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Interaction and transport of kynurenic acid via human organic anion transporters hOAT1 and hOAT3
Pharmacological Research 65 (2012) 254–260
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Interaction and transport of kynurenic acid via human organic anion transporters hOAT1 and hOAT3 Yuichi Uwai ∗ , Hiroaki Honjo, Kikuo Iwamoto Laboratory of Clinical Pharmacodynamics, School of Pharmacy, Aichi Gakuin University, 1-100, Kusumoto-cho, Chikusa-ku, Nagoya-shi 464-8650, Japan
a r t i c l e
i n f o
Article history: Received 12 October 2011 Received in revised form 2 November 2011 Accepted 2 November 2011 Keywords: hOAT1 hOAT3 Tryptophan metabolites Kynurenic acid Transport
a b s t r a c t Kynurenic acid, a catabolite of tryptophan, is suggested to be involved in schizophrenia, and is known to be a uremic toxin, although there is little information about the mechanism of its disposition. In this study, we performed uptake experiment using Xenopus laevis oocyte expression system to examine the transport of kynurenic acid by human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8), which mediate the transport of organic anions in the brain and kidney. The uptake of p-aminohippurate in hOAT1-expressing oocytes and of estrone sulfate in hOAT3-expressing oocytes was strongly inhibited by kynurenic acid, and other tryptophan catabolites, kynurenine and quinolinic acid, showed moderate and no inhibition, respectively. The apparent 50% inhibitory concentrations of kynurenic acid were estimated to be 12.9 M for hOAT1, and 7.76 M for hOAT3. Both hOAT1 and hOAT3 markedly stimulated the uptake of kynurenic acid into oocytes, and the Km values of the transport were calculated to be 5.06 M and 4.86 M, respectively. The transport efficiencies of kynurenic acid by hOAT1 and hOAT3 were comparable to those of p-aminohippurate and estrone sulfate, respectively. Probenecid inhibited kynurenic acid transport by hOAT1 and hOAT3. These findings show the interaction of kynurenic acid with hOAT1 and hOAT3, and that kynurenic acid is their substrate. It is suggested that these transporters are involved in the disposition of kynurenic acid. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Tryptophan is an essential amino acid, and the majority of tryptophan from food is metabolized to kynurenine, followed by conversion to quinolinic acid, kynurenic acid and so on [1], and their structures are represented in Fig. 1. These metabolites of tryptophan modulate several neurotransmitter systems, and impairment of their balance leads to a variety of diseases of the brain, such as neurotoxicity, Huntington’s disease, seizure and Alzheimer’s disease [1,2]. In particular, kynurenic acid is known to block N-methyl d-aspartate (NMDA) receptor and ␣7 nicotinic acetylcholine receptor in the brain under physiological conditions [2]. Several groups represented the increased concentration of kynurenic acid in the brain of schizophrenic patients, suggesting that kynurenic acid is one of the key molecules in schizophrenia [3–5]. In addition, it was shown that the serum concentration of kynurenic acid was elevated in patients with developed chronic renal failure [6,7]. Its accumulation was thought be related with certain uremic
Abbreviations: hOAT, human organic anion transporter; NMDA, N-methyl d-aspartate; NSAID, nonsteroidal anti-inflammatory drug. ∗ Corresponding author. Tel.: +81 52 757 6765; fax: +81 52 757 6799. E-mail address: [email protected] (Y. Uwai). 1043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2011.11.003
symptoms, including neurological disturbances, increased susceptibility to infectious disorders, lipid metabolism disorders and anemia [1,2,6,7]. Therefore, the molecular mechanism of the disposition of kynurenic acid is considered to be intriguing, although it remains to be elucidated. In the kidney, organic anion transporters mediate tubular secretion of a variety of endogenous metabolites, xenobiotics and drugs [8,9]. Human organic anion transporter 1 (hOAT1: SLC22A6) and 3 (hOAT3: SLC22A8) are expressed in the basolateral membrane of the renal proximal tubule, and are responsible for tubular uptake of organic anions from blood [8,9]. There are many reports exhibiting the transport characteristics of drugs by hOAT1 and hOAT3, and the number of reports showing the interaction of endogenous compounds with the transporters has recently increased. Several uremic toxins, including indoxyl sulfate, indoleacetate, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, hippurate and p-cresyl sulfate were shown to be transported by hOAT1 and/or hOAT3, in this decade, and it is accepted that hOAT1 and hOAT3 contribute to their basolateral entry into the kidney [10–13]. Furthermore, using rats, OAT3 was shown to be expressed at the abluminal membrane of brain capillary endothelial cells, and to play an important role in the transport of indoxyl sulfate and homovanillic acid, a major metabolite of dopamine, from the brain to blood [14,15].
Y. Uwai et al. / Pharmacological Research 65 (2012) 254–260
COOH
O
COOH
NH2 N H
NH2
Tryptophan
COOH N
NH2
COOH
Quinolinic acid
Kynurenine OH
N
255
T3 RNA polymerase. After 50 nl water or cRNA (25 ng) was injected into defolliculated oocytes, the oocytes were maintained in modified Barth’s medium (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3 )2 , 0.4 mM CaCl2 , 0.8 mM MgSO4 , 2.4 mM NaHCO3 and 5 mM HEPES; pH 7.4) containing 50 mg/l gentamicin at 18 ◦ C. Two or three days after injection, the uptake reaction was initiated by incubating the oocytes in 500 l uptake buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 , 1 mM MgCl2 and 5 mM HEPES; pH 7.4) with each radiolabeled compound at room temperature in the absence or presence of an unlabeled compound for the indicated periods. The uptake reaction was terminated by adding 2 ml ice-cold uptake buffer to each well, and the oocytes were washed three times with 2 ml ice-cold buffer. After washing, each oocyte was transferred to a scintillation counting vial, and solubilized in 150 l of 10% sodium lauryl sulfate. Two milliliters of scintillation cocktail Clear-sol II (Nacalai Tesque, Kyoto, Japan) were added to each solubilized oocyte, and radioactivity was determined using a liquid scintillation counter.
COOH
Kynurenic acid
Fig. 1. Chemical structures of tryptophan, kynurenine, quinolinic acid and kynurenic acid.
Kynurenic acid is one of the final metabolites of tryptophan, and hOAT1 and hOAT3 might be involved in its transport in the kidney and brain. Previously, Bahn et al. examined the interaction of tryptophan metabolites with murine OAT1 and OAT3, and represented the trans-stimulation of glutarate efflux via mOAT1 by kynurenic acid [16]. Although this finding suggests that OATs recognize kynurenic acid as a substrate, there is no information about whether hOAT1 and hOAT3 transport the metabolite, to our knowledge. In this study, we conducted uptake experiment with Xenopus laevis oocyte expression system to investigate the inhibitory effect of kynurenic acid on hOAT1 and hOAT3, and the transport characteristics. The obtained results provide useful information in considering the handling of kynurenic acid in the brain and kidney. 2. Materials and methods 2.1. Materials [3 H]p-Aminohippurate (4.53 Ci/mmol) and [3 H]estrone sulfate (57.3 Ci/mmol) were obtained from PerkinElmer Life Science (Boston, MA, USA). [3 H]Methotrexate (27.7 Ci/mmol) and [3 H]penciclovir (10.6 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA, USA). [3 H]Kynurenic acid (50 Ci/mmol) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). Unlabeled kynurenic acid and quinolinic acid were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Kynurenine and tryptophan were purchased from Alexis Corporation (Lausen, Switzerland) and Nacalai Tesque (Kyoto, Japan), respectively. Probenecid was obtained from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals used were of the highest purity available. 2.2. Uptake experiment using X. laevis oocytes expressing hOAT1 or hOAT3 pBK-CMV plasmid vectors containing cDNA of hOAT1 or hOAT3 were a kind gift from Prof. Ken-ichi Inui (Kyoto University Hospital, Kyoto, Japan). An uptake experiment using X. laevis oocytes was performed as previously reported [17]. Briefly, capped RNA encoding hOAT1 or hOAT3 was transcribed from XbaI-linearized pBK-CMV containing cDNA of hOAT1 or hOAT3, respectively, with
2.3. Kinetic analysis The apparent 50% inhibitory concentration (IC50 ) of kynurenic acid for hOAT1 and hOAT3 was estimated by non-linear least squares regression analysis of the competition curve with a one-compartment model according to the following equation: A = 100 × IC50 /(IC50 + [I]) + B, where A is the uptake amount of p-aminohippurate or estrone sulfate (% of control), [I] is the concentration of kynurenic acid, and B is the non-specific organic anion uptake (% of control). The kinetic parameters of kynurenic acid transport by hOAT1 and hOAT3 were calculated using non-linear least squares regression analysis from the following Michaelis–Menten equation: V = Vmax × [S]/(Km + [S]), where V is the transport rate (pmol/oocyte/h), Vmax is the maximum velocity by the saturable process (pmol/oocyte/h), [S] is the concentration of kynurenic acid (M), Km is the Michaelis–Menten constant (M). 2.4. Data analysis Eight to ten oocytes were used in each condition in one uptake experiment, and the same experiments were performed three times with different frogs. The mean ± S.E.M. was estimated using the data from these 3 experiments, and represented in tables and figures. Data were analyzed by the unpaired t-test or one-way analysis of variance followed by Dunnett’s test using GraphPad Prism, version 5.0 (GraphPad Software, San Diego, CA, USA), and by Scheffé’s test using KaleidaGraph (Synergy Software, Reading, PA, USA). Differences were considered significant at P < 0.05. 3. Results 3.1. Inhibitory effect of kynurenic acid on hOAT1 and hOAT3 First, we investigated the inhibitory effect of kynurenic acid on hOAT1 and hOAT3, and it was compared with tryptophan and its other catabolites, kynurenine and quinolinic acid. As shown in Fig. 2A, the injection of hOAT1 cRNA markedly increased paminohippurate uptake into oocytes, and it was reduced to the base level in water-injected oocytes by the coexistence of kynurenic acid at 300 M (P < 0.001). Modest but significant inhibition by kynurenine was observed (P < 0.001). The inhibitory effect of tryptophan and quinolinic acid was not recognized. The results for hOAT3 are shown in Fig. 2B. Kynurenic acid strongly inhibited not only hOAT1 but also estrone sulfate uptake by hOAT3. hOAT3 was inhibited moderately by tryptophan (P < 0.001), and slightly by kynurenine
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UPTAKE (% of control)
120
A
100 80 60 40 20 0 0.01
0.1
1
10
100
1000
UPTAKE (% of control)
Kynurenic acid (µM) 120
B
100 80 60 40 20 0 0.01
0.1
1
10
100
1000
Kynurenic acid (µM)
Fig. 2. Effect of tryptophan catabolites on uptake of p-aminohippurate by hOAT1 (A) and of estrone sulfate by hOAT3 (B). (A) Water-injected oocytes were incubated with 221 nM [3 H]p-aminohippurate for 1 h. Oocytes injected with hOAT1 cRNA were incubated with 221 nM [3 H]p-aminohippurate in the absence (control) or presence of tryptophan, kynurenine, quinolinic acid or kynurenic acid at 300 M for 1 h. (B) Water-injected oocytes (control) were incubated with 17.5 nM [3 H]estrone sulfate for 1 h. Oocytes injected with hOAT3 cRNA were incubated with 17.5 nM [3 H]estrone sulfate in the absence (control) or presence of tryptophan, kynurenine, quinolinic acid or kynurenic acid at 300 M for 1 h. The uptake amounts of [3 H]p-aminohippurate or [3 H]estrone sulfate in each oocyte were determined and shown as a percentage of the control. Each bar represents the mean ± S.E.M. of 25–30 oocytes from 3 experiments. **P < 0.01, significantly different from the control values. ***P < 0.001, significantly different from the control values.
