Evaluation of Platinum Anticancer Drug-Induced Kidney Injury in Primary Culture of Rat Kidney Tissue Slices by Using Gas-Permeable Plates

Abstract

The type of method adopted for the evaluation of drug-induced kidney injury (DIKI) plays an important role during the drug discovery process. In the present study, the usefulness of cultured rat kidney tissue slices maintained on gas-permeable poly(dimethylsiloxane) (PDMS) plates for DIKI was assessed by monitoring the ATP content as a marker of cell viability. The amount of ATP in the kidney slices cultured on the PDMS plates was higher than that in the slices cultured on gas-impermeable polystyrene plates. The protein expression of organic cation transporter-2 (Oct2) was maintained for 3 d. Cisplatin showed a time- and concentration-dependent reduction in ATP in the slices with a half-effective concentration value of 24 µM, which was alleviated by cimetidine, an Oct2 inhibitor, suggesting that cisplatin-induced kidney injury in the cultured slices was regulated by the basolateral uptake transporter Oct2. Furthermore, the intensity of platinum anticancer drug-induced nephrotoxicity in the cultured slices was consistent with that of the in vivo study. In conclusion, the primary culture of rat kidney tissue slices on gas-permeable plates is expected to aid in the prediction of the extent of nephrotoxicity of drugs, even when transporters are responsible for the accumulation of drugs in kidney tissues.

INTRODUCTION

Drug-induced kidney injury (DIKI) accounts for approximately 20% of acute kidney injury (AKI) cases in hospitalized patients and is associated with increased morbidity and mortality.¹⁾ During drug discovery, DIKI is generally evaluated using in vivo animal studies. The in vivo methods help in understanding the overall picture of DIKI by measuring the biomarkers and observing the histological changes; however, these methods have several drawbacks such as low throughput ability, species difference, and animal welfare. In contrast, in vitro cultured cells such as renal proximal tubular epithelial cells (RPTECs) elucidate the mechanism with high-throughput ability. However, extrapolation of the assay’s findings to in vivo DIKI is a challenge.²⁾ Therefore, an in vitro evaluation method for DIKI is required to overcome these problems.

There are several causes for the low predictability of DIKI in the conventional in vitro methods that use renal cell lines,²⁾ among which, the accumulation of drugs in kidney tissues is crucial. In addition to a high blood flow rate of 1.24 L/min/70 kg in humans,³⁾ drug transporters expressed in the plasma membrane of renal cells accelerate drug accumulation in the cells. RPTECs express abundant drug transporters such as basolateral organic anion transporter 1 and 3 (OAT1/3), organic cation transporter-2 (OCT2), and apical multidrug and toxin extrusion 1 and 2K (MATE1/2K), sodium-phosphate co-transporters 1 and 4 (NPT1/4), P-glycoprotein, multidrug resistance-associated proteins 2 and 4 (MRP2/4), and uric acid transporter 1 (URAT1).^4–10) These drug transporters are associated with the onset of DIKI. The accumulation of platinum anticancer drugs, such as cisplatin and oxaliplatin, in RPTECs is controlled by OCT2 and MATE1, which affects the extent of nephrotoxicity induced by these drugs.^11–13) Thus, drug transporters have been implicated in DIKI, although their expression levels were significantly low in kidney-derived cultured cells such as HK-2, HEK293, Madin–Darby canine kidney (MDCK), and primary cultured proximal tubular cells.^14,15) Furthermore, such in vitro renal cell lines consist of a single type of cells, RPTECs, although several types of cells in the kidney are involved in the onset and/or development of DIKI.¹⁶⁾ To overcome such concerns, the primary culture of kidney tissue slices is considered promising for the in vitro evaluation of DIKI.

Kidney tissue slices are widely used as tools for the characterization of renal accumulation of drugs and have been used to evaluate the basolateral uptake transporters such as OAT1, OAT3, and OCT2, as well as apically expressed transporters, including organic cation/carnitine transporter (Octn1/2), sodium-dependent glucose transporters (SGLTs), and peptide transporters in rats and mice.^17–22) Compared to in vitro cultured renal cells, kidney tissue slices have several advantages as follows: 1) the normal levels of transporters and metabolic enzymes associated with drug disposition and reaction are expressed when prepared freshly, 2) the renal tissue architecture is retained with all resident cell types available, 3) the number of tissue slices obtained per animal is 10 times higher than those obtained by in vivo animal studies, and 4) the handling of kidney tissue slices is technically simple. However, the primary culture of kidney slices remains challenging owing to the difficulty in maintaining viability.

