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Rat Kidney Slices for Evaluation of Apical Membrane Transporters in Proximal Tubular Cells
Journal of Pharmaceutical Sciences 108 (2019) 2798-2804
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Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism
Rat Kidney Slices for Evaluation of Apical Membrane Transporters in Proximal Tubular Cells Hiroshi Arakawa 1, Hikaru Kubo 1, Ikumi Washio 1, 2, Angelina Yukiko Staub 1, Shiho Nedachi 1, Naoki Ishiguro 2, Takeo Nakanishi 1, Ikumi Tamai 1, * 1 2
Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 December 2018 Revised 27 March 2019 Accepted 28 March 2019 Available online 5 April 2019
Kidney slice has been often used as a tool reflecting basolateral transport in renal tubular epithelial cells. Recently, we reported that several important apical reabsorptive transporters such as Octn1/2, Sglt1/2, and Pept1/2 were functional in mouse kidney slices as well as transporter activities in basolateral side, which have been well accepted. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. The present study investigates usefulness of kidney slices from rats for evaluation of apical membrane transporters for efflux (multidrug resistance 1a, mdr1a) as well as influx (Octn1/2, Sglt1/2, Pept1/2). Naþ-dependent uptake of ergothioneine (Octn1), carnitine (Octn2), and methyl-a-D-glucopyranoside (Sglt1/2) by rat kidney slices was observed, and the uptake was decreased by selective inhibitors. In addition, uptake of glycyl-sarcosine (Pept1/2) showed Hþdependence and was decreased by selective inhibitor. Furthermore, accumulation of mdr1a substrate azasetron was increased in the presence of zosuquidar, an mdr1a inhibitor, while strain differences existed. In conclusion, rat kidney slices should be useful for evaluation of renal drug disposition regulated by transporters in apical as well as basolateral membranes of rat renal proximal tubule cells. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
Keywords: renal transport transporter drug disposition P-gp reabsorption
Introduction Membrane transporters play important roles in renal handling, for example, renal reabsorption, secretion, and disposition of nutrients and xenobiotics, including drugs and their metabolites. For example, apically expressed sodium-dependent glucose transporter 2 (SGLT2) plays a major role in glucose reabsorption, of which inhibitors are used for treatment of diabetes. Apical efflux transporter MATE1 was involved in cisplatin-induced renal toxicity as demonstrated in mate1 knockout mice.1 Therefore, pharmacokinetic, Abbreviations used: OCTN, organic cation/carnitine transporter; SGLT, sodiumdependent glucose transporter; PEPT, oligopeptide transporter; OAT, organic anion transporter; OCT, organic cation transporter; aMG, methyl a-D-glucopyranoside; Gly-Sar, glycyl-sarcosine; Gly-Leu, glycyl-leucin; BSA, bovine serum albumin. Conflicts of interest: This study was supported partly by Nippon Boehringer Ingelheim Co., Ltd. The authors Hiroshi Arakawa and Hikaru Kubo equally contributed to the preparation of the study. This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.03.031. * Correspondence to: Ikumi Tamai, (Telephone: þ81-76-234-4479). E-mail address: [email protected] (I. Tamai).
pharmacodynamic, and toxicological demands exist in the evaluation of apical transporters as well as efflux transporters in the kidney. Several in vitro methods have been reported, including human renal cell line models and primary culture of renal epithelial cells.2-4 However, because they have limitations due to lowered expression of various genes compared with the intact kidney during cultivation, in vitro experimental method for apical uptake and efflux transporters using intact tissue or cells is desirable. Kidney slices are one of the tools to characterize renal disposition of drugs. This method has been used for evaluating basolateral uptake transporters, but we recently demonstrated that apically expressed transporters, including organic cation/carnitine transporter (Octn1/2), Sglt1/2, and peptide transporter (Pept)1/2, could be evaluated by kidney slices from mice.5 There are some advantages in this method: (1) biological macromolecules including functional transporter proteins and metabolizing enzymes are likely preserved in their intact form, (2) kidney tissue architecture is retained well with all resident cell types available and cell-cell contacts are preserved, and (3) the handling of kidney slices is quite simple. Therefore, this methodology should be promising to evaluate renal disposition of drugs and nutrients.
https://doi.org/10.1016/j.xphs.2019.03.031 0022-3549/© 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
H. Arakawa et al. / Journal of Pharmaceutical Sciences 108 (2019) 2798-2804
In addition to apically expressed influx transporters, efflux transporters such as multidrug resistance 1 (MDR1) contribute to drug disposition. In mdr1a knockout mice, renal accumulation of mdr1a substrates such as digoxin, ivermectin, and vinblastine were significantly higher than that in wild-type mice.6,7 Such an increase of tissue accumulation is possibly caused by a decreased apical efflux and should lead to drug-induced toxicity. However, it has not been clarified whether this method could apply for apically expressed efflux transporters. Accordingly, it needs to analyze them in kidney slices for prediction of drugs disposition. In the present study, we evaluated usefulness of kidney slices obtained from rats by measuring accumulation of substrates of each transporter. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice because of convenient handling such as tissue and blood collection, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. As there is species difference between rats and mice, Pept1 mRNA expression in the kidney of rats is much higher than that of mice.8 Moreover, the abundance of Sglt2 protein in the kidney was higher in male mice than in female mice, while that in female rats was higher than that in male rats.9 There is also species difference of affinity for med1a/1b-mediated transport of several compounds between rats and mice.10,11 In addition, several disease models are available only in rats, such as spontaneous hypertension12 and Zucker diabetes.13 In case apically expressed renal transporters in disease model rats were evaluated, basic data for evaluation of the transporters are necessary. The present study aimed to characterize apical influx transporters (Octn1/2, Sglt1/2, and Pept1/2) as well as efflux transporter (mdr1a) in rat kidney slices. Methods Materials [3H]Ergothioneine (100 mCi/mmol, custom-made) [methyl- C],-a-D-glucopyranoside (aMG, 39.3 mCi/mmol), and [3H]glycylsarcosine (Gly-Sar, 2.8 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA). L-[N-methyl-3H]carnitine (80 Ci/mmol) and [carboxyl-14C]inulin (2.5 mCi/g) were purchased from American Radiolabeled Chemicals Inc. (St, Louis, Mo). Azasetron HCl was obtained from Yoshitomi Pharmaceutical Industries (Osaka, Japan). All other chemicals and reagents were commercial products of reagent grade. 14
Animals Male Wistar (7 weeks) and Wistar/ST (7 weeks) rats were purchased from Sankyo Labo Service Co. Inc. (Tokyo, Japan). Rats were housed 3 per cage with free access to commercial chow and tap water and were maintained on a 12 h dark/light cycle in an aircontrolled room (temperature 24.0 ± 1 C; humidity 55 ± 5%). All animal studies were approved by the Kanazawa University Institutional Animal Care and Use Committee (AP-143148). Uptake Study by Rat Kidney Slices Uptake studies in rat kidney slices were carried out as described previously with slight modifications.5,14 Wistar rats were used for uptake study of ergothioneine, carnitine, aMG, and Gly-Sar, and Wistar/ST rats were used for uptake of azasetron. Briefly, rats were anesthetized with pentobarbital (50 mg/kg) and whole kidneys were removed after blood removal. Kidney slices (0.3 mm thick) were prepared with a microslicer (Zero 1; Dosaka EM, Kyoto, Japan) and were immediately placed in ice-cold oxygenated transport
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buffer (130 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 25 mM HEPES, adjusted to pH 7.4). To determine effect of Naþ on substrate uptake, NaCl was replaced with Nmethyl-D-glucamine in transport buffer. Three or four slices, weight of which ranged from 6 to 14 mg, were preincubated in a 12well plate with oxygenated buffer at 37 C for 5 min. To initiate uptake reaction, the slices were transferred into transport buffer containing radiolabeled or unlabeled test compounds. The uptake reaction was performed at 37 C for the designated period of time under oxygen supply, and then the reaction was terminated by removing slices from the incubating solution and rinsing them with fresh ice-cold transport buffer by 3 times. The washed slices were blotted on filter paper and weighed. To measure radioactivity, slices were dissolved by 1 N NaOH in water bath at 60 C for 20 min, and then the lysate was neutralized with 5 N HCl. All samples were mixed with liquid scintillation cocktail Cleasol I (Nacalai Tesque, Kyoto, Japan), and radioactivity was measured in a liquid scintillation counter (Hitachi Aloka Medical, Tokyo, Japan). The volume of water adhering to the kidney slices was estimated by the apparent uptake of [14C]inulin with 0.150 ± 0.022 mL/5 min/mg kidney (CV: 14.5% by 3 independent assay). To quantify azasetron, slices were homogenated by sonication in transport buffer, and proteins were removed by centrifugation after denatured adding acetonitrile. The resultant supernatants were subjected to LC-MS/MS analysis. Measurement of ATP Content in Kidney Slices Rat kidney slices were incubated for 10, 30, 60, 120, 240, and 360 min at pH 7.4 and 37 C in the presence or the absence of oxygen supply. For the condition without oxygen supply, bovine serum albumin (BSA) was added to the transport buffer to avoid drug adsorption. After incubation, the kidney slices were collected and immediately frozen and stored in liquid nitrogen for subsequent ATP analysis. The tissues were homogenized by bullet blender and centrifuged at 1000 g for 10 min at 4 C. The resultant supernatant was collected for analyzing ATP using “Tissue” ATP assay kit (TOYO B-Net Co., Ltd, Tokyo, Japan). Relative ATP content was calculated according to amount of luminescence versus the ATP standard curve. Uptake Study Using Transporter-Expressing Cells Rat Octn1-expressing and empty vector transfected (mock) HEK293 cells were established previously.15 The cells were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum (Life Technologies, Carlsbad, CA), 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 C in an atmosphere of 5% CO2. Octn1-expressing and mock HEK293 cells were seeded at 1.0 105 cells/well onto a 24-well poly-L-lysine-coated tissue culture plate. Two days later, cells were used for uptake study by preincubating with transport buffer (125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM HEPES, adjusted to pH 7.4) at 37 C for 15 min, followed by uptake reaction by adding transport buffer containing 0.05 mCi/mL [3H]ergothioneine. The uptake reaction was carried out at 37 C for 30 s. To terminate uptake reaction, the cells were washed with icecold transport buffer by 3 times, and then cells were lysed by 0.01% Triton X. The protein content of cells was measured with a protein assay kit (Bio-Rad, Hercules, CA) using BSA as the reference protein. Transport Studies Using Transporter-Expressing Cells Transport studies using LLC-PK1-expressing human MDR1 or rat mdr1a cells were carried out as described in a previous report.16,17 The cells were cultured in Medium 199 containing 10% fetal bovine
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Figure 1. Naþ- or Hþ-dependent uptake of substrates in rat kidney slices. Uptake of (a) [3H]ergothioneine (0.5 mM) was performed in the presence or in the absence of Naþ at pH 7.4 and 37 C for 1, 3, 5, 8, 10, and 20 min by rat kidney slices. (b) Uptake of [3H]carnitine (1.2 nM) was performed in the presence or in the absence of Naþ at pH 7.4 and 37 C for 1, 5, 10, 15, and 20 min by rat kidney slices. (c) Uptake of [14C]aMG (2.5 mM) was performed in the presence or in the absence of Naþ at pH 7.4 and 37 C for 1, 3, 5, 8, 10, and 20 min by rat kidney slices. Closed and open symbols indicate uptake in the presence or the absence of Naþ, respectively. (d) Uptake of [3H]Gly-Sar (36 nM) was performed at pH 6.0 or pH 7.4 and 37 C for 1, 5, 10, 15, and 20 min by rat kidney slices. Closed and open symbols indicate uptake at pH 6.0 or pH 7.4, respectively. Each result represents the mean ± SEM (n ¼ 4). All data show after subtraction of the uptake of [14C]inulin as the volume of water adhering to the kidney slices. *Indicates a significant difference from the uptake in the absence of Naþ or pH 7.4 at each point (p < 0.05) by Student's t-test.
