Role of OCT2 and MATE1 in renal disposition and toxicity of nitidine chloride

Abstract

Background and Purpose

Nitidine chloride (NC), a benzophenanthridine alkaloid, has various biological properties including anticancer and analgesic activities. The aim of the present study was to evaluate the role of organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 (MATE1) in the renal disposition and nephrotoxicity of NC.

Experimental Approach

MDCK cells stably expressing human OCT2 and/or hMATE1 were used to investigate the OCT2‐ and MATE1‐mediated transport of NC. In addition, the accumulation of NC and its potential toxicity were studied in rat primary‐cultured proximal tubular (rPCPT) cells and in rats in vivo.

Key Results

NC was found to be a high‐affinity substrate of both OCT2 and MATE1 with high cytotoxicity in MDCK‐hOCT2/hMATE1 and MDCK‐hOCT2 compared to mock cells. The OCT2 inhibitors, cimetidine and (+)‐tetrahydropalmatine ((+)‐THP), significantly reduced NC accumulation and cytotoxicity in MDCK‐hOCT2, MDCK‐hOCT2/hMATE1 and rPCPT cells. Severe kidney damage with high levels of blood urea nitrogen and lactate dehydrogenase (LDH), reduced levels of alkaline phosphatase (ALP) and pathological changes were found in rats after 20 days of successive i.v. doses of NC (5 mg·kg⁻¹·day⁻¹). Concomitantly, the concentration of NC in the kidney reached similar high levels at 2 h after the last dose of the 20 day treatment as those observed at 0.5 h after a single i.v. dose of 5 mg·kg⁻¹.

Conclusions and Implications

Our data indicate that NC‐induced nephrotoxicity might be mainly attributed to OCT2‐mediated extensive renal uptake and weak tubular secretion by MATE1.

Abbreviations

ASP⁺

4‐(4‐(dimethylamino)styryl)‐N‐methylpyridinium iodide

ESI

electrospray ionization

HPC

hydroxypropyl cellulose

MATE1

multidrug and toxin extrusion 1

MPP⁺

1‐methyl‐4‐phenylpyridinium iodide

MTT

3‐[4,5‐dimethylthiazol‐2‐yl]‐2, 5‐diphenyltetrazolium bromide

NC

nitidine chloride

OCT

organic cation transporter

(+)‐THP

(+)‐tetrahydropalmatine

Tables of Links

TARGETS
Enzymes a	Transporters b
CYP3A4	MATE1
CYP2D6	OCT1
	OCT2
	OCT3

LIGANDS
Cimetidine
MPP+

These Tables list key protein targets and ligands in this article which are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Southan et al., 2016) and are permanently archived in the Concise Guide to PHARMACOLOGY 2015/16 (^{a, b}Alexander et al., 2015a, 2015b).

Introduction

Nitidine chloride (NC, Figure 1), a benzo[c]phenanthridine alkaloid, was isolated from the traditional Chinese medicinal plant, Zanthoxylum nitidum (Roxb.) DC, and is found in several traditional remedies from diverse endemic areas. NC is commonly used as the major component of a traditional Kenyan antimalarial remedy (Gakunju et al., 1995). Liangmianzhen sustained–released tablet (Zhao et al., 2009), a traditional Chinese medicine containing NC, has been utilized as an analgesic in China for many years. Several studies have reported that NC displays various biological effects, including anti‐inflammatory, antifungal, antioxidant and anti‐HIV activities (Del Poeta et al., 1999; Hu et al., 2006). Recently, NC was proposed to be a potential anti‐tumour drug, as it inhibits the proliferation of hepatocellular carcinoma, renal cancer, human lung adenocarcinoma and the metastasis of breast cancer in vitro and in vivo (Liu et al., 2009; Iwasaki et al., 2010; Pan et al., 2011; Liao et al., 2013; Fang et al., 2014). These findings indicate that NC has potential as a therapeutic compound and should be subjected to further investigations.

Figure 1.

Chemical structure of nitidine chloride (NC).