Several studies have attempted the primary culture of kidney tissue slices.^23–25) A high concentration of oxygen around the tissue is required to maintain the viability of kidney tissues.²¹⁾ The conventional culture conditions which use a 5% CO₂ incubator and a culture dish made of gas-impermeable polystyrene cannot provide a sufficient amount of oxygen to tissues. Therefore, researchers have attempted to increase oxygen concentration by using an 80% O₂ incubator with a stirring culture medium.^23,24) However, there are several drawbacks such as the necessity of a special incubator, poor throughput ability, and low safety due to the ignition of oxygen. To solve such problems, we focused on gas-permeable culture equipment made of poly(dimethylsiloxane) (PDMS),²⁶⁾ which is generally used to make culture equipment prototypes. In the present study, we devised a method for the primary culture of rat kidney tissue slices using culture plates made of PDMS and investigated the utility of this method for the evaluation of DIKI, focusing on platinum drugs, which cause a high clinical incidence of DIKI and require renal transporters for regulating the accumulation of these drugs in the kidney tissue.²⁷⁾

MATERIALS AND METHODS

Materials

Cisplatin, cimetidine, and oxaliplatin were purchased from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Carboplatin, diethyldithiocarbamate, and 8-cyclopentyl-1,3-dipropylxanthine were obtained from Tokyo Chemical Industry (Tokyo, Japan), Sigma-Aldrich (St. Louis, MO, U.S.A.), and Abcam (Cambridge, U.K.), respectively. Hybrid VECELL 24-well H-plates were purchased from Vessel Inc. (Fukuoka, Japan). Rabbit polyclonal anti-Oct2 antibody and rabbit monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody were from Alpha Diagnostic International (San Antonio, TX, U.S.A.) and Cell Signaling (Danvers, MA, U.S.A.), respectively. All other chemicals and reagents were commercial products of reagent grade.

Animals

Male Wistar rats (7–8 weeks old) were purchased from Sankyo Labo Service Corporation, Inc. (Hamamatsu, Japan). The rats were housed two per cage with free access to commercial chow and tap water, and were maintained on a 12-h dark/light cycle in an air-controlled room (temperature, 24.0 ± 1 °C; humidity, 55 ± 5%). All the animal studies were approved by the Kanazawa University Institutional Animal Care and Use Committee (AP-183955) and were performed in accordance with the University’s guidelines.

Incubation of Rat Kidney Tissue Slices

The rat kidney tissue slices were prepared using a microslicer (Zero 1N; Dosaka EM, Kyoto, Japan) as described previously.²¹⁾ The prepared tissue slices (0.3 mm thickness) were transferred onto a Hybrid VECELL 24-well H-plate filled with 1.0 mL of Williams’ Medium E (Thermo Fisher Scientific, Waltham, MA, U.S.A.), supplemented with 5% (v/v) fetal bovine serum (FBS) (Thermo Fisher Scientific), 100 units/mL penicillin, 100 µg/mL streptomycin, 0.1 µM dexamethasone, 1% ITS Liquid Media Supplement (Sigma-Aldrich), and 2 mM alanine (Ala)-glutamine (Gln) (Nacalai Tesque, Kyoto, Japan) in the presence or absence of the drugs to be tested. The slices were incubated for up to 4 d at 37 °C in an atmosphere of 5% CO₂. The medium was refreshed every day. At specific time points during the experiments, the slices were blotted on filter paper, weighed, frozen in liquid nitrogen, and stored at −80 °C until the assay of drugs and ATP. The amount of ATP in the rat kidney tissue slices was measured using “Tissue” ATP assay kit (TOYO B-Net Co., LTD., Tokyo, Japan) as described previously.²¹⁾ The relative ATP content was calculated according to the intensity of luminescence versus the ATP standard curve. The accumulation of drugs in the slices was determined using liquid chromatography with tandem mass spectrometry (LC-MS/MS).