serum, 100 units/mL penicillin, 100 mg/mL streptomycin, and 500 mg/mL G418 at 37 C in an atmosphere of 5% CO2. LLC-PK1expressing human MDR1 and mock cells were seeded at 3.24 105 cells/well on cell culture inserts (3.0 mm high pore density; Falcon, Corning, NY) in a 12-well plate and were cultured for 6 days. Medium was replaced with fresh medium at 3 and 5 days after seeding. LLC-PK1-expressing rat mdr1a cells were seeded at 6.75 105 cells/well and were cultured for 4 days. Medium was replaced with fresh medium 3 days after seeding. For transport study, cells were preincubated in HBSS (137 mM NaCl, 5.4 mM KCl, 0.95 mM CaCl2, 0.81 mM MgSO4, 0.44 mM KH2PO4, 0.39 mM Na2HPO4, 25 mM D-glucose, and 10 mM HEPES, adjusted to pH 7.4) at 37 C for 15 min. Then, transport reaction was initiated by adding HBSS containing azasetron (20 mM), rhodamine 123 (10 mM), or a paracellular marker Lucifer Yellow (125 mM), and incubated at 37 C for designated period of time. The 200 mL aliquot of medium was taken as sample from the receiver side at 15, 30, 45, and 60 min and then replaced with the same amount of fresh HBSS. Lucifer Yellow was measured by ARVO (1420 ARVO MX/Light; PerkinElmer, Waltham, MA) at the excitation and emission wavelengths 405 and 535 nm, respectively.
1.5 min, was maintained at 70% solvent for 1.5 min, and then returned to the initial condition in 1.0 min. Electrospray positive ionization was used, and the mass transitions were monitored at m/z 350.3 > 224.1 for azasetron and m/z 294.3 > 184.2 for ondansetron as an internal standard. The lower limit of detection for both compounds was 10 nM. Data Analysis The uptake of compounds represents data after subtraction of extracellular space estimated by the apparent uptake of [14C]inulin, and the number (n) represents the number of kidney slices. All experiments were conducted at least twice. Kinetic parameters for carrier-mediated uptake of test substrates were estimated by fitting
Measurement of Azasetron by LC-MS/MS Azasetron was measured by LC-MS/MS (LCMS-8050; Shimadzu, Kyoto, Japan) coupled to an LC-30A system (Shimadzu). The analytical column was a Luna 5 mm C18(2) LC Column (50 mm 2 mm; Phenomenex, CA) maintained at 40 C. The mobile phase composed of 0.1% acetic acid aqueous solution (solvent A) and acetonitrile (solvent B) at a flow rate of 0.3 mL/min and the injection volume of 3 mL. The mobile phase gradient elution started at 10% solvent B for 0.5 min, ramped linearly to 70% solvent B in
Figure 2. Cellular ATP content of rat kidney slices in the presence or absence of O2 bubbling. ATP content in rat kidney tissue slices was measured after incubation at pH 7.4 and 37 C for 10, 30, 60, 120, 240, and 360 min. Closed and open symbols indicate ATP content in the presence or absence of O2 bubbling, respectively. Each point represents the mean ± SD (n ¼ 3).
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Figure 3. Concentration dependence of uptake by rat kidney slices. Uptake of (a) [3H]ergothioneine (1, 2, 10, 100, and 1000 mM) and (b) [3H]carnitine (1, 3, 10, 30, 100, 300, 1000, and 3000 mM) was measured at pH 7.4 and 37 C for 10 min by rat kidney slices. Each point represents the mean ± SD (n ¼ 3 or 4), after subtraction of the uptake of [3H]ergothioneine (10 mM) or [3H]carnitine (10 mM). (c) Uptake of [14C]aMG (50, 100, 300, 500, 1000, 3000, 5000, and 10,000 mM) was measured at pH 7.4 and 37 C for 5 min by rat kidney slices. Each point represents the mean ± SD (n ¼ 3), after subtraction of the uptake of [14C]aMG (30 mM). (d) Uptake of [3H]Gly-Sar (10, 20, 100, 200, 2000, and 10,000 mM) was measured at pH 7.4 and 37 C for 3 min by rat kidney slices. Each point represents the mean ± SD (n ¼ 4), after subtraction of the uptake of [3H]Gly-Sar in the presence of Gly-Leu (10 mM). The data were fitted to the Michaelis-Menten equation and Eadie-Hofstee plot by a nonlinear least-squares regression analysis.
to Equation 1, by means of a nonlinear least-squares regression analysis using KaleidaGraph 4 (HULINKS, Tokyo, Japan):
v ¼ Vmax s=ðKm þ sÞ
(1)
where v, s, Km, and Vmax are the uptake rate of substrate, the substrate concentration in the medium, Michaelis-Menten constant, and the maximal uptake rate, respectively. The half-inhibitory concentration (IC50) values of inhibitors were obtained by examining their inhibitory effects on uptake of test substrates according to following Equation 2:
% of control ¼ 100 IC50 =ðIC50 þ IÞ
(2)
where % of control is inhibition rate in the presence of inhibitors and I is inhibitor concentration in the medium. The apparent permeability coefficients (Papp) were calculated by Equation 3:
Papp ¼ dQ =dt=ðA C0 Þ
(3)
where dQ/dt, A, and C0 are the amount of substrate transported over time t, the area of membrane surface (0.9 cm2), and the initial concentration of substrate in the donor compartment, respectively. Results Uptake of Specific Substrates for Octn1, Octn2, Sglt1/2, and Pept1/2 by Rat Kidney Slices To distinguish activity of apically expressed transporters from that of basolaterally expressed ones in slices uptake, we focused on substrates of transporters that have specific driving forces for apically expressed ones, such as Naþ or Hþ. To evaluate Naþdependent transporter activity of Octn1, Octn2, and Sglt1/2, uptake of their selective substrates, ergothioneine, carnitine, and aMG,
respectively, by kidney slices was measured in the presence and absence of Naþ in transport buffer. Accumulation of [3H]ergothioneine, [3H]carnitine, and [14C]aMG by kidney slices was increased in a time-dependent manner, and uptake of [3H]ergothioneine over 20 min, [3H]carnitine for 10 and 20 min, and [14C] aMG over 20 min in the presence of Naþ was significantly higher than that in the absence of Naþ (Figs. 1a-1c). Because inward Hþ concentration gradient is a driving force of Pept1 and 2, effect of pH on Gly-Sar uptake by slices was examined (Fig. 1d). The uptake of [3H]Gly-Sar at pH 6.0 was significantly higher than that at pH 7.4. Because tissue uptake of substrates except for Gly-Sar increased linearly for the 10 min, following studies with [3H]ergothioneine, [3H]carnitine, and [14C]aMG were carried out within 10 min. For Gly-Sar, initial uptake was evaluated within 5 min. Furthermore, ATP content in slice was measured to evaluate viability of kidney slices during uptake study. The ATP content in kidney slices with and without oxygen supply was decreased to 85.9 ± 22.6% and 43.9 ± 6.3%, respectively, of the initial after 2 h incubation, and to 50.5 ± 10.9% and 38.0 ± 6.2 after 6 h, respectively. Therefore, following studies were carried out within 2 h after preparation of kidney slices in the presence of oxygen supply (Fig. 2). Kinetics of Uptakes by Rat Kidney Slices and Transporter-Transfected Cells Apparent uptake by rat kidney slices was further characterized to evaluate contributing transporters for each compound. Uptake of ergothioneine and carnitine was saturated with an increase in their concentrations from 10 to 1000 mM and from 300 to 3000 mM, respectively, as shown in Figure 3. Kinetic analysis showed that saturable uptake was monophasic with a Km value of 75.4 ± 12.1 mM and 530 ± 55 mM, for ergothioneine and carnitine, respectively (Figs. 3a and 3b). Although similar concentration-dependent
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Figure 4. Effect of inhibitors on the uptake of substrates in rat kidney slices and HEK293 cells. (a) Uptake of [3H]ergothioneine (0.5 mM) was performed in the absence or the presence of verapamil (3, 10, 30, 100, and 300 mM) at pH 7.4 and 37 C for 5 min by rat kidney slices. Each point represents the mean ± SD (n ¼ 3), after subtraction of the uptake [3H] ergothioneine in the absence of Naþ. (b) Uptake of [3H]ergothioneine (1.0 mM) by rOctn1-expressing HEK293 and mock cells was performed in the absence or the presence of verapamil (3, 10, 30, 100, and 300 mM) at pH 7.4 and 37 C for 30 s. Each point represents the mean ± SD (n ¼ 3) after subtraction of the uptake [3H]ergothioneine by the mock cells. (c) Uptake of [3H]Gly-Sar (36 nM) was performed at pH 7.4 and 37 C for 3 min in the absence or presence of Gly-Leu (1, 10, 100, and 1000 mM) by rat kidney slices. Each point represents the mean ± SEM (n ¼ 4), after subtraction of the uptake of [3H]Gly-Sar in the presence of 20 mM Gly-Sar. (d) Uptake of [3H]Gly-Sar (36 nM) was performed in the presence or the absence of the indicated compounds at pH 7.4 and 37 C for 3 min by rat kidney slices. Each bar represents the mean ± SD (n ¼ 4). *Indicates a significant difference from the control at each point (p < 0.05) by Tukey test. (e) Uptake of [3H]carnitine (1.2 nM) was performed at pH 7.4 and 37 C for 10 min in the absence or presence of verapamil (1, 10, 100, and 1000 mM) by rat kidney slices. Each point represents the mean ± SD (n ¼ 4), after subtraction of the uptake of [3H]carnitine in the absence of Naþ. (f) Uptake of [14C]aMG (2.5 mM) was performed at pH 7.4 and 37 C for 5 min in the absence or presence of phlorizin (10, 30, 100, 300, 1000, and 3000 nM) by rat kidney slices. Each point represents the mean ± SD (n ¼ 3), after subtraction of the uptake of [14C]aMG in the absence of Naþ.