It is well known that safety and efficacy are equally important in the development of drug candidates (Kola and Landis, 2004). Drug‐induced liver and kidney injury would probably preclude the use of a compound clinically. Cell membrane transporters and drug metabolic enzymes play crucial roles in the disposition of xenobiotics in vivo (Rappold et al., 2011; Yonezawa and Inui, 2011; Liu et al., 2015); thus, they might also contribute to drug‐induced toxicity. Our previous study demonstrated that NC is a high affinity substrate of human organic cation transporters 1 and 3 (hOCT1 and hOCT3), which mediated the hepatocellular uptake of NC and subsequently caused hepatotoxicity (Li et al., 2014). Additionally, we also found that NC was a substrate of CYPs, such as CYP3A4, which mediated NC metabolism and attenuated the toxicity of NC in hepatocytes. Because substrates of OCT1, OCT2 and OCT3 partially overlapped, we speculated that NC was also a substrate of OCT2. Considering the low level of CYPs and high level of OCT2 in kidney, we assumed that the kidney is probably a target for NC‐associated toxicity.

In addition, human multidrug and toxin extrusion 1 (hMATE1), an H⁺‐coupled organic cation exporter, is also expressed in the brush border membrane of proximal tubule cells (Omote et al., 2006; Kusuhara and Sugiyama, 2009). Because NC was also identified as a substrate of hMATE1 in our previous study (Li et al., 2014), MATE1 might mediate the efflux of NC from renal tubular epithelial cells and alleviate NC‐induced nephrotoxicity.

With this in mind, one aim of the present study was to investigate whether OCT2 and/or MATE1 mediate the transport of NC and subsequently cause cytotoxicity. To do this we used MDCK cells stably transfected with human OCT2 (MDCK‐hOCT2), hOCT2/pcDNA3.1 (MDCK‐hOCT2/pcDNA3.1) and co‐transfected with hOCT2 and hMATE1 (MDCK‐hOCT2/hMATE1). The other aim was to evaluate the potential role of OCT2 in NC‐induced renal injury by studying the uptake and toxicity of NC in rat primary‐cultured proximal tubular (rPCPT) cells, as well as its accumulation in the kidney and toxicity in rats after successive administration of NC. The results will give us more information about the possible mechanisms and risk factors associated with the nephrotoxicity of NC, which is vital for the future development and application of NC.

Methods

Animal welfare and ethical statements

Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny et al., 2010; McGrath and Lilley, 2015). The procedures were approved by Institutional Animal Care and Use Committee of Zhejiang University Medical Center (2015‐380‐01) and complied with the standards of animal welfare in China. All experimental procedures were conducted as humanely as possible. A total of 55 healthy male Sprague Dawley rats, aged 4–6 weeks (130–160 g), were obtained from the Experimental Animal Centre of the Zhejiang Academy of Medical Sciences [SCXK (Zhe) 2015‐0001] and were housed in cages at controlled temperature (22.0 ± 0.1°C) and humidity (50 ± 10) % with a 12 h light/dark cycle and free access to food and water. The animals were acclimatized to the laboratory environment for 1 week prior to the experiment, and each animal was examined for any gross signs of disease or injury.

Cell culture and rat proximal tubular cells isolation

MDCK cells stably transfected with plasmid vector pcDNA3.1(+) containing human OCT2 cDNA (MDCK‐hOCT2) and blank vector (mock cells) were established previously in our laboratory (Wang et al., 2014; Li et al., 2015). The co‐transfected MDCK‐hOCT2/hMATE1 and MDCK‐hOCT2/pcDNA3.1 cells were obtained by transfecting the single‐transfected MDCK‐hOCT2 cells with the plasmid pcDNA3.1 (+)‐Hygro vector containing human MATE1 cDNA and blank vector (Lei et al., 2015). The activity of hMATE1 in the co‐transfected cells was validated by the cellular uptake and transcellular transport of MPP⁺ and metformin, probe substrates for both OCT2 and MATE1 (Konig et al., 2011). Cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin in a humidified air/CO₂ incubator (5% v·v^‐1) at 37°C.

Rat primary proximal tubule cells were isolated from male rats by the previous method with minor modifications (Liu et al., 2010). Briefly, rats were anesthetized with diethyl ether, and kidneys were perfused with Ca^{2 +} free Hank's buffer (pH 7.4) containing 25 mM HEPES (HBSS) to remove blood, and then kidneys were removed and minced in a sterile cell culture dish on ice. The minced tissue was washed repeatedly in HBSS solution containing 1% penicillin/streptomycin and digested in HBSS solution containing 1 mg·mL⁻¹ collagenase IV and 1 mg·mL⁻¹ trypsin by shaking for 40 min at 37°C. The renal proximal tubular fragments were then isolated mechanically by sequential filtration through a cell strainer of 80 μm (BD Falcon, Bedford, MA, USA). The isolated fragments were resuspended in culture medium DMEM/F12 supplemented with 10% FBS, 1% penicillin/streptomycin and 1% insulin‐transferrin‐selenium and then seeded into collagen I coated (50 μg·mL⁻¹) 96‐ or 12‐well culture plates (Costar Corning Inc., Corning, NY, USA) and incubated in a humidified air/CO₂ incubator (5% v·v^‐1) at 37°C. Fragments of tubules adhered to the culture dish, in which the medium was changed every 2 days, and cells were observed to form confluent monolayers over a 4–5 day period.