Western Blot Analysis

The collected slices were homogenized in radioimmunoprecipitation assay buffer supplemented with a protease inhibitor cocktail (Merck Millipore, Burlington, MA, U.S.A.). The samples were centrifuged at 21600 × g for 10 min at 4 °C. The supernatant was collected, and the protein content was measured using the Protein Assay BCA kit (FUJIFILM Wako Pure Chemical Corporation). Twenty micrograms of protein were separated using 10% sodium dodecyl sulfate-polyacrylamide and transferred to a 0.45 µm polyvinylidene difluoride membrane (Merck Millipore). The membranes were blocked with 2% skimmed milk in phosphate-buffered saline-Tween 20 at room temperature for 1 h. The blots were probed with anti-Oct2 antibodies or anti-GAPDH antibody at 1 : 2000 dilution followed by appropriate secondary antibodies conjugated to horseradish peroxidase. The target protein was chemically luminescent after reaction with Immunostar Zeta (FUJIFILM Wako Pure Chemical Corporation) for GAPDH and Immunostar LD (FUJIFILM Wako Pure Chemical Corporation) for Oct2 using a Lumino image analyzer (LAS-4000; FUJIFILM, Tokyo, Japan). Densitometric analysis was performed using ImageJ software (National Institutes of Health, Bethesda, MD, U.S.A.).

Drug Uptake Studies Using Freshly Prepared Rat Kidney Tissue Slices

The uptake study was conducted according to a previous study with minor modifications.^21,22) Briefly, the freshly prepared rat kidney tissue slices were incubated in oxygenated transport buffer (130 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl₂, 1.2 mM MgSO₄, 1.2 mM KH₂PO_4, 11 mM glucose, and 25 mM N-(2-hydroxyethyl)piperazine-N′-2-ethanesulfonic acid (HEPES), adjusted to pH 7.4) containing cisplatin in the absence or presence of transporter inhibitors. The uptake reaction was carried out at 37 °C for the designated time period, and then, the reaction was terminated by removing the slices from the medium and washed twice in an ice-cold medium to remove the drugs that had adhered to cell surfaces. The washed slices were blotted on filter paper, weighed, frozen in liquid nitrogen, and stored at −80 °C until measurement of cisplatin in tissues.

LC-MS/MS Analysis

Cisplatin derivatization was conducted according to a previously reported method.²⁸⁾ Briefly, the tissues were homogenized in the transporter buffer using a bullet blender and centrifuged at 1000 × g for 10 min at 4 °C. Then, 5 µL of the resultant supernatant was mixed with 1% v/v diethyldithiocarbamate in 0.1 M NaOH solution. This mixture was incubated at 40 °C for 30 min, and 10 µM 8-cyclopentyl-1,3-dipropylxanthine in acetonitrile was added as an internal standard for LC-MS/MS analysis at the end of the incubation. The samples were mixed well and centrifuged at 21600 × g for 10 min at 4 °C. The resultant supernatant was collected and dried by evaporation under vacuum. The samples were reconstituted with a mixture of 0.1% formic acid in water and 0.1% formic acid in acetonitrile (80 : 20) and subjected to LC-MS/MS analysis. The derivatized cisplatin was measured using a triple quadrupole mass spectrometer LCMS8050 (Shimadzu, Kyoto, Japan) coupled with an LC30A system (Shimadzu). The analytical column was an ACQUITY UPLC BEH C18 column (130 Å, 1.7 µm, ID 2.1 ×50 mm; Waters Corporation, Waltham, MA, U.S.A.) maintained at 40 °C. The mobile phase was composed of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B) at a flow rate of 0.3 mL/min, and the injection volume was 10 µL. The mobile phase gradient elusion started at 5% solvent B for 0.5 min, followed by a linear gradient to 75% solvent B for 1 min, a linear gradient to 90% solvent B in 1.5 min, a linear gradient to 95% solvent B for 2.5 min, maintained at 95% solvent for 1.5 min, and then returned to the initial condition for 1 min. Electrospray positive ionization was used, and the mass transitions were monitored at m/z 639 > 491 for derivatized cisplatin and m/z 305 > 178 for 8-cyclopentyl-1,3-dipropylxanthine. LabSolutions software version 5.79 (Shimadzu) was used for data manipulation. The detection limit of each compound was 1 nM.

Data Analysis

The half-effective concentration (EC₅₀) values of ATP reduction were obtained based on the ratio of the ATP amount in the slices exposed to cisplatin and oxaliplatin to that in the non-exposed group, according to the following equation (1):

  

(1)

where, % of control represents the reduction rate in the presence of the tested drug at concentration (S).

All data are presented as mean ± standard error of the mean (S.E.M.). Statistical significance was evaluated using Student’s t-test or ANOVA followed by Tukey–Kramer test with a p-value <0.05.