uptake was observed for aMG and Gly-Sar, their Eadie-Hofstee plots imply involvement of more than 1 saturable component. Assuming biphasic uptakes, Km value of high- and low-affinity uptakes of aMG were 697 ± 123 mM and 6.81 ± 0.37 mM, respectively, and those for Gly-Sar were 46.4 ± 79.1 mM and 5.03 ± 1.43 mM, respectively (Figs. 3c and 3d). IC50 values of verapamil (an OCTN1/2 inhibitor) to [3H]ergothioneine uptake by the slices and
rOctn1-transfected HEK293 cells were 129 ± 25 mM and 30.8 ± 4.9 mM, respectively (Figs. 4a and 4b). IC50 values of verapamil (a Octn1/2 inhibitor), phlorizin (a SGLT1/2 inhibitor), and glycylleucine (Gly-Leu; a PEPT1/2 inhibitor) to tissue uptake of carnitine, aMG, and Gly-Sar were 268 ± 91 mM, 268 ± 82.0 nM, and 147 ± 11.0 mM, respectively (Figs. 4c, 4e, 4f). The Km and IC50 values for each substrate were mostly comparable to those obtained in
Table 1 Comparison of the Km and IC50 Values Between Kidney Slices and Expression Systems Transporter
Compound
Rat Kidney Slice (mM)
Expression System (mM)
Octn1
Ergothioneine (Km) Verapamil (IC50) Carnitine (Km) Verapamil (IC50) aMG (Km) Phlorizin (IC50) aMG (Km) Phlorizin (IC50) Gly-Sar (Km) Gly-Leu (IC50) Gly-Sar (Km) Gly-Leu (IC50)
75.4 ± 12.1 129 ± 25 530 ± 55 268 ± 91 697 ± 123 0.268 ± 0.082 6810 ± 370 0.268 ± 0.082 5030 ± 1430 147 ± 11 46.4 ± 79.1 147 ± 11
4.64 ± 0.67 30.8 ± 4.9 32.7 ± 5.11 46.7 397 0.179 3000 0.0497 1100 110 110 6
Octn2 Sglt1 Sglt2 Pept1 Pept2
The parameter represents the mean ± SD.
Reference for Expression System 15
This study 23 23 28 29 30 29 22 31 22 31
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Table 2 Transcellular Transport of Azasetron in LLC-PK1-Expressing Human or Rat P-gp, and Mock Cells Cell Lines
Compound
Papp (106 cm/s) Papp(A-B)
Human MDR1
Rat mdr1a
Mock
Azasetron Rhodamine 123 Lucifer Yellow Azasetron Rhodamine 123 Lucifer Yellow Azasetron Rhodamine 123 Lucifer Yellow
3.79 1.37 1.10 3.43 1.59 1.59 8.84 1.78 0.92
± 0.21 ± 0.22 ± 0.33 ± 0.39 ± 0.72 ± 0.27
Ratio Papp(B-A) 19.8 3.77 1.31 23.4 5.02 1.78 13.4 2.90 1.04
± 1.46 ± 0.16 ± 1.42 ± 0.33 ± 0.49 ± 0.12
5.22 2.78 1.19 6.83 3.16 1.12 1.51 1.62 1.13
Permeability data of azasetron and Lucifer Yellow represent the mean ± SD (n ¼ 3), and those of Rhodamine 123 represent the mean (n ¼ 2).
transporter-transfected cell models (Table 1). Moreover, [3H]GlySar uptake by rat kidney slices was decreased to 34.3% of control by 100 mM cefadroxil (a PEPT2 selective inhibitor) after correction of PEPT1-mediated uptake by subtracting the uptake of [3H]Gly-Sar in the presence of 5 mM Gly-Leu (Fig. 4d). Accumulation of P-gp Substrate in Rat Kidney Slices Papp(A-B) and Papp(B-A) values of azasetron were measured in LLCPK1-expressing human MDR1, LLC-PK1-expressing rat mdr1a, and mock cells (Table 2). The efflux ratio (Papp(B-A)/Papp(A-B)) of azasetron was 5.22, 6.83, and 1.51, respectively. Thus, these results suggest that azasetron is a substrate of human MDR1 and rat mdr1a. To evaluate mdr1a-mediated efflux of azasetron in rat kidney slices, accumulation of azasetron was examined in the presence and absence of zosuquidar, a mdr1a inhibitor. In case uptake of azasetron was performed by kidney slice from Wistar rats, the uptake was not increased by azasetron (Supplemental Fig. 1). On the other hand, that from Wistar/ST rats was significantly increased in the presence of 1 mM zosuquidar at 5 and 15 min compared with that in the absence of the inhibitor (Fig. 5). Discussion The present study provides experimental evidence that apical membrane transporters expressed in renal proximal tubular epithelial cells can be evaluated by kidney slices in rats as well as mice. As shown in Figure 1, uptake of ergothioneine, carnitine, and aMG by rat kidney slices was clearly dependent upon extracellular Naþ. Accordingly, it was clearly demonstrated that rat kidney slices are applied for apical influx transporters. Among 3 tested substrates, carnitine uptake in the absence of Naþ was somewhat higher than that of ergothioneine and aMG. This may be due to the presence of Naþ-independent carnitine transporter. We have previously shown that OCTN2 is essential renal reabsorptive transporter for carnitine by identifying OCTN2 as a causative molecule of systemic carnitine deficiency.18 On the other hand, carnitine is also transported by Naþ/Cl-dependent ATB0,þ/SLC6A14 and Naþ-independent CT2/SLC22A16.19,20 ATB0,þ was not expressed in the rat's kidney,21 and CT2 is specifically expressed in the testis.20 Uncharacterized transporters might be involved in the Naþ-independent uptake of carnitine in the kidney. Observed increase in Gly-Sar uptake by slices at pH 6.0 is mostly explained by Pept1 because Pept1 is more sensitive to alteration of pH between pH 6.0 and 7.4 than Pept2.22 We separately determined contribution of Pept1 and Pept2 to Gly-Sar uptake using rat Pept2 selective inhibitor cefadroxil with IC50 value of 2.17 mM for rat Pept1, and that of 3.0 mM for rat Pept2.22 Reduction of [3H]Gly-Sar uptake by cefadroxil suggested that contribution of rat PepT1 and
PepT2 for [3H]Gly-Sar uptake is about 30% and 70%, respectively. On the other hand, our previous study using mouse kidney slices showed that reduction of Gly-Sar uptake by cefadroxil was comparable to that by Gly-Leu. When it is assumed that transport capacity of Pept1 and Pept2 is comparable between rat and mouse, the difference of Pept1 contribution between rat and mouse was consistent with expression level of Pept1 and Pept2.8 Most affinity parameters using several substrates and inhibitors in rat kidney tissue slices were comparable to those in the cells that were transfected with each transporter gene (Table 1). By contrast, estimated Km values of ergothioneine for Octn1 (75.4 mM) and carnitine for Octn2 (530 mM) using rat kidney slices were approximately 20 times higher than those estimated by rat Octn1 (4.64 mM)- and Octn2 (32.7 mM)-expressing cells in the previous report.15,23 Although the reason for such a difference is not clear, Naþ-dependent uptake of ergothioneine and carnitine in kidney slices should reflect Octn1 and Octn2, respectively, because EadieHofstee plot for these substrates suggests that ergothioneine and carnitine transports are mediated via a single saturable component in kidney slices from mice and ergothioneine transport was diminished in Octn1/ knockout miceederived kidney slices.5 Because efflux transporters affect tissue accumulation of drugs, rat mdr1a activity was evaluated using human MDR1 substrate azasetron.24 Accumulation of azasetron in rat kidney slices was significantly increased in the presence of mdr1a inhibitor zosuquidar at 5 and 15 min and tended to increase at 30 and 60 min (Fig. 5). This suggests that kidney slice is used to evaluate apical efflux transporter mdr1a. Because the effect of zosuquidar on accumulation of azasetron was significant at initial phase, the
Figure 5. Time-dependent accumulation of azasetron into rat kidney slices. Accumulation of azasetron (2 mM) was performed in the absence or the presence of zosuquidar (1 mM) at pH 7.4 and 37 C for 5, 15, 30, and 60 min by rat kidney slices. Open and closed circles represent uptake of azasetron in the absence or presence of zosuquidar, respectively. Each point represents the mean ± SD (n ¼ 3). *Indicates a significant difference from the uptake in the absence of zosuquidar at each point (p < 0.05) by Student's t-test.