To confirm that rat (r) PCPT cells are a suitable model for studying kidney transporters, we quantified the mRNA expression of target genes, Slc22a1, Slc22a2 and Slc47a1 (encoding Oct1, Oct2 and Mate1, respectively) in rPCPT cells on day 5 by the real‐time quantitative PCR using SYBR Green with designed primers. Data showed that the mRNA level of Slc22a2 in rPCPT cells on day 5 was approximately twofold that of Slc47a1 with the 2^−ΔΔCT values (normalized to rGAPDH) of 0.0025 and 0.0014, respectively, but Slc22a1 mRNA was undetected, indicating that this cell model could be applied to explore the role of OCT2 or MATE1 in the renal disposition of drugs/compounds.

Cellular uptake experiment

The cellular accumulation of MPP⁺ and NC was performed using the method developed in our previous study (Wang et al., 2014). Briefly, the cells were pre‐incubated with HBSS (37°C, 10 min), and then 200 μL of HBSS containing MPP⁺ or NC in the absence or presence of inhibitors was added to initiate the uptake. The incubation was performed at 37°C; the uptake was terminated by removing the incubation buffer and adding ice‐cold PBS quickly at the designated time. Then the cells were washed three times with ice‐cold PBS and lysed with 100 μL of 0.1% sodium dodecyl sulfate (SDS). The intracellular accumulation of NC was obtained from MDCK‐hOCT2, MDCK‐hOCT2/hMATE1 or mock cells. All experiments were performed in triplicate in a typical experiment and at least two separate experiments were performed. The uptake of MPP⁺ and NC in the cells was quantified with LC–MS/MS and normalized to the total protein content in the lysates with a BCA protein assay kit. The hOCT2‐ or hOCT2/hMATE1‐mediated net uptake was obtained by subtracting the intracellular accumulation in mock cells from that in MDCK‐hOCT2 or MDCK‐hOCT2/hMATE1 cells respectively. The uptake in the presence of inhibitors was expressed as a percentage of the vehicle group (% of control).

Transcellular transport experiment

Transcellular transport studies were conducted as previously described (Muller et al., 2013). MDCK‐hOCT2/hMATE1 and MDCK‐hOCT2/pcDNA3.1 cells (passage: 13–17) were seeded on 12‐well transwell inserts (0.4 μm pore size; 12 mm insert; Corning Costar, Corning, NY, USA) at a density of 3 × 10⁵ cells per insert and grown for 3–5 days to form cell monolayers. Cells were washed once and equilibrated at 37°C for 30 min with incubation medium: HBSS containing HEPES (25 mM, pH 7.4). Trans‐epithelial electrical resistance (TEER) measurements were used to evaluate the integrity of the cell layer (Millicell‐ERS equipment; Millipore, MA, USA), and TEER ≥250 Ω·cm² was used as an acceptance criteria for further experiments. Then, 1.5 mL and 0.5 mL incubation medium were added to the basolateral and apical chamber, respectively, with or without NC (2.5 μM) at 37°C. Because the luminal pH is 7.4 at the initial proximal tubule, and becomes much lower at the distal portion (Vallon, 2009), the pH of the incubation medium in the basal chamber was 7.4, while in the apical chamber it was 6.0 to simulate the conditions of urine side and to maintain the higher efflux mediated by MATE1 (Konig et al., 2011; Muller et al., 2011). For the inhibition assay, (+)‐THP (50 μM) was co‐incubated with NC (2.5 μM) in the apical or basolateral side. A 100 μL aliquot was taken from the receiver chamber at 20, 40, 60, 80, 100 and 120 min for determination of NC, and the equivalent volume of fresh buffer was added. At the end of the incubation, the medium was removed, the inserts were rinsed with ice‐cold incubation medium, three times, and the cells were lysed with 0.1% SDS.