(P < 0.01). The significant inhibition of hOAT3 by quinolinic acid was not detected. Fig. 3 represents the concentration dependency in the inhibitory effect of kynurenic acid on hOAT1 and hOAT3. According to the increase in its concentration, uptake of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3 was reduced, and 1 mM kynurenic acid almost completely inhibited hOAT1 and hOAT3. The apparent 50% inhibitory concentrations of kynurenic acid were calculated to be 12.9 ± 2.1 M for hOAT1 and 7.76 ± 3.14 M for hOAT3 (mean ± S.E.M. from 3 experiments).
Fig. 3. Concentration dependence of inhibitory effect of kynurenic acid on paminohippurate uptake by hOAT1 (A) and on estrone sulfate uptake by hOAT3 (B). (A) Oocytes injected with hOAT1 cRNA were incubated with 221 nM [3 H]paminohippurate in the absence (control) or presence of kynurenic acid at various concentrations for 1 h. (B) Oocytes injected with hOAT3 cRNA were incubated with 17.5 nM [3 H]estrone sulfate in the absence (control) or presence of kynurenic acid at various concentrations for 1 h. The uptake amounts of [3 H]p-aminohippurate or [3 H]estrone sulfate in each oocyte were determined and shown as a percentage of the control. Each point represents the mean ± S.E.M. of 25–30 oocytes from 3 experiments. When an error bar is not shown, it is smaller than the symbol.
To examine the inhibitory effect of kynurenic acid on the transport of other compounds by hOAT1 and hOAT3, we determined the uptake amounts of antifolate methotrexate and antiviral penciclovir. As shown in Table 1, hOAT1 escalated the uptake of methotrexate and penciclovir, and 30 M kynurenic acid significantly inhibited them (P < 0.001). In addition, the strong inhibition by kynurenic acid was recognized in hOAT3-mediated transport of methotrexate and penciclovir (P < 0.001, Table 2). 3.2. Transport characteristics of kynurenic acid by hOAT1 and hOAT3 To examine whether hOAT1 and hOAT3 recognize kynurenic acid as a substrate, uptake amounts of kynurenic acid were measured in oocytes injected with water, hOAT1 cRNA or hOAT3 cRNA. As shown in Fig. 4, hOAT1 and hOAT3 increased the uptake of
Y. Uwai et al. / Pharmacological Research 65 (2012) 254–260 Table 1 Effect of kynurenic acid on uptake of methotrexate and penciclovir by hOAT1. Uptake (fmol/oocyte/h)
Methotrexate Penciclovir
Water-injected
Control
+Kynurenic acid
5.17 ± 0.26*** 3.48 ± 0.22***
20.1 ± 2.9 15.1 ± 1.1
8.94 ± 1.06*** 4.68 ± 0.24***
Water-injected oocytes were incubated with 41.7 nM [3 H]methotrexate or 94.3 nM [3 H]penciclovir for 1 h. hOAT1 cRNA-injected oocytes were incubated with these radiolabeled compounds in the absence (control) or presence of 30 M kynurenic acid for 1 h. The uptake amounts of the radiolabeled compounds in each oocyte were determined. Each value represents the mean ± S.E.M. of 28–30 oocytes from 3 experiments. *** P < 0.001, significantly different from the control values.
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Table 3 Uptake of p-aminohippurate, estrone sulfate and kynurenic acid by hOAT1 and hOAT3. Uptake (nl/oocyte/h) Control
hOAT1
71.0 ± 3.2 87.6 ± 3.2 51.2 ± 3.0
3055 ± 258 N.D. 3704 ± 387***
p-Aminohippurate Estrone sulfate Kynurenic acid
hOAT3 ***
N.D. 2951 ± 236*** 2785 ± 251***
Oocytes injected with water (control), hOAT1 cRNA or hOAT3 cRNA were incubated with 221 nM [3 H]p-aminohippurate, 17.5 nM [3 H]estrone sulfate or 20 nM [3 H]kynurenic acid for 1 h. The uptake amounts of the radiolabeled compounds in each oocyte were determined and divided by their concentrations in uptake buffer. Each value represents the mean ± S.E.M. of 27–30 oocytes from 3 experiments. N.D.: not determined. *** P < 0.001, significantly different from the control values.
Table 2 Effect of kynurenic acid on uptake of methotrexate and penciclovir by hOAT3. Uptake (fmol/oocyte/h)
Methotrexate Penciclovir
Water-injected
Control
+Kynurenic acid
6.02 ± 0.36*** 3.17 ± 0.17***
40.1 ± 6.6 23.5 ± 2.6
7.73 ± 0.63*** 5.97 ± 0.52***
Water-injected oocytes were incubated with 41.7 nM [3 H]methotrexate or 94.3 nM [3 H]penciclovir for 1 h. hOAT3 cRNA-injected oocytes were incubated with these radiolabeled compounds in the absence (control) or presence of 30 M kynurenic acid for 1 h. The uptake amounts of the radiolabeled compounds in each oocyte were determined. Each value represents the mean ± S.E.M. of 28–30 oocytes from 3 experiments. *** P < 0.001, significantly different from the control values.
kynurenic acid into oocytes markedly, and it was augmented timedependently. The linearity of kynurenic acid uptake by hOAT1 and hOAT3 was observed for up to 60 min. These findings indicate that kynurenic acid is a substrate of hOAT1 and hOAT3. Next, to compare the transport efficiency of kynurenic acid by hOAT1 and hOAT3 with those of their typical substrates p-aminohippurate and estrone sulfate, respectively, the uptake amounts of these compounds were measured, and are shown as clearance in Table 3. The uptake of kynurenic acid by hOAT1 was comparable to that of p-aminohippurate. Also in hOAT3, the
UPTAKE (fmol/oocyte)
150
Table 4 Kinetic parameters of transport of p-aminohippurate, estrone sulfate and kynurenic acid by hOAT1 and hOAT3. Km (M) hOAT1 a
p-Aminohippurate Estrone sulfatea Kynurenic acid a
3.58 – 5.06
Vmax (pmol/oocyte/h) hOAT3
hOAT1
hOAT3
– 4.71 4.86
7.05 – 16.9
– 6.18 10.5
Our previous study [18].
transport efficiencies of estrone sulfate and kynurenic acid were at the similar level. Fig. 5 illustrates the concentration dependency of kynurenic acid uptake by hOAT1 and hOAT3. hOAT1 and hOAT3 transported kynurenic acid dose-dependently, and saturation was observed. The apparent Km and Vmax values for hOAT1-mediated transport of kynurenic acid were calculated to be 5.06 ± 0.53 M and 16.9 ± 3.4 pmol/oocyte/h (mean ± S.E.M. from 3 experiments), respectively. In hOAT3, the apparent Km and Vmax values were estimated to be 4.86 ± 0.73 M and 10.5 ± 2.1 pmol/oocyte/h (mean ± S.E.M. from 3 experiments), respectively. Using these values, the kinetic parameters of the transport of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3, from our previous report [18], are shown in Table 4. Finally, the effect of probenecid, the representative inhibitor of organic anion transporters, on kynurenic acid transport by hOAT1 and hOAT3 was investigated. As shown in Fig. 6, uptake of kynurenic acid by hOAT1 and hOAT3 was reduced according to the increase in the concentration of probenecid, and 100 M probenecid almost completely inhibited the transport.