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inhibitor might affect tissue distribution (e.g., distribution into intracellular compartment and tissue binding) of azasetron. On the other hand, that from Wistar rats was not increased by azasetron. The precise reason is unclear, but strain differences might be existed in mdr1a/1b activity. In addition, we found some difficulties to fully characterize transport activity of P-gp using kidney slices, when digoxin a substrate of mdr1a was used due to fluctuation of measured initial concentration in transport buffer. This may be accounted for nonspecific adsorption of digoxin to use experimental apparatus and cell surface. Transport buffer containing BSA to decrease adsorption of drugs was difficult to be used for experiment due to severe foaming. Moreover, transport buffer containing BSA reduced tissue viability without supply of oxygen (Fig. 2). Further studies need to clarify whether efflux transporters affect accumulation of drugs in kidney slices. The Naþ-dependent or inhibitors-mediated uptake of each compounds considered to be mediated by transporters expressed in renal proximal tubular epithelial cells. On the other hand, the present study could not been molecularly supported, whereas our previous study indicated Naþ-dependent uptake of ergothioneine was mediated by Octn1.5 Although we preliminarily tried to perform in vivo knockdown for octn1 and gapdh genes by hydrodynamic perfusion method using siRNA in mice kidney (data not shown), efficient knockdown for mRNA expression of the genes was not observed. Recently, the knockdown studies using siRNA by a long-term culture method for kidney slices have been reported.25 Although there is a problem of knockdown efficiency, the methodology may enable to show the molecular involvement of various molecules including the tested genes in the present study. It was suggested that several renal transporters in disease model animals were changed compared with normal animals. For example, protein expression of Pept1 was increased in streptozotocininduced diabetes in rats.26 Moreover, protein expression of Pept1, Pept2, mdr1a, and mdr1b was increased in uninephrectomized rats.27 Because rats were used as disease model, the present study could provide important information to evaluate renal function in such disease models. In conclusion, we demonstrate that rat kidney slices are useful tools for evaluating transport activity of apically expressed transporters, for example, reabsorptive transporters Octn1/2, Sglt1/2, Pept1/2, and most likely efflux transporter mdr1a. Because this method has been used to examine basolaterally expressed transporters, kidney slices are useful technique for evaluation of both apical and basolateral transporters as long as tested substrate compounds exhibit different transport characteristics between both membranes. In addition, considering that activities of metabolic enzymes expressed in the kidney should be preserved, this method is considered useful in evaluating apparent disposition of drugs in the kidney. References 1. Nakamura T, Yonezawa A, Hashimoto S, Katsura T, Inui K. Disruption of multidrug and toxin extrusion MATE1 potentiates cisplatin-induced nephrotoxicity. Biochem Pharmacol. 2010;80(11):1762-1767. 2. Bajaj P, Chowdhury SK, Yucha R, Kelly EJ, Xiao G. Emerging kidney models to investigate metabolism, transport, and toxicity of drugs and xenobiotics. Drug Metab Dispos. 2018;46(11):1692-1702. 3. Brown CD, Sayer R, Windass AS, et al. Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling. Toxicol Appl Pharmacol. 2008;233(3):428-438. 4. Nakanishi T, Fukushi A, Sato M, et al. Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture. Mol Pharm. 2011;8(6):2142-2150.
5. Arakawa H, Washio I, Matsuoka N, et al. Usefulness of kidney slices for functional analysis of apical reabsorptive transporters. Sci Rep. 2017;7(1): 12814. 6. Schinkel AH, Smit JJ, van Tellingen O, et al. Disruption of the mouse mdr1a Pglycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell. 1994;77(4):491-502. 7. Schinkel AH, Wagenaar E, van Deemter L, Mol CA, Borst P. Absence of the mdr1a P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J Clin Invest. 1995;96(4): 1698-1705. 8. Lu H, Klaassen C. Tissue distribution and thyroid hormone regulation of Pept1 and Pept2 mRNA in rodents. Peptides. 2006;27(4):850-857. 9. Sabolic I, Vrhovac I, Eror DB, et al. Expression of Naþ-D-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences. Am J Physiol Cell Physiol. 2012;302(8):C1174-C1188. 10. Katoh M, Suzuyama N, Takeuchi T, Yoshitomi S, Asahi S, Yokoi T. Kinetic analyses for species differences in P-glycoprotein-mediated drug transport. J Pharm Sci. 2006;95(12):2673-2683. 11. Takeuchi T, Yoshitomi S, Higuchi T, et al. Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1. Pharm Res. 2006;23(7):1460-1472. 12. Okamoto K, Aoki K. Development of a strain of spontaneously hypertensive rats. Jpn Circ J. 1963;27:282-293. 13. Clark JB, Palmer CJ, Shaw WN. The diabetic Zucker fatty rat. Proc Soc Exp Biol Med. 1983;173(1):68-75. 14. Omote S, Matsuoka N, Arakawa H, Nakanishi T, Tamai I. Effect of tyrosine kinase inhibitors on renal handling of creatinine by MATE1. Sci Rep. 2018;8(1): 9237. 15. Nakamura T, Yoshida K, Yabuuchi H, Maeda T, Tamai I. Functional characterization of ergothioneine transport by rat organic cation/carnitine transporter Octn1 (slc22a4). Biol Pharm Bull. 2008;31(8):1580-1584. 16. Ueda K, Okamura N, Hirai M, et al. Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J Biol Chem. 1992;267(34):24248-24252. 17. Shirasaka Y, Suzuki K, Shichiri M, Nakanishi T, Tamai I. Intestinal absorption of HMG-CoA reductase inhibitor pitavastatin mediated by organic anion transporting polypeptide and P-glycoprotein/multidrug resistance 1. Drug Metab Pharmacokinet. 2011;26(2):171-179. 18. Nezu J, Tamai I, Oku A, et al. Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter. Nat Genet. 1999;21(1):91-94. 19. Nakanishi T, Hatanaka T, Huang W, et al. Naþ- and Cl–coupled active transport of carnitine by the amino acid transporter ATB(0,þ) from mouse colon expressed in HRPE cells and Xenopus oocytes. J Physiol. 2001;532(Pt 2):297-304. 20. Enomoto A, Wempe MF, Tsuchida H, et al. Molecular identification of a novel carnitine transporter specific to human testis. Insights into the mechanism of carnitine recognition. J Biol Chem. 2002;277(39):36262-36271. 21. The National Center for Biotechnology Information. Slc6a14 solute carrier family 6 member 14 [Rattus norvegicus (Norway rat)]. Available at: https:// www.ncbi.nlm.nih.gov/gene/298340. Accessed December 20, 2018. 22. Terada T, Saito H, Mukai M, Inui K. Recognition of beta-lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1 cells. Am J Physiol. 1997;273(5 Pt 2):F706-F711. 23. Szabo K, Nagy Z, Juhasz V, et al. Species specificity profiling of rat and human organic cation/carnitine transporter Slc22a5/SLC22A5 (Octn2/OCTN2). Drug Metab Pharmacokinet. 2017;32(3):165-171. 24. Tamai I, Saheki A, Saitoh R, Sai Y, Yamada I, Tsuji A. Nonlinear intestinal absorption of 5-hydroxytryptamine receptor antagonist caused by absorptive and secretory transporters. J Pharmacol Exp Ther. 1997;283(1):108-115. 25. Ruigrok MJR, Maggan N, Willaert D, et al. siRNA-mediated RNA interference in precision-cut tissue slices prepared from mouse lung and kidney. AAPS J. 2017;19(6):1855-1863. 26. Gangopadhyay A, Thamotharan M, Adibi SA. Regulation of oligopeptide transporter (Pept-1) in experimental diabetes. Am J Physiol Gastrointest Liver Physiol. 2002;283(1):G133-G138. 27. Tramonti G, Xie P, Wallner EI, Danesh FR, Kanwar YS. Expression and functional characteristics of tubular transporters: P-glycoprotein, PEPT1, and PEPT2 in renal mass reduction and diabetes. Am J Physiol Renal Physiol. 2006;291(5):F972-F980. 28. Lee WS, Kanai Y, Wells RG, Hediger MA. The high affinity Naþ/glucose cotransporter. Re-evaluation of function and distribution of expression. J Biol Chem. 1994;269(16):12032-12039. 29. Tahara A, Kurosaki E, Yokono M, et al. Pharmacological profile of ipragliflozin (ASP1941), a novel selective SGLT2 inhibitor, in vitro and in vivo. Naunyn Schmiedebergs Arch Pharmacol. 2012;385(4):423-436. 30. You G, Lee WS, Barros EJ, et al. Molecular characteristics of Na(þ)-coupled glucose transporters in adult and embryonic rat kidney. J Biol Chem. 1995;270(49):29365-29371. 31. Terada T, Sawada K, Irie M, Saito H, Hashimoto Y, Inui K. Structural requirements for determining the substrate affinity of peptide transporters PEPT1 and PEPT2. Pflugers Arch. 2000;440(5):679-684.