RESULTS

Viability of Rat Kidney Tissue Slices Cultured on Gas-Permeable Plates

Rat kidney tissue slices were cultured in gas-permeable PDMS or gas-impermeable polystyrene plates, and the ATP content was measured as a marker of tissue viability over 3 d. When cultured on PDMS plates, the ATP amount in the slices was decreased to 48.3 ± 7.1% of the initial amount (day 0) on day 1 and was maintained at a similar level up to day 3 (Fig. 1A). The ATP content on the PDMS plates on days 1 and 2 (31.8 ± 1.7% of initial) was significantly higher than that on conventional polystyrene plates, 23.3 ± 0.5 and 12.7 ± 1.4% of the initial amount on days 1 and 2, respectively (Fig. 1A). Cultivation of tissue slices at acidic pH is useful for the evaluation of DIKI for several drugs and metabolites such as acylglucuronide, which are unstable at neutral pH.²⁹⁾ Therefore, the effect of pH on the viability of kidney tissue slices was evaluated. As shown in Fig. 1B, the ATP amount in the slices cultured at pH 6.0 was significantly lower than that at pH 7.4. Although the content of ATP in the tissue was maintained for 4 d (Fig. 1B), apparent tissue damage was observed on day 4. Therefore, the following studies were performed for up to 3 d.

Fig. 1. Viability of Rat Kidney Slices by Primary Culture Using Gas-Permeable Plates

(A) Rat kidney tissue slices were incubated for 1, 2, and 3 d in gas-permeable PDMS (closed circle) plates and gas-impermeable polystyrene plates (open circle). Viability of kidney slices was represented by the ATP amount. *indicates a significant difference from the polystyrene plate (p < 0.05) based on Student’s t-test. (B) The slices were incubated for 1, 2, 3, and 4 d at pH 7.4 (closed circle) and pH 6.0 (open circle) in PDMS plates. Each symbol represents the mean ± S.E.M. (n = 3) value, and if not shown, are smaller than the symbol. *indicates a significant difference from pH 6.0 (p < 0.05) based on Student’s t-test.

Expression Level of Oct2 in Primary Cultured Rat Kidney Tissue Slices

The protein expression level of Oct2 in rat kidney tissue slices was measured on days 0, 1, 2, and 3 of the culture. It was found to increase transiently on day 1 compared to day 0, and the levels on days 2 and 3 were comparable to those of day 0 (Fig. 2). Accordingly, Oct2 was maintained for 3 d under the culture conditions used.

Fig. 2. Protein Expression Level of Oct2 in Primary Cultured Rat Kidney Tissue Slices

The protein expression level of Oct2 in the cultured rat kidney tissue slices was measured on days 0, 1, 2, and 3. The upper panel shows a representative blot. Band density was normalized to the density of GAPDH. Protein expression was observed to be relative to that on day 0. Each circle represents the mean ± S.E.M. (n = 3) value.

Evaluation of Platinum-Induced Nephrotoxicity

The effect of platinum drugs on the viability of the cultured slices was evaluated for understanding the usefulness of the primary culture of rat kidney tissue slices for DIKI analysis. Cisplatin caused a time-dependent decrease in ATP levels (Fig. 3A), and the effect was enhanced by increasing the cisplatin concentrations from 10 to 100 μM (Fig. 3A). As a concentration-dependent decrease in ATP was observed, the following experiments were conducted on day 2. The EC₅₀ value of cisplatin for the decrease in ATP amount in the cultured slices on day 2 was 23.9 ± 3.8 µM (Fig. 3B). Carboplatin and oxaliplatin also caused concentration-dependent reduction with the EC₅₀ values being 267 ± 47 and 78.8 ± 13.8 µM on day 2, respectively (Figs. 3C, D). The EC₅₀ values and clinical unbound maximum plasma concentrations (C_max,u) of platinum drugs are summarized in Table 1. The EC₅₀ value of cisplatin for ATP reduction in the slices was one-fifth of C_max,u at the clinical dose. On the contrary, EC₅₀ values of carboplatin and oxaliplatin were less than one-tenth of the clinically achievable C_max,u.

Fig. 3. Effect of Platinum Anticancer Drugs on the Viability of Primary Cultured Rat Kidney Tissue Slices

(A) The cultured kidney tissue slices were exposed to cisplatin for 1, 2, and 3 d at a dose of 0 (open circle), 10 (open triangle), 30 (closed circles), and 100 µM (closed triangles). (B) Concentration-dependent reduction in the ATP content by cisplatin in the cultured slices on day 2 was re-plotted. The experimental data were also used in Fig. 3A. The slices were exposed to oxaliplatin (C) and carboplatin (D) at 0, 10, 30, 100, 300, and 1000 µM for 2 d. The ATP amount in the slices was measured to determine their viability. Each value represents the mean ± S.E.M. (n = 3), and if not shown, are smaller than the symbol.