100 4. Discussion
50
0
30
60
120
TIME (min) Fig. 4. Time-dependent uptake of kynurenic acid by hOAT1 and hOAT3. Oocytes injected with water (open circle), hOAT1 cRNA (closed circle) or hOAT3 cRNA (open triangle) were incubated with 20 nM [3 H]kynurenic acid for the indicated periods. The uptake amounts of [3 H]kynurenic acid in each oocyte were determined. Each point represents the mean ± S.E.M. of 26–30 oocytes from 3 experiments. When an error bar is not shown, it is smaller than the symbol.
To elucidate the interaction of kynurenic acid with hOAT1 and hOAT3, we performed uptake experiments using X. laevis oocytes. The inhibitory effect of kynurenic acid on the transporters was exhibited in the first half of the results, and its transport characteristics were shown in the latter half. The most impressive finding was that kynurenic acid is a good substrate of both hOAT1 and hOAT3. As shown in Fig. 2, the inhibitory effect of kynurenic acid on the transport of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3 was the strongest among the tryptophan metabolites tested. In addition, its IC50 values were estimated to be 12.9 M for hOAT1 and 7.76 M for hOAT3. Previously, our laboratory showed that the IC50 values of caffeic acid were 16.6 M for hOAT1 and 5.4 M for hOAT3, and that the influence of chlorogenic acid and quinic acid was much weaker than caffeic acid [19]. The IC50 values or Ki values of most nonsteroidal anti-inflammatory drugs (NSAIDs)
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UPTAKE (pmol/oocyte/h)
20
A
15
10
5
0
10
20
40
Kynurenic acid (µM)
B UPTAKE (pmol/oocyte/h)
15
10
5
0
10
20
40
Kynurenic acid (µM) Fig. 5. Concentration-dependent uptake of kynurenic acid by hOAT1 (A) and hOAT3 (B). (A) Oocytes injected with hOAT1 cRNA were incubated with [3 H]kynurenic acid at various concentrations for 1 h. hOAT1-mediated uptake of [3 H]kynurenic acid was determined by subtracting its uptake amount in water-injected oocytes from that in oocytes injected with hOAT1 cRNA. (B) Oocytes injected with hOAT3 cRNA were incubated with [3 H]kynurenic acid at various concentrations for 1 h. hOAT3mediated uptake of [3 H]kynurenic acid was determined by subtracting its uptake amount in water-injected oocytes from that in oocytes injected with hOAT3 cRNA. Each point represents the mean ± S.E.M. of 26–30 oocytes from 3 experiments. When an error bar is not shown, it is smaller than the symbol.
were reported to be less than 20 M, but the Ki values of salicylate were exceptionally high, 407 M for hOAT1 and 111 M for hOAT3 [20–22]. The inhibitory effect of kynurenic acid on hOAT1 and hOAT3 is comparable to caffeic acid and the NSAIDs, except salicylate. Salicylate, chlorogenic acid and quinic acid have a typical anionic moiety carboxyl group that directly binds to the benzene ring or the six-membered ring, although there is a methylene group, methine group or vinylene group between the rings and the carboxyl group in caffeic acid and almost NSAIDs. From these findings, we speculated that the distance between a carboxyl group and a ring might influence the recognition of the compounds by hOAT1 and hOAT3. Because there is direct binding of a carboxyl group and an indole in the structure of kynurenic acid (Fig. 1), the strong inhibitory effect of kynurenic acid on hOAT1 and hOAT3 was not
Fig. 6. Effect of probenecid on kynurenic acid uptake by hOAT1 (A) and hOAT3 (B). (A) Water-injected oocytes were incubated with 20 nM [3 H]kynurenic acid for 1 h. Oocytes injected with hOAT1 cRNA were incubated with 20 nM [3 H]kynurenic acid in the absence (control) or presence of probenecid at 1, 10 and 100 M for 1 h. (B) Water-injected oocytes were incubated with 20 nM [3 H]kynurenic acid for 1 h. Oocytes injected with hOAT3 cRNA were incubated with 20 nM [3 H]kynurenic acid in the absence (control) or presence of probenecid at 1, 10 and 100 M for 1 h. The uptake amounts of [3 H]kynurenic acid in each oocyte were determined and shown as a percentage of the control. Each bar represents the mean ± S.E.M. of 27–30 oocytes from 3 experiments. *P < 0.05, significantly different. ***P < 0.001, significantly different.
anticipated. A significant effect of quinolinic acid was not observed, and the compound has two carboxyl groups, which directly bind to the pyridine (Fig. 1). This is consistent with our hypothesis, which is not perfect, but also does not seem to be irrelevant. The uptake of antifolate methotrexate and antiviral penciclovir by hOAT1 and hOAT3 was inhibited by kynurenic acid (Tables 1 and 2). Accordingly, drug–kynurenic acid interaction via hOAT1 and hOAT3 may occur. When considering this possibility, information about the serum concentration and protein binding ratio of kynurenic acid is required, and the IC50 values of kynurenic acid for the transporters should be compared with its free level in serum. Swan et al. reported that the normal serum concentration of kynurenic acid was less than 5.3 M, and that it reached
Y. Uwai et al. / Pharmacological Research 65 (2012) 254–260
approximately 50 M in uremic patients [6]. On the other hand, Pawlak et al. represented that the plasma concentration of kynurenic acid was 28.6 nM in healthy subjects, and 336.1 nM in renal insufficient patients [7]. There is a variance in these reports, and it is thought that the serum concentration of kynurenic acid is controversial. We could not find a report illustrating the protein binding of kynurenic acid. However, we think that it would be high because uremic toxins have been classified into free watersoluble low-molecular weight solutes, protein-bound solutes or middle solutes, according to the physico-chemical characteristics of the molecules, and kynurenic acid belongs to be the protein-bound solutes group [23]. From Fig. 3 and the IC50 values of kynurenic acid for hOAT1 and hOAT3, it is suggested that kynurenic acid does not influence hOAT1 and hOAT3 if the unbound serum concentration of kynurenic acid is less than 1 M. For assessing the inhibitory effect of kynurenic acid on hOAT1 and hOAT3 in clinical situations, its plasma level and protein binding ratio are missing. The most interesting findings of this study are shown in Fig. 4 and Table 3. Fig. 4 clearly demonstrates that both hOAT1 and hOAT3 stimulated the uptake of kynurenic acid into oocytes, indicating that kynurenic acid is their substrate. Table 3 shows the transport of kynurenic acid by hOAT1 and hOAT3 as clearance, by dividing its uptake amounts by the concentration in uptake buffer. It is noteworthy that the uptake clearance of kynurenic acid by hOAT1 and hOAT3 was comparable to that of their typical substrates, p-aminohippurate and estrone sulfate, respectively. This result shows two additional interesting discoveries. The first finding is that kynurenic acid was transported efficiently by both hOAT1 and hOAT3. Thus far, many compounds have been tested for the transport activities of OAT1 and OAT3, and the compounds transported were classified into three groups; compounds transported rapidly by OAT1; compounds transported rapidly by OAT3; compounds transported slowly by both OAT1 and OAT3 [8,9]. Accordingly, kynurenic acid, which was transported by both hOAT1 and hOAT3 at high activity, is an organic anion with no precedent. The second finding is the relationship between the molecular weight of kynurenic acid and its transport activities of hOAT1 and hOAT3. Previous studies examining drug transport by OAT suggest that OAT1 tends to prefer low-molecular-weight organic anions, including p-aminohippurate, acyclic nucleotide antivirals adefovir, cidofovir and tenofovir, but that OAT3 likes bulky amphipathic organic anions such as cephalosporins, benzylpenicillin and statins [9,24,25]. The molecular weight of kynurenic acid is low, 189 g/mol. Therefore, we were not surprised when high transport activity of hOAT1 for kynurenic acid was observed, although the marked uptake of kynurenic acid by hOAT3 was unexpected. Above, the transport characteristics of kynurenic acid by hOAT1 and hOAT3 are quite unique, and its transport in the body is speculated to be meaningful. Deguchi et al. characterized the transport of several uremic toxins by hOAT1 and hOAT3, and reported that the Km values of hOAT1-mediated transport of indoxyl sulfate, 3-carboxy-4methyl-5-propyl-2-furanpropionate, indoleacetate and hippurate were 20.5 M, 141 M, 14.0 M and 23.5 M, respectively [12]. The present study estimated the Km value of kynurenic acid transport by hOAT1 to be 5.06 M, meaning that this value is lower than the Km values of other uremic toxins. However, a difference is recognized in the Km value of p-aminohippurate transport by hOAT1. Deguchi et al. calculated this value to be 20.1 M, and our result was 3.58 M (Table 4). The reason for this difference in p-aminohippurate transport is thought to be due to the expression system used in the experiments. Deguchi et al. used HEK293 cells, a cell line derived from human embryonic kidney, and our experiment was performed with Xenopus oocytes. The ratios of Km values of uremic toxins with p-aminohippurate are similar for indoxyl sulfate, indoleacetate, hippurate and kynurenic
259