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Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism
Rat Kidney Slices for Evaluation of Apical Membrane Transporters in Proximal Tubular Cells Hiroshi Arakawa 1, Hikaru Kubo 1, Ikumi Washio 1, 2, Angelina Yukiko Staub 1, Shiho Nedachi 1, Naoki Ishiguro 2, Takeo Nakanishi 1, Ikumi Tamai 1, * 1 2
Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa, Japan Pharmacokinetics and Non-Clinical Safety Department, Nippon Boehringer Ingelheim Co., Ltd., Kobe, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 25 December 2018 Revised 27 March 2019 Accepted 28 March 2019 Available online 5 April 2019
Kidney slice has been often used as a tool reflecting basolateral transport in renal tubular epithelial cells. Recently, we reported that several important apical reabsorptive transporters such as Octn1/2, Sglt1/2, and Pept1/2 were functional in mouse kidney slices as well as transporter activities in basolateral side, which have been well accepted. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. The present study investigates usefulness of kidney slices from rats for evaluation of apical membrane transporters for efflux (multidrug resistance 1a, mdr1a) as well as influx (Octn1/2, Sglt1/2, Pept1/2). Naþ-dependent uptake of ergothioneine (Octn1), carnitine (Octn2), and methyl-a-D-glucopyranoside (Sglt1/2) by rat kidney slices was observed, and the uptake was decreased by selective inhibitors. In addition, uptake of glycyl-sarcosine (Pept1/2) showed Hþdependence and was decreased by selective inhibitor. Furthermore, accumulation of mdr1a substrate azasetron was increased in the presence of zosuquidar, an mdr1a inhibitor, while strain differences existed. In conclusion, rat kidney slices should be useful for evaluation of renal drug disposition regulated by transporters in apical as well as basolateral membranes of rat renal proximal tubule cells. © 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
Keywords: renal transport transporter drug disposition P-gp reabsorption
Introduction Membrane transporters play important roles in renal handling, for example, renal reabsorption, secretion, and disposition of nutrients and xenobiotics, including drugs and their metabolites. For example, apically expressed sodium-dependent glucose transporter 2 (SGLT2) plays a major role in glucose reabsorption, of which inhibitors are used for treatment of diabetes. Apical efflux transporter MATE1 was involved in cisplatin-induced renal toxicity as demonstrated in mate1 knockout mice.1 Therefore, pharmacokinetic, Abbreviations used: OCTN, organic cation/carnitine transporter; SGLT, sodiumdependent glucose transporter; PEPT, oligopeptide transporter; OAT, organic anion transporter; OCT, organic cation transporter; aMG, methyl a-D-glucopyranoside; Gly-Sar, glycyl-sarcosine; Gly-Leu, glycyl-leucin; BSA, bovine serum albumin. Conflicts of interest: This study was supported partly by Nippon Boehringer Ingelheim Co., Ltd. The authors Hiroshi Arakawa and Hikaru Kubo equally contributed to the preparation of the study. This article contains supplementary material available from the authors by request or via the Internet at https://doi.org/10.1016/j.xphs.2019.03.031. * Correspondence to: Ikumi Tamai, (Telephone: þ81-76-234-4479). E-mail address: [email protected] (I. Tamai).
pharmacodynamic, and toxicological demands exist in the evaluation of apical transporters as well as efflux transporters in the kidney. Several in vitro methods have been reported, including human renal cell line models and primary culture of renal epithelial cells.2-4 However, because they have limitations due to lowered expression of various genes compared with the intact kidney during cultivation, in vitro experimental method for apical uptake and efflux transporters using intact tissue or cells is desirable. Kidney slices are one of the tools to characterize renal disposition of drugs. This method has been used for evaluating basolateral uptake transporters, but we recently demonstrated that apically expressed transporters, including organic cation/carnitine transporter (Octn1/2), Sglt1/2, and peptide transporter (Pept)1/2, could be evaluated by kidney slices from mice.5 There are some advantages in this method: (1) biological macromolecules including functional transporter proteins and metabolizing enzymes are likely preserved in their intact form, (2) kidney tissue architecture is retained well with all resident cell types available and cell-cell contacts are preserved, and (3) the handling of kidney slices is quite simple. Therefore, this methodology should be promising to evaluate renal disposition of drugs and nutrients.
https://doi.org/10.1016/j.xphs.2019.03.031 0022-3549/© 2019 Published by Elsevier Inc. on behalf of the American Pharmacists Association.
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In addition to apically expressed influx transporters, efflux transporters such as multidrug resistance 1 (MDR1) contribute to drug disposition. In mdr1a knockout mice, renal accumulation of mdr1a substrates such as digoxin, ivermectin, and vinblastine were significantly higher than that in wild-type mice.6,7 Such an increase of tissue accumulation is possibly caused by a decreased apical efflux and should lead to drug-induced toxicity. However, it has not been clarified whether this method could apply for apically expressed efflux transporters. Accordingly, it needs to analyze them in kidney slices for prediction of drugs disposition. In the present study, we evaluated usefulness of kidney slices obtained from rats by measuring accumulation of substrates of each transporter. Because rats are often used for preclinical pharmacodynamic and pharmacokinetic studies as well as mice because of convenient handling such as tissue and blood collection, it is important to confirm applicability of rat kidney slices for evaluation of apically expressed transporters. As there is species difference between rats and mice, Pept1 mRNA expression in the kidney of rats is much higher than that of mice.8 Moreover, the abundance of Sglt2 protein in the kidney was higher in male mice than in female mice, while that in female rats was higher than that in male rats.9 There is also species difference of affinity for med1a/1b-mediated transport of several compounds between rats and mice.10,11 In addition, several disease models are available only in rats, such as spontaneous hypertension12 and Zucker diabetes.13 In case apically expressed renal transporters in disease model rats were evaluated, basic data for evaluation of the transporters are necessary. The present study aimed to characterize apical influx transporters (Octn1/2, Sglt1/2, and Pept1/2) as well as efflux transporter (mdr1a) in rat kidney slices. Methods Materials [3H]Ergothioneine (100 mCi/mmol, custom-made) [methyl- C],-a-D-glucopyranoside (aMG, 39.3 mCi/mmol), and [3H]glycylsarcosine (Gly-Sar, 2.8 Ci/mmol) were purchased from Moravek Biochemicals (Brea, CA). L-[N-methyl-3H]carnitine (80 Ci/mmol) and [carboxyl-14C]inulin (2.5 mCi/g) were purchased from American Radiolabeled Chemicals Inc. (St, Louis, Mo). Azasetron HCl was obtained from Yoshitomi Pharmaceutical Industries (Osaka, Japan). All other chemicals and reagents were commercial products of reagent grade. 14
Animals Male Wistar (7 weeks) and Wistar/ST (7 weeks) rats were purchased from Sankyo Labo Service Co. Inc. (Tokyo, Japan). Rats were housed 3 per cage with free access to commercial chow and tap water and were maintained on a 12 h dark/light cycle in an aircontrolled room (temperature 24.0 ± 1 C; humidity 55 ± 5%). All animal studies were approved by the Kanazawa University Institutional Animal Care and Use Committee (AP-143148). Uptake Study by Rat Kidney Slices Uptake studies in rat kidney slices were carried out as described previously with slight modifications.5,14 Wistar rats were used for uptake study of ergothioneine, carnitine, aMG, and Gly-Sar, and Wistar/ST rats were used for uptake of azasetron. Briefly, rats were anesthetized with pentobarbital (50 mg/kg) and whole kidneys were removed after blood removal. Kidney slices (0.3 mm thick) were prepared with a microslicer (Zero 1; Dosaka EM, Kyoto, Japan) and were immediately placed in ice-cold oxygenated transport
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buffer (130 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4, and 25 mM HEPES, adjusted to pH 7.4). To determine effect of Naþ on substrate uptake, NaCl was replaced with Nmethyl-D-glucamine in transport buffer. Three or four slices, weight of which ranged from 6 to 14 mg, were preincubated in a 12well plate with oxygenated buffer at 37 C for 5 min. To initiate uptake reaction, the slices were transferred into transport buffer containing radiolabeled or unlabeled test compounds. The uptake reaction was performed at 37 C for the designated period of time under oxygen supply, and then the reaction was terminated by removing slices from the incubating solution and rinsing them with fresh ice-cold transport buffer by 3 times. The washed slices were blotted on filter paper and weighed. To measure radioactivity, slices were dissolved by 1 N NaOH in water bath at 60 C for 20 min, and then the lysate was neutralized with 5 N HCl. All samples were mixed with liquid scintillation cocktail Cleasol I (Nacalai Tesque, Kyoto, Japan), and radioactivity was measured in a liquid scintillation counter (Hitachi Aloka Medical, Tokyo, Japan). The volume of water adhering to the kidney slices was estimated by the apparent uptake of [14C]inulin with 0.150 ± 0.022 mL/5 min/mg kidney (CV: 14.5% by 3 independent assay). To quantify azasetron, slices were homogenated by sonication in transport buffer, and proteins were removed by centrifugation after denatured adding acetonitrile. The resultant supernatants were subjected to LC-MS/MS analysis. Measurement of ATP Content in Kidney Slices Rat kidney slices were incubated for 10, 30, 60, 120, 240, and 360 min at pH 7.4 and 37 C in the presence or the absence of oxygen supply. For the condition without oxygen supply, bovine serum albumin (BSA) was added to the transport buffer to avoid drug adsorption. After incubation, the kidney slices were collected and immediately frozen and stored in liquid nitrogen for subsequent ATP analysis. The tissues were homogenized by bullet blender and centrifuged at 1000 g for 10 min at 4 C. The resultant supernatant was collected for analyzing ATP using “Tissue” ATP assay kit (TOYO B-Net Co., Ltd, Tokyo, Japan). Relative ATP content was calculated according to amount of luminescence versus the ATP standard curve. Uptake Study Using Transporter-Expressing Cells Rat Octn1-expressing and empty vector transfected (mock) HEK293 cells were established previously.15 The cells were cultured in Dulbecco's modified Eagle's medium containing 10% (v/v) fetal bovine serum (Life Technologies, Carlsbad, CA), 100 units/mL penicillin, and 100 mg/mL streptomycin at 37 C in an atmosphere of 5% CO2. Octn1-expressing and mock HEK293 cells were seeded at 1.0 105 cells/well onto a 24-well poly-L-lysine-coated tissue culture plate. Two days later, cells were used for uptake study by preincubating with transport buffer (125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl2, 1.2 mM KH2PO4, 1.2 mM MgSO4, and 25 mM HEPES, adjusted to pH 7.4) at 37 C for 15 min, followed by uptake reaction by adding transport buffer containing 0.05 mCi/mL [3H]ergothioneine. The uptake reaction was carried out at 37 C for 30 s. To terminate uptake reaction, the cells were washed with icecold transport buffer by 3 times, and then cells were lysed by 0.01% Triton X. The protein content of cells was measured with a protein assay kit (Bio-Rad, Hercules, CA) using BSA as the reference protein. Transport Studies Using Transporter-Expressing Cells Transport studies using LLC-PK1-expressing human MDR1 or rat mdr1a cells were carried out as described in a previous report.16,17 The cells were cultured in Medium 199 containing 10% fetal bovine
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Figure 1. Naþ- or Hþ-dependent uptake of substrates in rat kidney slices. Uptake of (a) [3H]ergothioneine (0.5 mM) was performed in the presence or in the absence of Naþ at pH 7.4 and 37 C for 1, 3, 5, 8, 10, and 20 min by rat kidney slices. (b) Uptake of [3H]carnitine (1.2 nM) was performed in the presence or in the absence of Naþ at pH 7.4 and 37 C for 1, 5, 10, 15, and 20 min by rat kidney slices. (c) Uptake of [14C]aMG (2.5 mM) was performed in the presence or in the absence of Naþ at pH 7.4 and 37 C for 1, 3, 5, 8, 10, and 20 min by rat kidney slices. Closed and open symbols indicate uptake in the presence or the absence of Naþ, respectively. (d) Uptake of [3H]Gly-Sar (36 nM) was performed at pH 6.0 or pH 7.4 and 37 C for 1, 5, 10, 15, and 20 min by rat kidney slices. Closed and open symbols indicate uptake at pH 6.0 or pH 7.4, respectively. Each result represents the mean ± SEM (n ¼ 4). All data show after subtraction of the uptake of [14C]inulin as the volume of water adhering to the kidney slices. *Indicates a significant difference from the uptake in the absence of Naþ or pH 7.4 at each point (p < 0.05) by Student's t-test.