Table 1. Renal Toxicity of Platinum Anticancer Drugs in Primary Cultured Rat Kidney Slices

Drug	Dose (mg/m2)	Cmax,u (µM)	EC50 (µM)	Cmax,u/EC50
Carboplatin	125	12a)	267 ± 47	0.045
Cisplatin	30	4.5b)	23.9 ± 3.8	0.19
Oxaliplatin	130	4.1c)	78.8 ± 13.8	0.052

a) C_max,u was calculated from 4.48 µg/mL at a dose of 125 mg/m².⁴⁰⁾ b) C_max,u was obtained from Souid et al.⁴¹⁾ c) Calculated from 1.612 µg/mL at a dose of 130 mg/m².⁴²⁾

Figure 4A shows the effect of cimetidine, a non-specific OCT inhibitor, on the cisplatin-induced decrease in ATP content on day 2. The decrease in ATP due to cisplatin exposure was alleviated by cimetidine by increasing the EC₅₀ value from 49.3 ± 10.1 to 122 ± 33 μM (Fig. 4A). The accumulation of cisplatin in the slices tended to be decreased by cimetidine to 78.3 ± 9.5% of control, while the decrease was not statistically significant (Fig. 4B). The representative remaining concentration of cisplatin at 30 μM after 24-h-exposure was 45.4 ± 10.4 and 40.1 ± 6.4% of initial concentration in the absence and presence of cimetidine at 1 mM. OCT inhibitors such as procainamide, N-methylnicotinamide (NMN), imipramine, and tetra-butylammonium (TBA) also decreased the accumulation of cisplatin although the effect was not statistically significant (Fig. 4C).

Fig. 4. Effect of Cimetidine on Cisplatin-Induced Reduction of Tissue ATP Content and Cisplatin Accumulation in Rat Kidney Tissue Slices

(A) The slices were exposed to cisplatin (0, 10, 30, 100, and 300 µM) in the absence (open circles) or presence (closed circles) of cimetidine (1 mM) for 2 d. The ATP content in the slices was measured for viability of the slices. Each value represents the mean ± S.E.M. (n = 3 or 4), and if not shown, are smaller than the symbol. (B) Accumulation of cisplatin in the primary cultured rat kidney tissue slices for 2 d was measured in the absence or presence of cimetidine (1 mM). Bar indicates mean ± S.E.M. (n = 4). (C) Accumulation of cisplatin in the freshly prepared rat kidney tissue slices was measured in the absence (open bar) or presence (closed bar) of OCT inhibitors (1 mM procainamide, 2 mM N-methylnicotinamide (NMN), 50 µM imipramine, and 100 µM tetra-butylammonium (TBA)) for 60 min. Each value represents mean ± S.E.M. (n = 3), and if not shown, are smaller than the symbol.

DISCUSSION

Primary culture of rat kidney tissue slices is a promising in vitro tool for DIKI evaluation. However, it is technically difficult to maintain tissue viability using previously reported methods for culturing kidney slices.^23–25) Therefore, in the present study, we introduced gas-permeable PDMS plates that facilitate oxygen supply to the tissues, thereby maintaining tissue viability for a longer time, to evaluate the usefulness of primary culture of the tissue slices as an in vitro tool for DIKI evaluation. First, the viability of the cultured slices on PDMS plates was higher than that on polystyrene plates and was maintained over 3 d (Fig. 1A). This method was further examined to assess the nephrotoxicity of platinum anticancer drugs. Before conducting the toxicity studies, the expression level of Oct2 transporter protein was examined, as it has been demonstrated that OCT2 in RPTECs is a prerequisite for cisplatin-induced nephrotoxicity as it regulates intracellular cisplatin accumulation in humans as well as rodents.^11,12) The protein expression of Oct2 was maintained for 3 d (Fig. 2) in the rat kidney tissue slices culture on gas-permeable plates, suggesting that the cultured slices are more useful than in vitro cell lines such as HK-2 cells, which exhibit lower expression of drug transporters.¹⁴⁾ The mechanism by which protein expression of Oct2 was maintained is unclear, but it might be due to the oxygen supply by gas-permeable plates, since the expression of metabolic enzymes in primary cultured rat hepatocytes was increased by oxygen supply using PDMS plates compared with the gas-impermeable cultures.³⁰⁾

