for rOat3 but is a relatively low-affinity substrate for rOat1.