acid, suggesting that the affinity of kynurenic acid to hOAT1 would be comparable to indoxyl sulfate, indoleacetate and hippurate. On one hand, the Km value of kynurenic acid transport by hOAT3 was estimated to be 4.86 M, and the Km values of hOAT3-mediated transport of indoxyl sulfate and 3-carboxy-4methyl-5-propyl-2-furanpropionate were reported to 263 M and 26.5 M, respectively [12]. The Km value of kynurenic acid is much lower than that of indoxyl sulfate, but it is difficult to say that the affinity of kynurenic acid for hOAT3 is higher than 3carboxy-4-methyl-5-propyl-2-furanpropionate, because there is the example of hOAT1. As standard substrates, benzylpenicillin and estrone sulfate were used by Deguchi et al. and us, respectively, so it is impossible to compare the ratio of the Km values with the typical substrate. They estimated the inhibition constant, Ki value of 3-carboxy-4-methyl-5-propyl-2-furanpropionate for hOAT3 to be 27.9 M. Using this equation: Ki value = IC50 value/[1 + (concentration of tracer in uptake buffer/its Km value)], the Ki value of kynurenic acid was estimated to be 7.73 M. These Ki values suggest that hOAT3 would have higher affinity for kynurenic acid than 3-carboxy-4-methyl-5-propyl-2-furanpropionate. It was reported that the renal excretion of indoxyl sulfate was hampered by p-aminohippurate and probenecid in rats [26]. In addition, Taki et al. demonstrated the accumulation of indoxyl sulfate in renal tubular cells expressing hOAT1 and hOAT3 of patients with chronic renal failure by immunohistochemistry [27]. From these reports, it is suggested that OAT1 and OAT3 are responsible for the renal disposition of indoxyl sulfate. In the case of kynurenic acid, Goralski et al. showed that its renal clearance was greater than that of creatinine in rats, implying that renal tubular secretion contributes to the urinary excretion of kynurenic acid [28]. The present study represents not only that kynurenic acid is a substrate of hOAT1 and hOAT3, but also that they transport kynurenic acid efficiently. In addition, the mRNA expression levels of hOAT1 and hOAT3 were reported to be predominant in the kidney [29]. Taking these findings together, it is highly possible that hOAT1 and hOAT3 play important roles in the renal tubular uptake of kynurenic acid from blood. In future, an uptake experiment with kidney slices and a pharmacokinetic study with knockout mice would reveal their contribution. Kynurenic acid is an endogenous antagonist of the NMDA receptor, and increased levels of kynurenic acid were observed in cerebrospinal fluid and brain tissue specimens from schizophrenic patients [3–5]. It was reported that probenecid escalated the concentration of kynurenic acid in rat brain, proposing that organic anion transporters might be responsible for the efflux of kynurenic acid from the brain to blood as one of the mechanisms [30,31]. Using rats, OAT3 was shown to be expressed at the abluminal membrane of brain capillary endothelial cells, and be involved in the elimination of indoxyl sulfate and homovanillic acid from the brain [14,15]. The present study shows that hOAT3 transported kynurenic acid and that it was inhibited by probenecid (Fig. 6B). Therefore, hOAT3 might play a role in the uptake of kynurenic acid from the brain into brain capillary endothelial cells. In OAT1, the expression was detected in neurons of the cortex cerebri and hippocampus in addition to the ependymal cell layer of the choroid plexus in the mouse by immunohistochemical analysis [16]. However, no report has shown the existence of OAT1 in the brain from a functional aspect. Because kynurenic acid is a good substrate of hOAT1 as well as hOAT3, the functional expression of OAT1 in the brain may be demonstrated by using kynurenic acid as a tracer. Our work does not show the role of hOAT1 and hOAT3 in regulating the cerebral concentration of kynurenic acid, and its elucidation may provide useful information in considering the pathology of schizophrenia. In conclusion, we investigated the interaction of kynurenic acid with hOAT1 and hOAT3. It was clearly demonstrated that kynurenic acid is a substrate of hOAT1 and hOAT3, and both hOAT1 and hOAT3
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transported kynurenic acid as efficiently as their typical substrates. To our knowledge, this is the first report presenting the transport of kynurenic acid by hOAT1 and hOAT3. Acknowledgements We thank Prof. Ken-ichi Inui (Kyoto University Hospital, Kyoto, Japan) for kindly providing pBK-CMV plasmid vectors containing hOAT1 cDNA or hOAT3 cDNA. References [1] Stone TW. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev 1993;45:309–79. [2] Schwarcz R, Pellicciari R. Manipulation of brain kynurenines: glial targets, neuronal effects, and clinical opportunities. J Pharmacol Exp Ther 2002;303:1–10. [3] Schwarcz R, Rassoulpour A, Wu HQ, Medoff D, Tamminga CA, Roberts RC. Increased cortical kynurenate content in schizophrenia. Biol Psychiatry 2001;50:521–30. [4] Erhardt S, Blennow K, Nordin C, Skogh E, Lindström LH, Engberg G. Kynurenic acid levels are elevated in the cerebrospinal fluid of patients with schizophrenia. Neurosci Lett 2001;313:96–8. [5] Nilsson LK, Linderholm KR, Engberg G, Paulson L, Blennow K, Lindström LH, et al. Elevated levels of kynurenic acid in the cerebrospinal fluid of male patients with schizophrenia. Schizophr Res 2005;80:315–22. [6] Swan JS, Kragten EY, Veening H. Liquid-chromatographic study of fluorescent materials in uremic fluids. Clin Chem 1983;29:1082–4. [7] Pawlak D, Pawlak K, Malyszko J, Mysliwiec M, Buczko W. Accumulation of toxic products degradation of kynurenine in hemodialyzed patients. Int Urol Nephrol 2001;33:399–404. [8] El-Sheikh AA, Masereeuw R, Russel FG. Mechanisms of renal anionic drug transport. Eur J Pharmacol 2008;585:245–55. [9] Kusuhara H, Sugiyama Y. In vitro–in vivo extrapolation of transporter-mediated clearance in the liver and kidney. Drug Metab Pharmacokinet 2009;24:37–52. [10] Motojima M, Hosokawa A, Yamato H, Muraki T, Yoshioka T. Uraemic toxins induce proximal tubular injury via organic anion transporter 1-mediated uptake. Br J Pharmacol 2002;135:555–63. [11] Enomoto A, Takeda M, Tojo A, Sekine T, Cha SH, Khamdang S, et al. Role of organic anion transporters in the tubular transport of indoxyl sulfate and the induction of its nephrotoxicity. J Am Soc Nephrol 2002;13:1711–20. [12] Deguchi T, Kusuhara H, Takadate A, Endou H, Otagiri M, Sugiyama Y. Characterization of uremic toxin transport by organic anion transporters in the kidney. Kidney Int 2004;65:162–74. [13] Miyamoto Y, Watanabe H, Noguchi T, Kotani S, Nakajima M, Kadowaki D, et al. Organic anion transporters play an important role in the uptake of p-cresyl sulfate, a uremic toxin, in the kidney. Nephrol Dial Transplant 2011;26:2498–502. [14] Ohtsuki S, Asaba H, Takanaga H, Deguchi T, Hosoya K, Otagiri M, et al. Role of blood–brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain. J Neurochem 2002;83:57–66.
[15] Mori S, Takanaga H, Ohtsuki S, Deguchi T, Kang YS, Hosoya K, et al. Rat organic anion transporter 3 (rOAT3) is responsible for brain-to-blood efflux of homovanillic acid at the abluminal membrane of brain capillary endothelial cells. J Cereb Blood Flow Metab 2003;23:432–40. [16] Bahn A, Ljubojevic´ M, Lorenz H, Schultz C, Ghebremedhin E, Ugele B, et al. Murine renal organic anion transporters mOAT1 and mOAT3 facilitate the transport of neuroactive tryptophan metabolites. Am J Physiol Cell Physiol 2005;289:C1075–84. [17] Uwai Y, Iwamoto K. Transport of aminopterin by human organic anion transporters hOAT1 and hOAT3: comparison with methotrexate. Drug Metab Pharmacokinet 2010;25:163–9. [18] Uwai Y, Honjo H, Iwamoto K. Inhibitory effect of selective cyclooxygenase-2 inhibitor lumiracoxib on human organic anion transporters hOAT1 and hOAT3. Drug Metab Pharmacokinet 2010;25:450–5. [19] Uwai Y, Ozeki Y, Isaka T, Honjo H, Iwamoto K. Inhibitory effect of caffeic acid on human organic anion transporters hOAT1 and hOAT3: a novel candidate for food–drug interaction. Drug Metab Pharmacokinet 2011;26:486–93. [20] Mulato AS, Ho ES, Cihlar T. Nonsteroidal anti-inflammatory drugs efficiently reduce the transport and cytotoxicity of adefovir mediated by the human renal organic anion transporter 1. J Pharmacol Exp Ther 2000;295:10–5. [21] Uwai Y, Taniguchi R, Motohashi H, Saito H, Okuda M, Inui K. Methotrexate–loxoprofen interaction: involvement of human organic anion transporters hOAT1 and hOAT3. Drug Metab Pharmacokinet 2004;19:369–74. [22] Nozaki Y, Kusuhara H, Kondo T, Iwaki M, Shiroyanagi Y, Nakayama H, et al. Species difference in the inhibitory effect of nonsteroidal anti-inflammatory drugs on the uptake of methotrexate by human kidney slices. J Pharmacol Exp Ther 2007;322:1162–70. [23] Vanholder R, De Smet R, Glorieux G, Argilés A, Baurmeister U, Brunet P, et al. Review on uremic toxins: classification, concentration, and interindividual variability. Kidney Int 2003;63:1934–43. [24] Uwai Y, Ida H, Tsuji Y, Katsura T, Inui K. Renal transport of adefovir, cidofovir, and tenofovir by SLC22A family members (hOAT1, hOAT3, and hOCT2). Pharm Res 2007;24:811–5. [25] Ueo H, Motohashi H, Katsura T, Inui K. Human organic anion transporter hOAT3 is a potent transporter of cephalosporin antibiotics, in comparison with hOAT1. Biochem Pharmacol 2005;70:1104–13. [26] Deguchi T, Nakamura M, Tsutsumi Y, Suenaga A, Otagiri M. Pharmacokinetics and tissue distribution of uraemic indoxyl sulphate in rats. Biopharm Drug Dispos 2003;24:345–55. [27] Taki K, Nakamura S, Miglinas M, Enomoto A, Niwa T. Accumulation of indoxyl sulfate in OAT1/3-positive tubular cells in kidneys of patients with chronic renal failure. J Ren Nutr 2006;16:199–203. [28] Goralski KB, Smyth DD, Sitar DS. In vivo analysis of amantadine renal clearance in the uninephrectomized rat: functional significance of in vitro bicarbonate-dependent amantadine renal tubule transport. J Pharmacol Exp Ther 1999;290:496–504. [29] Motohashi H, Sakurai Y, Saito H, Masuda S, Urakami Y, Goto M, et al. Gene expression levels and immunolocalization of organic ion transporters in the human kidney. J Am Soc Nephrol 2002;13:866–74. [30] Moroni F, Russi P, Lombardi G, Beni M, Carlà V. Presence of kynurenic acid in the mammalian brain. J Neurochem 1988;51:177–80. [31] Miller JM, MacGarvey U, Beal MF. The effect of peripheral loading with kynurenine and probenecid on extracellular striatal kynurenic acid concentrations. Neurosci Lett 1992;146:115–8.