serum, 100 units/mL penicillin, 100 mg/mL streptomycin, and 500 mg/mL G418 at 37 C in an atmosphere of 5% CO2. LLC-PK1expressing human MDR1 and mock cells were seeded at 3.24 105 cells/well on cell culture inserts (3.0 mm high pore density; Falcon, Corning, NY) in a 12-well plate and were cultured for 6 days. Medium was replaced with fresh medium at 3 and 5 days after seeding. LLC-PK1-expressing rat mdr1a cells were seeded at 6.75 105 cells/well and were cultured for 4 days. Medium was replaced with fresh medium 3 days after seeding. For transport study, cells were preincubated in HBSS (137 mM NaCl, 5.4 mM KCl, 0.95 mM CaCl2, 0.81 mM MgSO4, 0.44 mM KH2PO4, 0.39 mM Na2HPO4, 25 mM D-glucose, and 10 mM HEPES, adjusted to pH 7.4) at 37 C for 15 min. Then, transport reaction was initiated by adding HBSS containing azasetron (20 mM), rhodamine 123 (10 mM), or a paracellular marker Lucifer Yellow (125 mM), and incubated at 37 C for designated period of time. The 200 mL aliquot of medium was taken as sample from the receiver side at 15, 30, 45, and 60 min and then replaced with the same amount of fresh HBSS. Lucifer Yellow was measured by ARVO (1420 ARVO MX/Light; PerkinElmer, Waltham, MA) at the excitation and emission wavelengths 405 and 535 nm, respectively.
1.5 min, was maintained at 70% solvent for 1.5 min, and then returned to the initial condition in 1.0 min. Electrospray positive ionization was used, and the mass transitions were monitored at m/z 350.3 > 224.1 for azasetron and m/z 294.3 > 184.2 for ondansetron as an internal standard. The lower limit of detection for both compounds was 10 nM. Data Analysis The uptake of compounds represents data after subtraction of extracellular space estimated by the apparent uptake of [14C]inulin, and the number (n) represents the number of kidney slices. All experiments were conducted at least twice. Kinetic parameters for carrier-mediated uptake of test substrates were estimated by fitting
Measurement of Azasetron by LC-MS/MS Azasetron was measured by LC-MS/MS (LCMS-8050; Shimadzu, Kyoto, Japan) coupled to an LC-30A system (Shimadzu). The analytical column was a Luna 5 mm C18(2) LC Column (50 mm 2 mm; Phenomenex, CA) maintained at 40 C. The mobile phase composed of 0.1% acetic acid aqueous solution (solvent A) and acetonitrile (solvent B) at a flow rate of 0.3 mL/min and the injection volume of 3 mL. The mobile phase gradient elution started at 10% solvent B for 0.5 min, ramped linearly to 70% solvent B in
Figure 2. Cellular ATP content of rat kidney slices in the presence or absence of O2 bubbling. ATP content in rat kidney tissue slices was measured after incubation at pH 7.4 and 37 C for 10, 30, 60, 120, 240, and 360 min. Closed and open symbols indicate ATP content in the presence or absence of O2 bubbling, respectively. Each point represents the mean ± SD (n ¼ 3).
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Figure 3. Concentration dependence of uptake by rat kidney slices. Uptake of (a) [3H]ergothioneine (1, 2, 10, 100, and 1000 mM) and (b) [3H]carnitine (1, 3, 10, 30, 100, 300, 1000, and 3000 mM) was measured at pH 7.4 and 37 C for 10 min by rat kidney slices. Each point represents the mean ± SD (n ¼ 3 or 4), after subtraction of the uptake of [3H]ergothioneine (10 mM) or [3H]carnitine (10 mM). (c) Uptake of [14C]aMG (50, 100, 300, 500, 1000, 3000, 5000, and 10,000 mM) was measured at pH 7.4 and 37 C for 5 min by rat kidney slices. Each point represents the mean ± SD (n ¼ 3), after subtraction of the uptake of [14C]aMG (30 mM). (d) Uptake of [3H]Gly-Sar (10, 20, 100, 200, 2000, and 10,000 mM) was measured at pH 7.4 and 37 C for 3 min by rat kidney slices. Each point represents the mean ± SD (n ¼ 4), after subtraction of the uptake of [3H]Gly-Sar in the presence of Gly-Leu (10 mM). The data were fitted to the Michaelis-Menten equation and Eadie-Hofstee plot by a nonlinear least-squares regression analysis.
to Equation 1, by means of a nonlinear least-squares regression analysis using KaleidaGraph 4 (HULINKS, Tokyo, Japan):
v ¼ Vmax s=ðKm þ sÞ
(1)
where v, s, Km, and Vmax are the uptake rate of substrate, the substrate concentration in the medium, Michaelis-Menten constant, and the maximal uptake rate, respectively. The half-inhibitory concentration (IC50) values of inhibitors were obtained by examining their inhibitory effects on uptake of test substrates according to following Equation 2:
% of control ¼ 100 IC50 =ðIC50 þ IÞ
(2)
where % of control is inhibition rate in the presence of inhibitors and I is inhibitor concentration in the medium. The apparent permeability coefficients (Papp) were calculated by Equation 3:
Papp ¼ dQ =dt=ðA C0 Þ
(3)
where dQ/dt, A, and C0 are the amount of substrate transported over time t, the area of membrane surface (0.9 cm2), and the initial concentration of substrate in the donor compartment, respectively. Results Uptake of Specific Substrates for Octn1, Octn2, Sglt1/2, and Pept1/2 by Rat Kidney Slices To distinguish activity of apically expressed transporters from that of basolaterally expressed ones in slices uptake, we focused on substrates of transporters that have specific driving forces for apically expressed ones, such as Naþ or Hþ. To evaluate Naþdependent transporter activity of Octn1, Octn2, and Sglt1/2, uptake of their selective substrates, ergothioneine, carnitine, and aMG,
respectively, by kidney slices was measured in the presence and absence of Naþ in transport buffer. Accumulation of [3H]ergothioneine, [3H]carnitine, and [14C]aMG by kidney slices was increased in a time-dependent manner, and uptake of [3H]ergothioneine over 20 min, [3H]carnitine for 10 and 20 min, and [14C] aMG over 20 min in the presence of Naþ was significantly higher than that in the absence of Naþ (Figs. 1a-1c). Because inward Hþ concentration gradient is a driving force of Pept1 and 2, effect of pH on Gly-Sar uptake by slices was examined (Fig. 1d). The uptake of [3H]Gly-Sar at pH 6.0 was significantly higher than that at pH 7.4. Because tissue uptake of substrates except for Gly-Sar increased linearly for the 10 min, following studies with [3H]ergothioneine, [3H]carnitine, and [14C]aMG were carried out within 10 min. For Gly-Sar, initial uptake was evaluated within 5 min. Furthermore, ATP content in slice was measured to evaluate viability of kidney slices during uptake study. The ATP content in kidney slices with and without oxygen supply was decreased to 85.9 ± 22.6% and 43.9 ± 6.3%, respectively, of the initial after 2 h incubation, and to 50.5 ± 10.9% and 38.0 ± 6.2 after 6 h, respectively. Therefore, following studies were carried out within 2 h after preparation of kidney slices in the presence of oxygen supply (Fig. 2). Kinetics of Uptakes by Rat Kidney Slices and Transporter-Transfected Cells Apparent uptake by rat kidney slices was further characterized to evaluate contributing transporters for each compound. Uptake of ergothioneine and carnitine was saturated with an increase in their concentrations from 10 to 1000 mM and from 300 to 3000 mM, respectively, as shown in Figure 3. Kinetic analysis showed that saturable uptake was monophasic with a Km value of 75.4 ± 12.1 mM and 530 ± 55 mM, for ergothioneine and carnitine, respectively (Figs. 3a and 3b). Although similar concentration-dependent
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Figure 4. Effect of inhibitors on the uptake of substrates in rat kidney slices and HEK293 cells. (a) Uptake of [3H]ergothioneine (0.5 mM) was performed in the absence or the presence of verapamil (3, 10, 30, 100, and 300 mM) at pH 7.4 and 37 C for 5 min by rat kidney slices. Each point represents the mean ± SD (n ¼ 3), after subtraction of the uptake [3H] ergothioneine in the absence of Naþ. (b) Uptake of [3H]ergothioneine (1.0 mM) by rOctn1-expressing HEK293 and mock cells was performed in the absence or the presence of verapamil (3, 10, 30, 100, and 300 mM) at pH 7.4 and 37 C for 30 s. Each point represents the mean ± SD (n ¼ 3) after subtraction of the uptake [3H]ergothioneine by the mock cells. (c) Uptake of [3H]Gly-Sar (36 nM) was performed at pH 7.4 and 37 C for 3 min in the absence or presence of Gly-Leu (1, 10, 100, and 1000 mM) by rat kidney slices. Each point represents the mean ± SEM (n ¼ 4), after subtraction of the uptake of [3H]Gly-Sar in the presence of 20 mM Gly-Sar. (d) Uptake of [3H]Gly-Sar (36 nM) was performed in the presence or the absence of the indicated compounds at pH 7.4 and 37 C for 3 min by rat kidney slices. Each bar represents the mean ± SD (n ¼ 4). *Indicates a significant difference from the control at each point (p < 0.05) by Tukey test. (e) Uptake of [3H]carnitine (1.2 nM) was performed at pH 7.4 and 37 C for 10 min in the absence or presence of verapamil (1, 10, 100, and 1000 mM) by rat kidney slices. Each point represents the mean ± SD (n ¼ 4), after subtraction of the uptake of [3H]carnitine in the absence of Naþ. (f) Uptake of [14C]aMG (2.5 mM) was performed at pH 7.4 and 37 C for 5 min in the absence or presence of phlorizin (10, 30, 100, 300, 1000, and 3000 nM) by rat kidney slices. Each point represents the mean ± SD (n ¼ 3), after subtraction of the uptake of [14C]aMG in the absence of Naþ.