Cytotoxicity assay

Cells were incubated with medium containing NC or 0.2% DMSO with or without OCT2 inhibitors at 37°C for 24 h. At the end of the incubation, the medium was collected for measurement of LDH activity using an LDH assay kit (Jiancheng Co., Ltd., Nanjing, China). Then 20 μL of MTT reagent was added to each well, and the mixture was incubated for another 4 h. Subsequently, the incubation medium was removed and 150 μL of DMSO was added to dissolve the formazan. The absorbance of each well was measured at 570 nm with 630 nm as a reference by a microplate reader (Spectra Max M2; Molecular Devices, Sunnyvale, CA, USA), and the cell viability was expressed as a percentage of the vehicle group (% control).

Toxicity study in rats

After a 7 day period of quarantine and acclimatization, sex‐matched healthy rats were randomly allocated into the designated groups. The animals were fasted overnight before the experiment was conducted. Rats received 10% HPC containing 0.5% DMSO solution (0.2 mL·100 g^‐1) for the vehicle group (n = 6) or 5 mg·kg⁻¹ NC for single‐dose (n = 6) or repeated‐dose (20 consecutive days, n = 12) treatment by tail i.v. injection. The following items were examined by a person blind to the treatment allocation of each rat during the experimental period: macroscopic observations, body weights, serum biochemistry, necropsy findings, organ weights and histopathology.

Macroscopic observations and body weights

Macroscopic signs and mortality were observed continuously for the first 1 h after administration of the drugs. Each animal was checked daily for general condition throughout the 20 day experimental period. Abnormal type and severity of signs, as well as the observation day and time, were recorded. Individual body weight was recorded on each day before treatment.

Serum biochemistry

Before (day 0) and at treatment days 5 and 20, blood samples were collected from the orbital venous plexus of each rat into sterile tubes without anticoagulant and centrifuged at 3500 g for 10 min to obtain serum for biochemical tests. The serum biochemistry indexes, including blood urea nitrogen (BUN), alkaline phosphatase (ALP), LDH, creatinine (CRE), uric acid (URA), aspartate aminotransferase (AST) and alanine aminotransferase (ALT), were measured with an electrolyte autoanalyser (model 7070; Hitachi Ltd., Tokyo, Japan).

Necropsy and organ weights

All surviving animals were killed with diethyl ether inhalation and exsanguination at the end of the treatment period. Macroscopic observations were conducted at autopsy, then kidneys (right and left) and liver were removed and weighed (Mettler‐Toledo XS4002S; Mettler Toledo, Switzerland); the paired organs were weighed separately. Relative organ weights were calculated based on the body weights of the fasted animals (ratio of organ weights/body weights).

Histopathological study

After the macroscopic study, the kidney and liver of the vehicle and treatment group were fixed in neutral 10% buffered formalin, and slides were prepared for histopathological examination. Histopathological examination was conducted through routine paraffin embedding. Tissue samples were sectioned, stained with haematoxylin and eosin and then examined microscopically. Microscopic examinations were performed in the Experimental Animal Centre of the Zhejiang University using the Pristima™ and Path/Tox System (version 6.3.0; Xybion Medical Systems Co., Cedar Knolls, NJ, USA).

Tissue distribution studies in rats

Blood and tissues (liver and kidney) were collected at 0.25, 0.5 and 2 h after a single i.v. dose of 5 mg·kg⁻¹ NC or at 2 h after repeated doses for 20 days. Tissue samples were rinsed with normal saline solution to remove the blood, blotted with the filter paper, weighed accurately, then minced and homogenized thoroughly with 1:32 (w v^‐1) 80% acetonitrile solution. The separated plasma and tissue homogenates were frozen at −80°C until analysis.

LC–MS/MS determination of MPP⁺ and NC

The concentrations of MPP⁺ and NC in the cellular uptake and tissue samples were determined by the modified LC–MS/MS method (Li et al., 2014) using an Agilent 1290/6460 LC–MS with a triple quadrupole mass spectrometer. Two or eight volumes of acetonitrile containing 90 ng mL^‐1 loratadine as an internal standard (IS) were added to the cell lysates or tissue samples, respectively, to precipitate proteins. After being vortex mixed for 2 min, the mixture was centrifuged at 15 000 g for 15 min, and 2.0 μL of the supernatant was analysed by LC–MS/MS. The mass spectrometric analysis was carried out on an electrospray ionization (ESI) source in positive ion mode, and the quantification was performed using multiple reaction monitoring (MRM) mode (the ion pair of MPP⁺ at m/z 170.1 > 128.0, NC at m/z 348.1 > 332.1 and IS at m/z 383.1 > 337.1).