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Interaction and transport of kynurenic acid via human organic anion transporters hOAT1 and hOAT3 Yuichi Uwai ∗ , Hiroaki Honjo, Kikuo Iwamoto Laboratory of Clinical Pharmacodynamics, School of Pharmacy, Aichi Gakuin University, 1-100, Kusumoto-cho, Chikusa-ku, Nagoya-shi 464-8650, Japan
a r t i c l e
i n f o
Article history: Received 12 October 2011 Received in revised form 2 November 2011 Accepted 2 November 2011 Keywords: hOAT1 hOAT3 Tryptophan metabolites Kynurenic acid Transport
a b s t r a c t Kynurenic acid, a catabolite of tryptophan, is suggested to be involved in schizophrenia, and is known to be a uremic toxin, although there is little information about the mechanism of its disposition. In this study, we performed uptake experiment using Xenopus laevis oocyte expression system to examine the transport of kynurenic acid by human organic anion transporters hOAT1 (SLC22A6) and hOAT3 (SLC22A8), which mediate the transport of organic anions in the brain and kidney. The uptake of p-aminohippurate in hOAT1-expressing oocytes and of estrone sulfate in hOAT3-expressing oocytes was strongly inhibited by kynurenic acid, and other tryptophan catabolites, kynurenine and quinolinic acid, showed moderate and no inhibition, respectively. The apparent 50% inhibitory concentrations of kynurenic acid were estimated to be 12.9 M for hOAT1, and 7.76 M for hOAT3. Both hOAT1 and hOAT3 markedly stimulated the uptake of kynurenic acid into oocytes, and the Km values of the transport were calculated to be 5.06 M and 4.86 M, respectively. The transport efficiencies of kynurenic acid by hOAT1 and hOAT3 were comparable to those of p-aminohippurate and estrone sulfate, respectively. Probenecid inhibited kynurenic acid transport by hOAT1 and hOAT3. These findings show the interaction of kynurenic acid with hOAT1 and hOAT3, and that kynurenic acid is their substrate. It is suggested that these transporters are involved in the disposition of kynurenic acid. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction Tryptophan is an essential amino acid, and the majority of tryptophan from food is metabolized to kynurenine, followed by conversion to quinolinic acid, kynurenic acid and so on [1], and their structures are represented in Fig. 1. These metabolites of tryptophan modulate several neurotransmitter systems, and impairment of their balance leads to a variety of diseases of the brain, such as neurotoxicity, Huntington’s disease, seizure and Alzheimer’s disease [1,2]. In particular, kynurenic acid is known to block N-methyl d-aspartate (NMDA) receptor and ␣7 nicotinic acetylcholine receptor in the brain under physiological conditions [2]. Several groups represented the increased concentration of kynurenic acid in the brain of schizophrenic patients, suggesting that kynurenic acid is one of the key molecules in schizophrenia [3–5]. In addition, it was shown that the serum concentration of kynurenic acid was elevated in patients with developed chronic renal failure [6,7]. Its accumulation was thought be related with certain uremic
Abbreviations: hOAT, human organic anion transporter; NMDA, N-methyl d-aspartate; NSAID, nonsteroidal anti-inflammatory drug. ∗ Corresponding author. Tel.: +81 52 757 6765; fax: +81 52 757 6799. E-mail address: [email protected] (Y. Uwai). 1043-6618/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.phrs.2011.11.003
symptoms, including neurological disturbances, increased susceptibility to infectious disorders, lipid metabolism disorders and anemia [1,2,6,7]. Therefore, the molecular mechanism of the disposition of kynurenic acid is considered to be intriguing, although it remains to be elucidated. In the kidney, organic anion transporters mediate tubular secretion of a variety of endogenous metabolites, xenobiotics and drugs [8,9]. Human organic anion transporter 1 (hOAT1: SLC22A6) and 3 (hOAT3: SLC22A8) are expressed in the basolateral membrane of the renal proximal tubule, and are responsible for tubular uptake of organic anions from blood [8,9]. There are many reports exhibiting the transport characteristics of drugs by hOAT1 and hOAT3, and the number of reports showing the interaction of endogenous compounds with the transporters has recently increased. Several uremic toxins, including indoxyl sulfate, indoleacetate, 3-carboxy-4-methyl-5-propyl-2-furanpropionate, hippurate and p-cresyl sulfate were shown to be transported by hOAT1 and/or hOAT3, in this decade, and it is accepted that hOAT1 and hOAT3 contribute to their basolateral entry into the kidney [10–13]. Furthermore, using rats, OAT3 was shown to be expressed at the abluminal membrane of brain capillary endothelial cells, and to play an important role in the transport of indoxyl sulfate and homovanillic acid, a major metabolite of dopamine, from the brain to blood [14,15].
Y. Uwai et al. / Pharmacological Research 65 (2012) 254–260
COOH
O
COOH
NH2 N H
NH2
Tryptophan
COOH N
NH2
COOH
Quinolinic acid
Kynurenine OH
N
255
T3 RNA polymerase. After 50 nl water or cRNA (25 ng) was injected into defolliculated oocytes, the oocytes were maintained in modified Barth’s medium (88 mM NaCl, 1 mM KCl, 0.33 mM Ca(NO3 )2 , 0.4 mM CaCl2 , 0.8 mM MgSO4 , 2.4 mM NaHCO3 and 5 mM HEPES; pH 7.4) containing 50 mg/l gentamicin at 18 ◦ C. Two or three days after injection, the uptake reaction was initiated by incubating the oocytes in 500 l uptake buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2 , 1 mM MgCl2 and 5 mM HEPES; pH 7.4) with each radiolabeled compound at room temperature in the absence or presence of an unlabeled compound for the indicated periods. The uptake reaction was terminated by adding 2 ml ice-cold uptake buffer to each well, and the oocytes were washed three times with 2 ml ice-cold buffer. After washing, each oocyte was transferred to a scintillation counting vial, and solubilized in 150 l of 10% sodium lauryl sulfate. Two milliliters of scintillation cocktail Clear-sol II (Nacalai Tesque, Kyoto, Japan) were added to each solubilized oocyte, and radioactivity was determined using a liquid scintillation counter.
COOH
Kynurenic acid
Fig. 1. Chemical structures of tryptophan, kynurenine, quinolinic acid and kynurenic acid.