uptake was observed for aMG and Gly-Sar, their Eadie-Hofstee plots imply involvement of more than 1 saturable component. Assuming biphasic uptakes, Km value of high- and low-affinity uptakes of aMG were 697 ± 123 mM and 6.81 ± 0.37 mM, respectively, and those for Gly-Sar were 46.4 ± 79.1 mM and 5.03 ± 1.43 mM, respectively (Figs. 3c and 3d). IC50 values of verapamil (an OCTN1/2 inhibitor) to [3H]ergothioneine uptake by the slices and
rOctn1-transfected HEK293 cells were 129 ± 25 mM and 30.8 ± 4.9 mM, respectively (Figs. 4a and 4b). IC50 values of verapamil (a Octn1/2 inhibitor), phlorizin (a SGLT1/2 inhibitor), and glycylleucine (Gly-Leu; a PEPT1/2 inhibitor) to tissue uptake of carnitine, aMG, and Gly-Sar were 268 ± 91 mM, 268 ± 82.0 nM, and 147 ± 11.0 mM, respectively (Figs. 4c, 4e, 4f). The Km and IC50 values for each substrate were mostly comparable to those obtained in
Table 1 Comparison of the Km and IC50 Values Between Kidney Slices and Expression Systems Transporter
Compound
Rat Kidney Slice (mM)
Expression System (mM)
Octn1
Ergothioneine (Km) Verapamil (IC50) Carnitine (Km) Verapamil (IC50) aMG (Km) Phlorizin (IC50) aMG (Km) Phlorizin (IC50) Gly-Sar (Km) Gly-Leu (IC50) Gly-Sar (Km) Gly-Leu (IC50)
75.4 ± 12.1 129 ± 25 530 ± 55 268 ± 91 697 ± 123 0.268 ± 0.082 6810 ± 370 0.268 ± 0.082 5030 ± 1430 147 ± 11 46.4 ± 79.1 147 ± 11
4.64 ± 0.67 30.8 ± 4.9 32.7 ± 5.11 46.7 397 0.179 3000 0.0497 1100 110 110 6
Octn2 Sglt1 Sglt2 Pept1 Pept2
The parameter represents the mean ± SD.
Reference for Expression System 15
This study 23 23 28 29 30 29 22 31 22 31
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Table 2 Transcellular Transport of Azasetron in LLC-PK1-Expressing Human or Rat P-gp, and Mock Cells Cell Lines
Compound
Papp (106 cm/s) Papp(A-B)
Human MDR1
Rat mdr1a
Mock
Azasetron Rhodamine 123 Lucifer Yellow Azasetron Rhodamine 123 Lucifer Yellow Azasetron Rhodamine 123 Lucifer Yellow
3.79 1.37 1.10 3.43 1.59 1.59 8.84 1.78 0.92
± 0.21 ± 0.22 ± 0.33 ± 0.39 ± 0.72 ± 0.27
Ratio Papp(B-A) 19.8 3.77 1.31 23.4 5.02 1.78 13.4 2.90 1.04
± 1.46 ± 0.16 ± 1.42 ± 0.33 ± 0.49 ± 0.12
5.22 2.78 1.19 6.83 3.16 1.12 1.51 1.62 1.13
Permeability data of azasetron and Lucifer Yellow represent the mean ± SD (n ¼ 3), and those of Rhodamine 123 represent the mean (n ¼ 2).
transporter-transfected cell models (Table 1). Moreover, [3H]GlySar uptake by rat kidney slices was decreased to 34.3% of control by 100 mM cefadroxil (a PEPT2 selective inhibitor) after correction of PEPT1-mediated uptake by subtracting the uptake of [3H]Gly-Sar in the presence of 5 mM Gly-Leu (Fig. 4d). Accumulation of P-gp Substrate in Rat Kidney Slices Papp(A-B) and Papp(B-A) values of azasetron were measured in LLCPK1-expressing human MDR1, LLC-PK1-expressing rat mdr1a, and mock cells (Table 2). The efflux ratio (Papp(B-A)/Papp(A-B)) of azasetron was 5.22, 6.83, and 1.51, respectively. Thus, these results suggest that azasetron is a substrate of human MDR1 and rat mdr1a. To evaluate mdr1a-mediated efflux of azasetron in rat kidney slices, accumulation of azasetron was examined in the presence and absence of zosuquidar, a mdr1a inhibitor. In case uptake of azasetron was performed by kidney slice from Wistar rats, the uptake was not increased by azasetron (Supplemental Fig. 1). On the other hand, that from Wistar/ST rats was significantly increased in the presence of 1 mM zosuquidar at 5 and 15 min compared with that in the absence of the inhibitor (Fig. 5). Discussion The present study provides experimental evidence that apical membrane transporters expressed in renal proximal tubular epithelial cells can be evaluated by kidney slices in rats as well as mice. As shown in Figure 1, uptake of ergothioneine, carnitine, and aMG by rat kidney slices was clearly dependent upon extracellular Naþ. Accordingly, it was clearly demonstrated that rat kidney slices are applied for apical influx transporters. Among 3 tested substrates, carnitine uptake in the absence of Naþ was somewhat higher than that of ergothioneine and aMG. This may be due to the presence of Naþ-independent carnitine transporter. We have previously shown that OCTN2 is essential renal reabsorptive transporter for carnitine by identifying OCTN2 as a causative molecule of systemic carnitine deficiency.18 On the other hand, carnitine is also transported by Naþ/Cl-dependent ATB0,þ/SLC6A14 and Naþ-independent CT2/SLC22A16.19,20 ATB0,þ was not expressed in the rat's kidney,21 and CT2 is specifically expressed in the testis.20 Uncharacterized transporters might be involved in the Naþ-independent uptake of carnitine in the kidney. Observed increase in Gly-Sar uptake by slices at pH 6.0 is mostly explained by Pept1 because Pept1 is more sensitive to alteration of pH between pH 6.0 and 7.4 than Pept2.22 We separately determined contribution of Pept1 and Pept2 to Gly-Sar uptake using rat Pept2 selective inhibitor cefadroxil with IC50 value of 2.17 mM for rat Pept1, and that of 3.0 mM for rat Pept2.22 Reduction of [3H]Gly-Sar uptake by cefadroxil suggested that contribution of rat PepT1 and
PepT2 for [3H]Gly-Sar uptake is about 30% and 70%, respectively. On the other hand, our previous study using mouse kidney slices showed that reduction of Gly-Sar uptake by cefadroxil was comparable to that by Gly-Leu. When it is assumed that transport capacity of Pept1 and Pept2 is comparable between rat and mouse, the difference of Pept1 contribution between rat and mouse was consistent with expression level of Pept1 and Pept2.8 Most affinity parameters using several substrates and inhibitors in rat kidney tissue slices were comparable to those in the cells that were transfected with each transporter gene (Table 1). By contrast, estimated Km values of ergothioneine for Octn1 (75.4 mM) and carnitine for Octn2 (530 mM) using rat kidney slices were approximately 20 times higher than those estimated by rat Octn1 (4.64 mM)- and Octn2 (32.7 mM)-expressing cells in the previous report.15,23 Although the reason for such a difference is not clear, Naþ-dependent uptake of ergothioneine and carnitine in kidney slices should reflect Octn1 and Octn2, respectively, because EadieHofstee plot for these substrates suggests that ergothioneine and carnitine transports are mediated via a single saturable component in kidney slices from mice and ergothioneine transport was diminished in Octn1/ knockout miceederived kidney slices.5 Because efflux transporters affect tissue accumulation of drugs, rat mdr1a activity was evaluated using human MDR1 substrate azasetron.24 Accumulation of azasetron in rat kidney slices was significantly increased in the presence of mdr1a inhibitor zosuquidar at 5 and 15 min and tended to increase at 30 and 60 min (Fig. 5). This suggests that kidney slice is used to evaluate apical efflux transporter mdr1a. Because the effect of zosuquidar on accumulation of azasetron was significant at initial phase, the
Figure 5. Time-dependent accumulation of azasetron into rat kidney slices. Accumulation of azasetron (2 mM) was performed in the absence or the presence of zosuquidar (1 mM) at pH 7.4 and 37 C for 5, 15, 30, and 60 min by rat kidney slices. Open and closed circles represent uptake of azasetron in the absence or presence of zosuquidar, respectively. Each point represents the mean ± SD (n ¼ 3). *Indicates a significant difference from the uptake in the absence of zosuquidar at each point (p < 0.05) by Student's t-test.