Data analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Experimental subjects/preparations were randomized to groups, and group assignments, data recording and data analysis were blinded to the operator. The values of IC₅₀ and median lethal concentration (LC₅₀) were estimated, respectively, by sigmoidal curve fitting of the log₁₀ inhibitor concentrations versus the uptake of MPP⁺ (% of control) and by sigmoidal curve fitting of the log₁₀ NC concentrations versus the cells’ viability using graphpad prism 5.0 (GraphPad Software Inc., San Diego, CA, USA). The Michaelis–Menten constants V _max and K _M were calculated by fitting the data to the Michaelis–Menten equation V = V _max/(1 + (K _M/[S])), where V is the initial uptake velocity and [S] is the concentration of substrate. For in vitro data, each point represents the mean ± SD of at least five wells or monolayers, and in vivo data are shown as mean ± SD from at least six animals.

Statistical analyses and significance were determined by Student's unpaired two‐tailed t‐test. One‐way ANOVA with Dunnett's or Bonferroni post hoc test was applied to the data if more than two groups were analysed, but only if F achieved the level of significance P < 0.05 and no significant variance inhomogeneity was observed. All of the statistical analyses were performed using graphpad prism 5.0. Some data were displayed as a percentage of the vehicle group (% of control). P values < 0.05 were considered statistically significant.

Materials

FBS, trypsin, insulin‐transferrin‐selenium, DMEM and DMEM/F12 were purchased from Gibico Invitrogen Corporation (Carlsbad, CA, USA). SYBR Green was obtained from Takara Bio Inc. (Otsu, Japan). Collagenase IV, collagen (type I solution from rat tail), 4‐(4‐(dimethylamino)styryl)‐N‐methylpyridinium iodide (ASP⁺), hydroxypropyl cellulose (HPC), 1‐methyl‐4‐phenylpyridinium (MPP⁺) and loratadine were obtained from Sigma‐Aldrich (St. Louis, MO, USA). 3‐[4,5‐Dimethylthiazol‐2‐yl]‐2, 5‐diphenyltetrazolium bromide (MTT) was obtained from Sangon Biotech Co., Ltd. (Shanghai, China). NC and (+)‐tetrahydropalmatine ((+)‐THP) were purchased from Nanjing Zelang Medical Technology Co., Ltd. (Nanjing, China) and Huatuo Co., Ltd. (Shanghai, China) respectively. LDH activity assay kit and bicinchoninic acid (BCA) protein assay kit were obtained from Jiancheng Co., Ltd. (Nanjing, China) and Beyotime Institute of Biotechnology (Nangjing, China) respectively. Acetonitrile was obtained from Tedia (Fairfield, TX, USA). All other chemicals were purchased from commercial sources and were of analytical grade.

Results

Inhibitory effect of NC on hOCT2 and hMAT

The inhibitory effect of NC (0.001–100 μM) on the intracellular accumulation of MPP⁺ (1 μM, 3 min) in MDCK‐hOCT2 and MDCK‐hMATE1 cells was evaluated. NC and cimetidine (a known inhibitor of OCT2 and MATE1) inhibited the uptake of MPP⁺ with IC₅₀ values of 2.41 ± 0.64 μM and 3.62 ± 1.1 μM for MDCK‐hOCT2 cells, and 0.15 ± 0.03 μM and 0.093 ± 0.05 μM for MDCK‐hMATE1 cells, respectively (Figure 2).

Figure 2.

Inhibitory effect of NC and cimetidine on the intracellular accumulation of MPP⁺ in MDCK‐hOCT2 (A) and MDCK‐hMATE1 (B) cells. n = 6. The uptake is expressed as a percentage of MPP⁺ uptake without inhibitors (% of control).

Uptake of NC mediated by hOCT2

To determine whether NC is a substrate of hOCT2, cellular uptake of NC in MDCK‐hOCT2 cells and mock cells were compared. The uptake of NC in MDCK‐hOCT2 cells was much higher than that in mock cells. The uptake of NC in mock cells was linear within the concentration of 0.1–4.0 μM, while the hOCT2‐mediated uptake followed Michaelis–Menten kinetics with the K _M (0.97 ± 0.10 μM), V _max (288.1 ± 12.8 pmol·mg^‐1·protein·min^‐1) and Cl _int (V _max/K _M: 297.0 ± 17.0 μL mg^‐1·protein·min^‐1) (Figure 3A), indicating that NC is a high‐affinity substrate of hOCT2.