Cisplatin-induced kidney injury was evaluated in cultured slices in this study. The concentration-dependent reduction in tissue ATP levels by cisplatin exposure was observed on day 2 (Fig. 3A), while almost no reduction was observed on day 1. This finding suggests that a long-term culture of slices is needed for the evaluation of DIKI. Moreover, the lower values of EC₅₀/C_max,u ratios of carboplatin and oxaliplatin than that of cisplatin (Table 1) were consistent with those of an earlier study which reported that they were less toxic than cisplatin in rats.³¹⁾ Furthermore, the decreased ATP level after cisplatin exposure was clearly alleviated by co-treatment with the OCT inhibitor cimetidine in the cultured slices (Fig. 3A) in accordance with the significant role of Oct2 in cisplatin-induced renal injury in humans and mice.^11,12) The accumulation of cisplatin tended to be decreased by co-treatment with cimetidine, as well as OCT inhibitors such as procainamide, NMN, imipramine, and TBA (Figs. 4B, C) although the effect was not statistically significant. The reason for the non-significant reduction in cisplatin uptake was uncertain, as the concentrations used for each compound were higher than the reported IC₅₀ values for reducing rat Oct2 activity.^20,32) Moreover, as drug medium was replaced every 24 h, effect of drug concentration changes by 40–50% in culture medium on the cisplatin accumulation considered to be limited. One possible reason could be that the accumulation of cisplatin was not limited to RPTECs, as kidney slices include all types of renal cells, whereas Oct2-mediated accumulation is limited to RPTECs.³³⁾ On the other hand, structural and functional damage to mitochondria suggested the nephrotoxic mechanism of cisplatin.³⁴⁾ Since MATE1 is localized not only at the cell membrane but also at mitochondria,³⁵⁾ there is a possibility that mitochondrial MATE1 mediates the uptake of cisplatin into mitochondria. Therefore, cimetidine might inhibit the MATE1-mediated accumulation of cisplatin into mitochondria leading to attenuation of the ATP reduction.

In addition to OCT2, MATE1 plays an important role in cisplatin accumulation in cultured slices, because it was reported that Mate1-deficient mice exhibited increased cisplatin-induced nephrotoxicity.¹³⁾ Our previous studies showed that renal transporters at apical membranes of RPTECs such as SLGT1/2 can be evaluated by the kidney slices,^21,22) suggesting that such transporters could affect the DIKI in the cultured slices. On the other hand, detection of MATE1-mediated transport by kidney slices considered to be difficult, because it was reported that uptake of metformin, a dual substrate of OCT2 and MATE1, by mouse kidney slices was not affected by pyrimethamine at 1 µM, which inhibits selectively mouse Mate1 but not Oct2.³⁶⁾ Therefore, further studies are required to evaluate the involvement of MATE1 in observed toxicity in the cultured kidney slices.

Several drug transporters other than OCT2 and MATE1 are expected to play a significant role in the onset of DIKI, for example, aristolochic acid-induced kidney injury is mediated by OAT1 and OAT3.³⁷⁾ Moreover, gentamicin-induced kidney injury is mediated by renal reabsorption via megalin-mediated endocytosis.^38,39) Although the present study investigated only Oct2, a measurement of protein expression of other transporters such as Oat3 and megalin will be useful for an in vitro evaluation of DIKI caused by other drugs and chemicals. Furthermore, it has been reported that various types of cells other than RPTECs in the kidney are associated with DIKI.¹⁶⁾ Therefore, the usefulness of kidney slices is not limited to RPTECs, and other types of cell-specific toxicity can be evaluated by measuring the respective cell-specific markers.²⁾ Further studies are required to clarify the usefulness of the primary culture of kidney slices on gas-permeable plates.

CONCLUSION

The primary culture of rat kidney slices can be useful in the prediction of nephrotoxicity of drugs, even when transporters are responsible for the accumulation of drugs in kidney tissues.

Acknowledgments

This research was supported by Japan Agency for Medical Research and Development (AMED) (Grant No. 20mk0101182h0001), Kanazawa University SAKIGAKE Project 2020, Grant-in-Aid for Transformative Research Areas (B) (Grant No. 20H05745), and Grant-in-Aid for Scientific Research (B) (Grant No. 21H02641).

Conflict of Interest

The authors declare no conflict of interest.

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