Kynurenic acid is one of the final metabolites of tryptophan, and hOAT1 and hOAT3 might be involved in its transport in the kidney and brain. Previously, Bahn et al. examined the interaction of tryptophan metabolites with murine OAT1 and OAT3, and represented the trans-stimulation of glutarate efflux via mOAT1 by kynurenic acid [16]. Although this finding suggests that OATs recognize kynurenic acid as a substrate, there is no information about whether hOAT1 and hOAT3 transport the metabolite, to our knowledge. In this study, we conducted uptake experiment with Xenopus laevis oocyte expression system to investigate the inhibitory effect of kynurenic acid on hOAT1 and hOAT3, and the transport characteristics. The obtained results provide useful information in considering the handling of kynurenic acid in the brain and kidney. 2. Materials and methods 2.1. Materials [3 H]p-Aminohippurate (4.53 Ci/mmol) and [3 H]estrone sulfate (57.3 Ci/mmol) were obtained from PerkinElmer Life Science (Boston, MA, USA). [3 H]Methotrexate (27.7 Ci/mmol) and [3 H]penciclovir (10.6 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA, USA). [3 H]Kynurenic acid (50 Ci/mmol) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO, USA). Unlabeled kynurenic acid and quinolinic acid were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA). Kynurenine and tryptophan were purchased from Alexis Corporation (Lausen, Switzerland) and Nacalai Tesque (Kyoto, Japan), respectively. Probenecid was obtained from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals used were of the highest purity available. 2.2. Uptake experiment using X. laevis oocytes expressing hOAT1 or hOAT3 pBK-CMV plasmid vectors containing cDNA of hOAT1 or hOAT3 were a kind gift from Prof. Ken-ichi Inui (Kyoto University Hospital, Kyoto, Japan). An uptake experiment using X. laevis oocytes was performed as previously reported [17]. Briefly, capped RNA encoding hOAT1 or hOAT3 was transcribed from XbaI-linearized pBK-CMV containing cDNA of hOAT1 or hOAT3, respectively, with
2.3. Kinetic analysis The apparent 50% inhibitory concentration (IC50 ) of kynurenic acid for hOAT1 and hOAT3 was estimated by non-linear least squares regression analysis of the competition curve with a one-compartment model according to the following equation: A = 100 × IC50 /(IC50 + [I]) + B, where A is the uptake amount of p-aminohippurate or estrone sulfate (% of control), [I] is the concentration of kynurenic acid, and B is the non-specific organic anion uptake (% of control). The kinetic parameters of kynurenic acid transport by hOAT1 and hOAT3 were calculated using non-linear least squares regression analysis from the following Michaelis–Menten equation: V = Vmax × [S]/(Km + [S]), where V is the transport rate (pmol/oocyte/h), Vmax is the maximum velocity by the saturable process (pmol/oocyte/h), [S] is the concentration of kynurenic acid (M), Km is the Michaelis–Menten constant (M). 2.4. Data analysis Eight to ten oocytes were used in each condition in one uptake experiment, and the same experiments were performed three times with different frogs. The mean ± S.E.M. was estimated using the data from these 3 experiments, and represented in tables and figures. Data were analyzed by the unpaired t-test or one-way analysis of variance followed by Dunnett’s test using GraphPad Prism, version 5.0 (GraphPad Software, San Diego, CA, USA), and by Scheffé’s test using KaleidaGraph (Synergy Software, Reading, PA, USA). Differences were considered significant at P < 0.05. 3. Results 3.1. Inhibitory effect of kynurenic acid on hOAT1 and hOAT3 First, we investigated the inhibitory effect of kynurenic acid on hOAT1 and hOAT3, and it was compared with tryptophan and its other catabolites, kynurenine and quinolinic acid. As shown in Fig. 2A, the injection of hOAT1 cRNA markedly increased paminohippurate uptake into oocytes, and it was reduced to the base level in water-injected oocytes by the coexistence of kynurenic acid at 300 M (P < 0.001). Modest but significant inhibition by kynurenine was observed (P < 0.001). The inhibitory effect of tryptophan and quinolinic acid was not recognized. The results for hOAT3 are shown in Fig. 2B. Kynurenic acid strongly inhibited not only hOAT1 but also estrone sulfate uptake by hOAT3. hOAT3 was inhibited moderately by tryptophan (P < 0.001), and slightly by kynurenine
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UPTAKE (% of control)
120
A
100 80 60 40 20 0 0.01
0.1
1
10
100
1000
UPTAKE (% of control)
Kynurenic acid (µM) 120
B
100 80 60 40 20 0 0.01
0.1
1
10
100
1000
Kynurenic acid (µM)
Fig. 2. Effect of tryptophan catabolites on uptake of p-aminohippurate by hOAT1 (A) and of estrone sulfate by hOAT3 (B). (A) Water-injected oocytes were incubated with 221 nM [3 H]p-aminohippurate for 1 h. Oocytes injected with hOAT1 cRNA were incubated with 221 nM [3 H]p-aminohippurate in the absence (control) or presence of tryptophan, kynurenine, quinolinic acid or kynurenic acid at 300 M for 1 h. (B) Water-injected oocytes (control) were incubated with 17.5 nM [3 H]estrone sulfate for 1 h. Oocytes injected with hOAT3 cRNA were incubated with 17.5 nM [3 H]estrone sulfate in the absence (control) or presence of tryptophan, kynurenine, quinolinic acid or kynurenic acid at 300 M for 1 h. The uptake amounts of [3 H]p-aminohippurate or [3 H]estrone sulfate in each oocyte were determined and shown as a percentage of the control. Each bar represents the mean ± S.E.M. of 25–30 oocytes from 3 experiments. **P < 0.01, significantly different from the control values. ***P < 0.001, significantly different from the control values.
(P < 0.01). The significant inhibition of hOAT3 by quinolinic acid was not detected. Fig. 3 represents the concentration dependency in the inhibitory effect of kynurenic acid on hOAT1 and hOAT3. According to the increase in its concentration, uptake of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3 was reduced, and 1 mM kynurenic acid almost completely inhibited hOAT1 and hOAT3. The apparent 50% inhibitory concentrations of kynurenic acid were calculated to be 12.9 ± 2.1 M for hOAT1 and 7.76 ± 3.14 M for hOAT3 (mean ± S.E.M. from 3 experiments).
Fig. 3. Concentration dependence of inhibitory effect of kynurenic acid on paminohippurate uptake by hOAT1 (A) and on estrone sulfate uptake by hOAT3 (B). (A) Oocytes injected with hOAT1 cRNA were incubated with 221 nM [3 H]paminohippurate in the absence (control) or presence of kynurenic acid at various concentrations for 1 h. (B) Oocytes injected with hOAT3 cRNA were incubated with 17.5 nM [3 H]estrone sulfate in the absence (control) or presence of kynurenic acid at various concentrations for 1 h. The uptake amounts of [3 H]p-aminohippurate or [3 H]estrone sulfate in each oocyte were determined and shown as a percentage of the control. Each point represents the mean ± S.E.M. of 25–30 oocytes from 3 experiments. When an error bar is not shown, it is smaller than the symbol.
To examine the inhibitory effect of kynurenic acid on the transport of other compounds by hOAT1 and hOAT3, we determined the uptake amounts of antifolate methotrexate and antiviral penciclovir. As shown in Table 1, hOAT1 escalated the uptake of methotrexate and penciclovir, and 30 M kynurenic acid significantly inhibited them (P < 0.001). In addition, the strong inhibition by kynurenic acid was recognized in hOAT3-mediated transport of methotrexate and penciclovir (P < 0.001, Table 2). 3.2. Transport characteristics of kynurenic acid by hOAT1 and hOAT3 To examine whether hOAT1 and hOAT3 recognize kynurenic acid as a substrate, uptake amounts of kynurenic acid were measured in oocytes injected with water, hOAT1 cRNA or hOAT3 cRNA. As shown in Fig. 4, hOAT1 and hOAT3 increased the uptake of
Y. Uwai et al. / Pharmacological Research 65 (2012) 254–260 Table 1 Effect of kynurenic acid on uptake of methotrexate and penciclovir by hOAT1. Uptake (fmol/oocyte/h)
Methotrexate Penciclovir
Water-injected
Control
+Kynurenic acid
5.17 ± 0.26*** 3.48 ± 0.22***
20.1 ± 2.9 15.1 ± 1.1
8.94 ± 1.06*** 4.68 ± 0.24***
Water-injected oocytes were incubated with 41.7 nM [3 H]methotrexate or 94.3 nM [3 H]penciclovir for 1 h. hOAT1 cRNA-injected oocytes were incubated with these radiolabeled compounds in the absence (control) or presence of 30 M kynurenic acid for 1 h. The uptake amounts of the radiolabeled compounds in each oocyte were determined. Each value represents the mean ± S.E.M. of 28–30 oocytes from 3 experiments. *** P < 0.001, significantly different from the control values.
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Table 3 Uptake of p-aminohippurate, estrone sulfate and kynurenic acid by hOAT1 and hOAT3. Uptake (nl/oocyte/h) Control
hOAT1
71.0 ± 3.2 87.6 ± 3.2 51.2 ± 3.0
3055 ± 258 N.D. 3704 ± 387***
p-Aminohippurate Estrone sulfate Kynurenic acid
hOAT3 ***
N.D. 2951 ± 236*** 2785 ± 251***
Oocytes injected with water (control), hOAT1 cRNA or hOAT3 cRNA were incubated with 221 nM [3 H]p-aminohippurate, 17.5 nM [3 H]estrone sulfate or 20 nM [3 H]kynurenic acid for 1 h. The uptake amounts of the radiolabeled compounds in each oocyte were determined and divided by their concentrations in uptake buffer. Each value represents the mean ± S.E.M. of 27–30 oocytes from 3 experiments. N.D.: not determined. *** P < 0.001, significantly different from the control values.
