Characterization of mouse organic anion transporter 5 as a renal steroid sulfate transporter

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Abstract

A family of organic anion transporters (OAT) recently identified has important roles for the excretion or reabsorption of endogenous and exogenous compounds, and several new isoforms have been reported in this decade. Although the transepithelial transport properties of organic anions are gradually being understood, many portions of their functional characteristics in functions remain to be elucidated. A recently reported new cDNA encoding a mouse OAT5 (mOAT5) was constructed, using 3′-RACE PCR, with the total RNA isolated from a mouse kidney. When mOAT5 was expressed in Xenopus oocytes, mOAT5 transported estrone sulfate, dehydroepiandrosterone sulfate and ochratoxin A. Estrone sulfate uptake by mOAT5 displayed a time-dependent and sodium-independent manner. The Km values of estrone sulfate and dehydroepiandrosterone sulfate were 2.2 and 3.8 μM, respectively. mOAT5 interacted with chemically heterogeneous steroid or organic sulfates, such as nitrophenyl sulfate, methylumbelliferyl sulfate and estradiol sulfates. In contrast to the sulfate conjugates, mOAT5-mediated estrone sulfate uptake was not inhibited by the steroid or organic glucuronides. The mOAT5 protein having about 85 kDa molecular weight was shown to be mainly localized in the apical membrane of the proximal tubules of the outer medulla. These results suggest an important role of mOAT5 for the excretion or reabsorption of steroid sulfates in the kidney.

Introduction

The kidney, as well as the liver, plays a primary role for the excretion of endogenous/exogenous compounds and their metabolites [1], [2], [3], [4], [5]. Not only is it responsible for glomerular filtration, the kidney also excretes charged compounds via carrier-mediated pathways that are organic anion and organic cation transport pathways in the renal proximal tubular cells [6], [7], [8], [9]. Particularly, the organic anion transport pathway has been shown to mediate the elimination of various drugs, including anti-human immunodeficiency virus therapeutics, non-steroidal anti-inflammatory drugs, antitumor drugs, antibiotics, diuretics and antihypertensives [10].

Transepithelial transport of organic anions in the proximal tubules is carried out by two distinct transporters. The organic anions are first transported from the blood by basolateral organic anion transporter(s), and then they are subsequently effluxed into the tubular lumen by the luminal transporter(s). Physiologically, renal organic anion transport has been considered to be carried out by several carrier proteins that showed wide substrate specificity. Various anionic compounds have been shown to be taken up into the proximal tubular cells by the “classical PAH (p-aminohippurate) transporter”. Several organic anion transporters have recently been discovered from the kidney, liver and olfactory mucosa [10], [11], [12]. In 2004, an additional fifth isoform, mOAT5, was reported. By using ochratoxin A, part of the functional assessment for mOAT5 has been performed. Research has shown that mOAT5-mediated ochratoxin A uptake was strongly inhibited by estrone sulfate [11]. Among the reported OATs, the third and fourth isoforms of hOAT have been shown to transport steroid sulfate conjugates transepithelially, and the inhibitory profile of these hOATs by sulfate conjugates has also been demonstrated [13], [14]. The amino acid sequence of mOAT5 shows the highest homology with that of hOAT4. From this comparison, we could presume that the substrate specificity of mOAT5 is similar to that of hOAT4. Therefore, we performed the characterization of mOAT5-mediated steroid sulfate uptake and we determined the localization of mOAT5 in the kidney using anti-sera induced by the synthesized C-terminus oligopeptidyl antigen.

We report here on the extended functional characterization of mOAT5 as a renal steroid sulfate transporter and we also report on the localization of mOAT5. The results indicate that mOAT5 functions for steroid sulfate transport, and is localized in the apical membranes of late proximal tubular cells.

Section snippets

Chemicals and solutions

The radiolabeled compounds we used were purchased from the following sources: [14C] p-aminohippurate (2.0 GBq/mmol), [3H] dehydroepiandrosterone sulfate (520 GBq/mmol), [3H] estrone sulfate (2.0 TBq/mmol), [14C] carnitine (370 kBq/mmol), [3H] prostaglandin E2 (925 kBq/mmol), [3H] cyclic-adenosine monophosphate (9.25 MBq/mmol), [3H] estradiol 17 β-d-glucuronide (1.85 MBq/mmol) and [14C] tetraethyl ammonium bromide (9.25 MBq/mmol) were from Perkin-Elmer Life Science Products (Boston, MA); [14C] uric acid

mOAT5 cDNA construction and substrate profile

Using 3′-rapid amplification of the cDNA ends (RACE), a mOAT5 cDNA that consisted of 1925 base pairs and encoded a 551 amino acid residue protein was constructed (sequence data not shown). Using the Xenopus oocyte expression system, we investigated the transport characteristics by using various substances. The uptake of various organic tracers via mOAT5 was investigated (Table 1). The uptake rates of [3H] estrone sulfate, [3H] dehydroepiandrosterone sulfate and [3H] ochratoxin A in the oocytes

Discussion

In the present study, we reported the functional characterization of the recently added fifth member of the organic anion transporter (mOAT5) as a renal steroid sulfate transporter. Several lines of evidences provided in the current study support this conclusion: (1) mOAT5 transported estrone sulfate and dehydroepiandrosterone sulfate in a sodium-independent manner; (2) mOAT5-mediated estrone sulfate transport was strongly inhibited by steroid sulfate conjugates but not by the steroid

Acknowledgement

This study was supported by a grant of the Korea Science and Engineering Foundation (R05-2004-000-11002).

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