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inhibitor might affect tissue distribution (e.g., distribution into intracellular compartment and tissue binding) of azasetron. On the other hand, that from Wistar rats was not increased by azasetron. The precise reason is unclear, but strain differences might be existed in mdr1a/1b activity. In addition, we found some difficulties to fully characterize transport activity of P-gp using kidney slices, when digoxin a substrate of mdr1a was used due to fluctuation of measured initial concentration in transport buffer. This may be accounted for nonspecific adsorption of digoxin to use experimental apparatus and cell surface. Transport buffer containing BSA to decrease adsorption of drugs was difficult to be used for experiment due to severe foaming. Moreover, transport buffer containing BSA reduced tissue viability without supply of oxygen (Fig. 2). Further studies need to clarify whether efflux transporters affect accumulation of drugs in kidney slices. The Naþ-dependent or inhibitors-mediated uptake of each compounds considered to be mediated by transporters expressed in renal proximal tubular epithelial cells. On the other hand, the present study could not been molecularly supported, whereas our previous study indicated Naþ-dependent uptake of ergothioneine was mediated by Octn1.5 Although we preliminarily tried to perform in vivo knockdown for octn1 and gapdh genes by hydrodynamic perfusion method using siRNA in mice kidney (data not shown), efficient knockdown for mRNA expression of the genes was not observed. Recently, the knockdown studies using siRNA by a long-term culture method for kidney slices have been reported.25 Although there is a problem of knockdown efficiency, the methodology may enable to show the molecular involvement of various molecules including the tested genes in the present study. It was suggested that several renal transporters in disease model animals were changed compared with normal animals. For example, protein expression of Pept1 was increased in streptozotocininduced diabetes in rats.26 Moreover, protein expression of Pept1, Pept2, mdr1a, and mdr1b was increased in uninephrectomized rats.27 Because rats were used as disease model, the present study could provide important information to evaluate renal function in such disease models. In conclusion, we demonstrate that rat kidney slices are useful tools for evaluating transport activity of apically expressed transporters, for example, reabsorptive transporters Octn1/2, Sglt1/2, Pept1/2, and most likely efflux transporter mdr1a. Because this method has been used to examine basolaterally expressed transporters, kidney slices are useful technique for evaluation of both apical and basolateral transporters as long as tested substrate compounds exhibit different transport characteristics between both membranes. In addition, considering that activities of metabolic enzymes expressed in the kidney should be preserved, this method is considered useful in evaluating apparent disposition of drugs in the kidney. References 1. Nakamura T, Yonezawa A, Hashimoto S, Katsura T, Inui K. Disruption of multidrug and toxin extrusion MATE1 potentiates cisplatin-induced nephrotoxicity. Biochem Pharmacol. 2010;80(11):1762-1767. 2. Bajaj P, Chowdhury SK, Yucha R, Kelly EJ, Xiao G. Emerging kidney models to investigate metabolism, transport, and toxicity of drugs and xenobiotics. Drug Metab Dispos. 2018;46(11):1692-1702. 3. Brown CD, Sayer R, Windass AS, et al. Characterisation of human tubular cell monolayers as a model of proximal tubular xenobiotic handling. Toxicol Appl Pharmacol. 2008;233(3):428-438. 4. Nakanishi T, Fukushi A, Sato M, et al. Functional characterization of apical transporters expressed in rat proximal tubular cells (PTCs) in primary culture. Mol Pharm. 2011;8(6):2142-2150.
5. Arakawa H, Washio I, Matsuoka N, et al. Usefulness of kidney slices for functional analysis of apical reabsorptive transporters. Sci Rep. 2017;7(1): 12814. 6. Schinkel AH, Smit JJ, van Tellingen O, et al. Disruption of the mouse mdr1a Pglycoprotein gene leads to a deficiency in the blood-brain barrier and to increased sensitivity to drugs. Cell. 1994;77(4):491-502. 7. Schinkel AH, Wagenaar E, van Deemter L, Mol CA, Borst P. Absence of the mdr1a P-Glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin A. J Clin Invest. 1995;96(4): 1698-1705. 8. Lu H, Klaassen C. Tissue distribution and thyroid hormone regulation of Pept1 and Pept2 mRNA in rodents. Peptides. 2006;27(4):850-857. 9. Sabolic I, Vrhovac I, Eror DB, et al. Expression of Naþ-D-glucose cotransporter SGLT2 in rodents is kidney-specific and exhibits sex and species differences. Am J Physiol Cell Physiol. 2012;302(8):C1174-C1188. 10. Katoh M, Suzuyama N, Takeuchi T, Yoshitomi S, Asahi S, Yokoi T. Kinetic analyses for species differences in P-glycoprotein-mediated drug transport. J Pharm Sci. 2006;95(12):2673-2683. 11. Takeuchi T, Yoshitomi S, Higuchi T, et al. Establishment and characterization of the transformants stably-expressing MDR1 derived from various animal species in LLC-PK1. Pharm Res. 2006;23(7):1460-1472. 12. Okamoto K, Aoki K. Development of a strain of spontaneously hypertensive rats. Jpn Circ J. 1963;27:282-293. 13. Clark JB, Palmer CJ, Shaw WN. The diabetic Zucker fatty rat. Proc Soc Exp Biol Med. 1983;173(1):68-75. 14. Omote S, Matsuoka N, Arakawa H, Nakanishi T, Tamai I. Effect of tyrosine kinase inhibitors on renal handling of creatinine by MATE1. Sci Rep. 2018;8(1): 9237. 15. Nakamura T, Yoshida K, Yabuuchi H, Maeda T, Tamai I. Functional characterization of ergothioneine transport by rat organic cation/carnitine transporter Octn1 (slc22a4). Biol Pharm Bull. 2008;31(8):1580-1584. 16. Ueda K, Okamura N, Hirai M, et al. Human P-glycoprotein transports cortisol, aldosterone, and dexamethasone, but not progesterone. J Biol Chem. 1992;267(34):24248-24252. 17. Shirasaka Y, Suzuki K, Shichiri M, Nakanishi T, Tamai I. Intestinal absorption of HMG-CoA reductase inhibitor pitavastatin mediated by organic anion transporting polypeptide and P-glycoprotein/multidrug resistance 1. Drug Metab Pharmacokinet. 2011;26(2):171-179. 18. Nezu J, Tamai I, Oku A, et al. Primary systemic carnitine deficiency is caused by mutations in a gene encoding sodium ion-dependent carnitine transporter. Nat Genet. 1999;21(1):91-94. 19. Nakanishi T, Hatanaka T, Huang W, et al. Naþ- and Cl–coupled active transport of carnitine by the amino acid transporter ATB(0,þ) from mouse colon expressed in HRPE cells and Xenopus oocytes. J Physiol. 2001;532(Pt 2):297-304. 20. Enomoto A, Wempe MF, Tsuchida H, et al. Molecular identification of a novel carnitine transporter specific to human testis. Insights into the mechanism of carnitine recognition. J Biol Chem. 2002;277(39):36262-36271. 21. The National Center for Biotechnology Information. Slc6a14 solute carrier family 6 member 14 [Rattus norvegicus (Norway rat)]. Available at: https:// www.ncbi.nlm.nih.gov/gene/298340. Accessed December 20, 2018. 22. Terada T, Saito H, Mukai M, Inui K. Recognition of beta-lactam antibiotics by rat peptide transporters, PEPT1 and PEPT2, in LLC-PK1 cells. Am J Physiol. 1997;273(5 Pt 2):F706-F711. 23. Szabo K, Nagy Z, Juhasz V, et al. Species specificity profiling of rat and human organic cation/carnitine transporter Slc22a5/SLC22A5 (Octn2/OCTN2). Drug Metab Pharmacokinet. 2017;32(3):165-171. 24. Tamai I, Saheki A, Saitoh R, Sai Y, Yamada I, Tsuji A. Nonlinear intestinal absorption of 5-hydroxytryptamine receptor antagonist caused by absorptive and secretory transporters. J Pharmacol Exp Ther. 1997;283(1):108-115. 25. Ruigrok MJR, Maggan N, Willaert D, et al. siRNA-mediated RNA interference in precision-cut tissue slices prepared from mouse lung and kidney. AAPS J. 2017;19(6):1855-1863. 26. Gangopadhyay A, Thamotharan M, Adibi SA. Regulation of oligopeptide transporter (Pept-1) in experimental diabetes. Am J Physiol Gastrointest Liver Physiol. 2002;283(1):G133-G138. 27. Tramonti G, Xie P, Wallner EI, Danesh FR, Kanwar YS. Expression and functional characteristics of tubular transporters: P-glycoprotein, PEPT1, and PEPT2 in renal mass reduction and diabetes. Am J Physiol Renal Physiol. 2006;291(5):F972-F980. 28. Lee WS, Kanai Y, Wells RG, Hediger MA. The high affinity Naþ/glucose cotransporter. Re-evaluation of function and distribution of expression. J Biol Chem. 1994;269(16):12032-12039. 29. Tahara A, Kurosaki E, Yokono M, et al. Pharmacological profile of ipragliflozin (ASP1941), a novel selective SGLT2 inhibitor, in vitro and in vivo. Naunyn Schmiedebergs Arch Pharmacol. 2012;385(4):423-436. 30. You G, Lee WS, Barros EJ, et al. Molecular characteristics of Na(þ)-coupled glucose transporters in adult and embryonic rat kidney. J Biol Chem. 1995;270(49):29365-29371. 31. Terada T, Sawada K, Irie M, Saito H, Hashimoto Y, Inui K. Structural requirements for determining the substrate affinity of peptide transporters PEPT1 and PEPT2. Pflugers Arch. 2000;440(5):679-684.