Table 2 Effect of kynurenic acid on uptake of methotrexate and penciclovir by hOAT3. Uptake (fmol/oocyte/h)
Methotrexate Penciclovir
Water-injected
Control
+Kynurenic acid
6.02 ± 0.36*** 3.17 ± 0.17***
40.1 ± 6.6 23.5 ± 2.6
7.73 ± 0.63*** 5.97 ± 0.52***
Water-injected oocytes were incubated with 41.7 nM [3 H]methotrexate or 94.3 nM [3 H]penciclovir for 1 h. hOAT3 cRNA-injected oocytes were incubated with these radiolabeled compounds in the absence (control) or presence of 30 M kynurenic acid for 1 h. The uptake amounts of the radiolabeled compounds in each oocyte were determined. Each value represents the mean ± S.E.M. of 28–30 oocytes from 3 experiments. *** P < 0.001, significantly different from the control values.
kynurenic acid into oocytes markedly, and it was augmented timedependently. The linearity of kynurenic acid uptake by hOAT1 and hOAT3 was observed for up to 60 min. These findings indicate that kynurenic acid is a substrate of hOAT1 and hOAT3. Next, to compare the transport efficiency of kynurenic acid by hOAT1 and hOAT3 with those of their typical substrates p-aminohippurate and estrone sulfate, respectively, the uptake amounts of these compounds were measured, and are shown as clearance in Table 3. The uptake of kynurenic acid by hOAT1 was comparable to that of p-aminohippurate. Also in hOAT3, the
UPTAKE (fmol/oocyte)
150
Table 4 Kinetic parameters of transport of p-aminohippurate, estrone sulfate and kynurenic acid by hOAT1 and hOAT3. Km (M) hOAT1 a
p-Aminohippurate Estrone sulfatea Kynurenic acid a
3.58 – 5.06
Vmax (pmol/oocyte/h) hOAT3
hOAT1
hOAT3
– 4.71 4.86
7.05 – 16.9
– 6.18 10.5
Our previous study [18].
transport efficiencies of estrone sulfate and kynurenic acid were at the similar level. Fig. 5 illustrates the concentration dependency of kynurenic acid uptake by hOAT1 and hOAT3. hOAT1 and hOAT3 transported kynurenic acid dose-dependently, and saturation was observed. The apparent Km and Vmax values for hOAT1-mediated transport of kynurenic acid were calculated to be 5.06 ± 0.53 M and 16.9 ± 3.4 pmol/oocyte/h (mean ± S.E.M. from 3 experiments), respectively. In hOAT3, the apparent Km and Vmax values were estimated to be 4.86 ± 0.73 M and 10.5 ± 2.1 pmol/oocyte/h (mean ± S.E.M. from 3 experiments), respectively. Using these values, the kinetic parameters of the transport of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3, from our previous report [18], are shown in Table 4. Finally, the effect of probenecid, the representative inhibitor of organic anion transporters, on kynurenic acid transport by hOAT1 and hOAT3 was investigated. As shown in Fig. 6, uptake of kynurenic acid by hOAT1 and hOAT3 was reduced according to the increase in the concentration of probenecid, and 100 M probenecid almost completely inhibited the transport.
100 4. Discussion
50
0
30
60
120
TIME (min) Fig. 4. Time-dependent uptake of kynurenic acid by hOAT1 and hOAT3. Oocytes injected with water (open circle), hOAT1 cRNA (closed circle) or hOAT3 cRNA (open triangle) were incubated with 20 nM [3 H]kynurenic acid for the indicated periods. The uptake amounts of [3 H]kynurenic acid in each oocyte were determined. Each point represents the mean ± S.E.M. of 26–30 oocytes from 3 experiments. When an error bar is not shown, it is smaller than the symbol.
To elucidate the interaction of kynurenic acid with hOAT1 and hOAT3, we performed uptake experiments using X. laevis oocytes. The inhibitory effect of kynurenic acid on the transporters was exhibited in the first half of the results, and its transport characteristics were shown in the latter half. The most impressive finding was that kynurenic acid is a good substrate of both hOAT1 and hOAT3. As shown in Fig. 2, the inhibitory effect of kynurenic acid on the transport of p-aminohippurate by hOAT1 and of estrone sulfate by hOAT3 was the strongest among the tryptophan metabolites tested. In addition, its IC50 values were estimated to be 12.9 M for hOAT1 and 7.76 M for hOAT3. Previously, our laboratory showed that the IC50 values of caffeic acid were 16.6 M for hOAT1 and 5.4 M for hOAT3, and that the influence of chlorogenic acid and quinic acid was much weaker than caffeic acid [19]. The IC50 values or Ki values of most nonsteroidal anti-inflammatory drugs (NSAIDs)
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UPTAKE (pmol/oocyte/h)
20
A
15
10
5
0
10
20
40
Kynurenic acid (µM)
B UPTAKE (pmol/oocyte/h)
15
10
5
0
10
20
40
Kynurenic acid (µM) Fig. 5. Concentration-dependent uptake of kynurenic acid by hOAT1 (A) and hOAT3 (B). (A) Oocytes injected with hOAT1 cRNA were incubated with [3 H]kynurenic acid at various concentrations for 1 h. hOAT1-mediated uptake of [3 H]kynurenic acid was determined by subtracting its uptake amount in water-injected oocytes from that in oocytes injected with hOAT1 cRNA. (B) Oocytes injected with hOAT3 cRNA were incubated with [3 H]kynurenic acid at various concentrations for 1 h. hOAT3mediated uptake of [3 H]kynurenic acid was determined by subtracting its uptake amount in water-injected oocytes from that in oocytes injected with hOAT3 cRNA. Each point represents the mean ± S.E.M. of 26–30 oocytes from 3 experiments. When an error bar is not shown, it is smaller than the symbol.
were reported to be less than 20 M, but the Ki values of salicylate were exceptionally high, 407 M for hOAT1 and 111 M for hOAT3 [20–22]. The inhibitory effect of kynurenic acid on hOAT1 and hOAT3 is comparable to caffeic acid and the NSAIDs, except salicylate. Salicylate, chlorogenic acid and quinic acid have a typical anionic moiety carboxyl group that directly binds to the benzene ring or the six-membered ring, although there is a methylene group, methine group or vinylene group between the rings and the carboxyl group in caffeic acid and almost NSAIDs. From these findings, we speculated that the distance between a carboxyl group and a ring might influence the recognition of the compounds by hOAT1 and hOAT3. Because there is direct binding of a carboxyl group and an indole in the structure of kynurenic acid (Fig. 1), the strong inhibitory effect of kynurenic acid on hOAT1 and hOAT3 was not
Fig. 6. Effect of probenecid on kynurenic acid uptake by hOAT1 (A) and hOAT3 (B). (A) Water-injected oocytes were incubated with 20 nM [3 H]kynurenic acid for 1 h. Oocytes injected with hOAT1 cRNA were incubated with 20 nM [3 H]kynurenic acid in the absence (control) or presence of probenecid at 1, 10 and 100 M for 1 h. (B) Water-injected oocytes were incubated with 20 nM [3 H]kynurenic acid for 1 h. Oocytes injected with hOAT3 cRNA were incubated with 20 nM [3 H]kynurenic acid in the absence (control) or presence of probenecid at 1, 10 and 100 M for 1 h. The uptake amounts of [3 H]kynurenic acid in each oocyte were determined and shown as a percentage of the control. Each bar represents the mean ± S.E.M. of 27–30 oocytes from 3 experiments. *P < 0.05, significantly different. ***P < 0.001, significantly different.
anticipated. A significant effect of quinolinic acid was not observed, and the compound has two carboxyl groups, which directly bind to the pyridine (Fig. 1). This is consistent with our hypothesis, which is not perfect, but also does not seem to be irrelevant. The uptake of antifolate methotrexate and antiviral penciclovir by hOAT1 and hOAT3 was inhibited by kynurenic acid (Tables 1 and 2). Accordingly, drug–kynurenic acid interaction via hOAT1 and hOAT3 may occur. When considering this possibility, information about the serum concentration and protein binding ratio of kynurenic acid is required, and the IC50 values of kynurenic acid for the transporters should be compared with its free level in serum. Swan et al. reported that the normal serum concentration of kynurenic acid was less than 5.3 M, and that it reached
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approximately 50 M in uremic patients [6]. On the other hand, Pawlak et al. represented that the plasma concentration of kynurenic acid was 28.6 nM in healthy subjects, and 336.1 nM in renal insufficient patients [7]. There is a variance in these reports, and it is thought that the serum concentration of kynurenic acid is controversial. We could not find a report illustrating the protein binding of kynurenic acid. However, we think that it would be high because uremic toxins have been classified into free watersoluble low-molecular weight solutes, protein-bound solutes or middle solutes, according to the physico-chemical characteristics of the molecules, and kynurenic acid belongs to be the protein-bound solutes group [23]. From Fig. 3 and the IC50 values of kynurenic acid for hOAT1 and hOAT3, it is suggested that kynurenic acid does not influence hOAT1 and hOAT3 if the unbound serum concentration of kynurenic acid is less than 1 M. For assessing the inhibitory effect of kynurenic acid on hOAT1 and hOAT3 in clinical situations, its plasma level and protein binding ratio are missing. The most interesting findings of this study are shown in Fig. 4 and Table 3. Fig. 4 clearly demonstrates that both hOAT1 and hOAT3 stimulated the uptake of kynurenic acid into oocytes, indicating that kynurenic acid is their substrate. Table 3 shows the transport of kynurenic acid by hOAT1 and hOAT3 as clearance, by dividing its uptake amounts by the concentration in uptake buffer. It is noteworthy that the uptake clearance of kynurenic acid by hOAT1 and hOAT3 was comparable to that of their typical substrates, p-aminohippurate and estrone sulfate, respectively. This result shows two additional interesting discoveries. The first finding is that kynurenic acid was transported efficiently by both hOAT1 and hOAT3. Thus far, many compounds have been tested for the transport activities of OAT1 and OAT3, and the compounds transported were classified into three groups; compounds transported rapidly by OAT1; compounds transported rapidly by OAT3; compounds transported slowly by both OAT1 and OAT3 [8,9]. Accordingly, kynurenic acid, which was transported by both hOAT1 and hOAT3 at high activity, is an organic anion with no precedent. The second finding is the relationship between the molecular weight of kynurenic acid and its transport activities of hOAT1 and hOAT3. Previous studies examining drug transport by OAT suggest that OAT1 tends to prefer low-molecular-weight organic anions, including p-aminohippurate, acyclic nucleotide antivirals adefovir, cidofovir and tenofovir, but that OAT3 likes bulky amphipathic organic anions such as cephalosporins, benzylpenicillin and statins [9,24,25]. The molecular weight of kynurenic acid is low, 189 g/mol. Therefore, we were not surprised when high transport activity of hOAT1 for kynurenic acid was observed, although the marked uptake of kynurenic acid by hOAT3 was unexpected. Above, the transport characteristics of kynurenic acid by hOAT1 and hOAT3 are quite unique, and its transport in the body is speculated to be meaningful. Deguchi et al. characterized the transport of several uremic toxins by hOAT1 and hOAT3, and reported that the Km values of hOAT1-mediated transport of indoxyl sulfate, 3-carboxy-4methyl-5-propyl-2-furanpropionate, indoleacetate and hippurate were 20.5 M, 141 M, 14.0 M and 23.5 M, respectively [12]. The present study estimated the Km value of kynurenic acid transport by hOAT1 to be 5.06 M, meaning that this value is lower than the Km values of other uremic toxins. However, a difference is recognized in the Km value of p-aminohippurate transport by hOAT1. Deguchi et al. calculated this value to be 20.1 M, and our result was 3.58 M (Table 4). The reason for this difference in p-aminohippurate transport is thought to be due to the expression system used in the experiments. Deguchi et al. used HEK293 cells, a cell line derived from human embryonic kidney, and our experiment was performed with Xenopus oocytes. The ratios of Km values of uremic toxins with p-aminohippurate are similar for indoxyl sulfate, indoleacetate, hippurate and kynurenic
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acid, suggesting that the affinity of kynurenic acid to hOAT1 would be comparable to indoxyl sulfate, indoleacetate and hippurate. On one hand, the Km value of kynurenic acid transport by hOAT3 was estimated to be 4.86 M, and the Km values of hOAT3-mediated transport of indoxyl sulfate and 3-carboxy-4methyl-5-propyl-2-furanpropionate were reported to 263 M and 26.5 M, respectively [12]. The Km value of kynurenic acid is much lower than that of indoxyl sulfate, but it is difficult to say that the affinity of kynurenic acid for hOAT3 is higher than 3carboxy-4-methyl-5-propyl-2-furanpropionate, because there is the example of hOAT1. As standard substrates, benzylpenicillin and estrone sulfate were used by Deguchi et al. and us, respectively, so it is impossible to compare the ratio of the Km values with the typical substrate. They estimated the inhibition constant, Ki value of 3-carboxy-4-methyl-5-propyl-2-furanpropionate for hOAT3 to be 27.9 M. Using this equation: Ki value = IC50 value/[1 + (concentration of tracer in uptake buffer/its Km value)], the Ki value of kynurenic acid was estimated to be 7.73 M. These Ki values suggest that hOAT3 would have higher affinity for kynurenic acid than 3-carboxy-4-methyl-5-propyl-2-furanpropionate. It was reported that the renal excretion of indoxyl sulfate was hampered by p-aminohippurate and probenecid in rats [26]. In addition, Taki et al. demonstrated the accumulation of indoxyl sulfate in renal tubular cells expressing hOAT1 and hOAT3 of patients with chronic renal failure by immunohistochemistry [27]. From these reports, it is suggested that OAT1 and OAT3 are responsible for the renal disposition of indoxyl sulfate. In the case of kynurenic acid, Goralski et al. showed that its renal clearance was greater than that of creatinine in rats, implying that renal tubular secretion contributes to the urinary excretion of kynurenic acid [28]. The present study represents not only that kynurenic acid is a substrate of hOAT1 and hOAT3, but also that they transport kynurenic acid efficiently. In addition, the mRNA expression levels of hOAT1 and hOAT3 were reported to be predominant in the kidney [29]. Taking these findings together, it is highly possible that hOAT1 and hOAT3 play important roles in the renal tubular uptake of kynurenic acid from blood. In future, an uptake experiment with kidney slices and a pharmacokinetic study with knockout mice would reveal their contribution. Kynurenic acid is an endogenous antagonist of the NMDA receptor, and increased levels of kynurenic acid were observed in cerebrospinal fluid and brain tissue specimens from schizophrenic patients [3–5]. It was reported that probenecid escalated the concentration of kynurenic acid in rat brain, proposing that organic anion transporters might be responsible for the efflux of kynurenic acid from the brain to blood as one of the mechanisms [30,31]. Using rats, OAT3 was shown to be expressed at the abluminal membrane of brain capillary endothelial cells, and be involved in the elimination of indoxyl sulfate and homovanillic acid from the brain [14,15]. The present study shows that hOAT3 transported kynurenic acid and that it was inhibited by probenecid (Fig. 6B). Therefore, hOAT3 might play a role in the uptake of kynurenic acid from the brain into brain capillary endothelial cells. In OAT1, the expression was detected in neurons of the cortex cerebri and hippocampus in addition to the ependymal cell layer of the choroid plexus in the mouse by immunohistochemical analysis [16]. However, no report has shown the existence of OAT1 in the brain from a functional aspect. Because kynurenic acid is a good substrate of hOAT1 as well as hOAT3, the functional expression of OAT1 in the brain may be demonstrated by using kynurenic acid as a tracer. Our work does not show the role of hOAT1 and hOAT3 in regulating the cerebral concentration of kynurenic acid, and its elucidation may provide useful information in considering the pathology of schizophrenia. In conclusion, we investigated the interaction of kynurenic acid with hOAT1 and hOAT3. It was clearly demonstrated that kynurenic acid is a substrate of hOAT1 and hOAT3, and both hOAT1 and hOAT3
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transported kynurenic acid as efficiently as their typical substrates. To our knowledge, this is the first report presenting the transport of kynurenic acid by hOAT1 and hOAT3. Acknowledgements We thank Prof. Ken-ichi Inui (Kyoto University Hospital, Kyoto, Japan) for kindly providing pBK-CMV plasmid vectors containing hOAT1 cDNA or hOAT3 cDNA. References [1] Stone TW. Neuropharmacology of quinolinic and kynurenic acids. Pharmacol Rev 1993;45:309–79. [2] Schwarcz R, Pellicciari R. Manipulation of brain kynurenines: glial targets, neuronal effects, and clinical opportunities. J Pharmacol Exp Ther 2002;303:1–10. [3] Schwarcz R, Rassoulpour A, Wu HQ, Medoff D, Tamminga CA, Roberts RC. Increased cortical kynurenate content in schizophrenia. Biol Psychiatry 2001;50:521–30. [4] Erhardt S, Blennow K, Nordin C, Skogh E, Lindström LH, Engberg G. Kynurenic acid levels are elevated in the cerebrospinal fluid of patients with schizophrenia. Neurosci Lett 2001;313:96–8. [5] Nilsson LK, Linderholm KR, Engberg G, Paulson L, Blennow K, Lindström LH, et al. Elevated levels of kynurenic acid in the cerebrospinal fluid of male patients with schizophrenia. Schizophr Res 2005;80:315–22. [6] Swan JS, Kragten EY, Veening H. Liquid-chromatographic study of fluorescent materials in uremic fluids. Clin Chem 1983;29:1082–4. [7] Pawlak D, Pawlak K, Malyszko J, Mysliwiec M, Buczko W. Accumulation of toxic products degradation of kynurenine in hemodialyzed patients. Int Urol Nephrol 2001;33:399–404. [8] El-Sheikh AA, Masereeuw R, Russel FG. Mechanisms of renal anionic drug transport. Eur J Pharmacol 2008;585:245–55. [9] Kusuhara H, Sugiyama Y. In vitro–in vivo extrapolation of transporter-mediated clearance in the liver and kidney. Drug Metab Pharmacokinet 2009;24:37–52. [10] Motojima M, Hosokawa A, Yamato H, Muraki T, Yoshioka T. Uraemic toxins induce proximal tubular injury via organic anion transporter 1-mediated uptake. Br J Pharmacol 2002;135:555–63. [11] Enomoto A, Takeda M, Tojo A, Sekine T, Cha SH, Khamdang S, et al. Role of organic anion transporters in the tubular transport of indoxyl sulfate and the induction of its nephrotoxicity. J Am Soc Nephrol 2002;13:1711–20. [12] Deguchi T, Kusuhara H, Takadate A, Endou H, Otagiri M, Sugiyama Y. Characterization of uremic toxin transport by organic anion transporters in the kidney. Kidney Int 2004;65:162–74. [13] Miyamoto Y, Watanabe H, Noguchi T, Kotani S, Nakajima M, Kadowaki D, et al. Organic anion transporters play an important role in the uptake of p-cresyl sulfate, a uremic toxin, in the kidney. Nephrol Dial Transplant 2011;26:2498–502. [14] Ohtsuki S, Asaba H, Takanaga H, Deguchi T, Hosoya K, Otagiri M, et al. Role of blood–brain barrier organic anion transporter 3 (OAT3) in the efflux of indoxyl sulfate, a uremic toxin: its involvement in neurotransmitter metabolite clearance from the brain. J Neurochem 2002;83:57–66.
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