Figure 3.

Cellular accumulation of NC mediated by hOCT2. (A) Concentration‐dependence of NC accumulation in MDCK‐hOCT2 and mock cells. Cells were incubated at 37°C for 2 min with NC (0.1–4 μM). (B) Inhibition of hOCT2‐mediated NC uptake by OCT2 inhibitors. Cells were incubated with 0.2 μM NC in the absence or presence of 50 μM cimetidine or 50 μM (+)‐THP for 2 min. n = 5. *P < 0.05 in comparison with the vehicle group (0.2% DMSO, control); one‐way ANOVA with Dunnett's post hoc test.

To further confirm the effect of OCT2 on the uptake of NC, the cellular accumulation of NC in MDCK‐hOCT2 with or without OCT2 inhibitors was compared. The results revealed that cimetidine (50 μM) and (+)‐THP (50 μM) reduced NC uptake in MDCK‐hOCT2 cells to 16.0% and 26.0% of the control (P < 0.05, Figure 3B), further indicating that NC is a substrate of hOCT2.

Intracellular accumulation and transcellular transport of NC

Because OCT2 is located on the basolateral membrane of renal proximal tubules, whereas MATE1 is mainly expressed in the apical membrane, and NC is also a substrate of MATE1, intracellular accumulation and transcellular transport of NC (2.5 μM) were investigated in MDCK‐hOCT2/hMATE1 and MDCK‐hOCT2/pcDNA3.1 cells. NC transcellular transport from the basal to the apical chamber was significantly higher in MDCK‐hOCT2/hMATE1 cells compared with the MDCK‐hOCT2/pcDNA3.1 cell monolayers (Figure 4A). Because intracellular NC accumulation in MDCK‐hOCT2/pcDNA3.1 cells was approximately 1.2‐fold of that in MDCK‐hOCT2/hMATE1 cells (Figure 4B), we deduced that MATE1 plays a minor role in NC transcellular transport.

Figure 4.

Transcellular transport (A) and cellular accumulation (B) of NC in MDCK‐hOCT2/pcDNA3.1 and MDCK‐hOCT2/hMATE1 cell monolayers. NC (2.5 μM) with or without 50 μM (+)‐THP was administered to the basal [basolateral (BL) → apical (AP)] or apical (AP → BL) side of the cell monolayers, and the uptake and transcellular transport were determined within 120 min. Values are displayed as percentage of the vehicle group (% of control). n = 5. #P < 0.05 in comparison with MDCK‐hOCT2/pcDNA3.1 cells; *P < 0.05 in comparison with BL → AP; one‐way ANOVA with Bonferroni post hoc test.

We also investigated the effect of (+)‐THP on the transcellular transport of NC in MDCK‐hOCT2/hMATE1 and MDCK‐hOCT2/pcDNA3.1 cell monolayers. When NC was co‐incubated with 50 μM (+)‐THP, an inhibitor of bidirectional transport, intracellular accumulation of NC was reduced to 28.6% and 30.1% of the vehicle in respective cells (P < 0.05, Figure 4B).

Contribution of OCT2 and MATE1 to NC‐induced cytotoxicity

Because OCT2 mediated the influx of NC into cells, while MATE1 mediated NC efflux, we assessed the contribution of OCT2 and MATE1 to NC‐induced cytotoxicity. The cell viability was evaluated by incubating MDCK‐hOCT2, MDCK‐hOCT2/hMATE1 and mock cells with NC (0.01–100 μM) at 37°C for 24 h. As shown in Figure 5A, MDCK‐hOCT2 and MDCK‐hOCT2/hMATE1 cells were much more sensitive to NC‐induced toxicity than mock cells, and the LC₅₀ values of NC on MDCK‐hOCT2 and MDCK‐hOCT2/hMATE1 cells were 0.47 ± 0.12 μM and 1.60 ± 0.03 μM, respectively, versus 29.1 ± 7.1 μM on mock cells, which indicated that OCT2 played a crucial role in NC‐induced cytotoxicity, while MATE1 slightly attenuated the toxicity. In addition, (+)‐THP and cimetidine markedly increased the cell viability of MDCK‐hOCT2 and MDCK‐hOCT2/hMATE1 cells, reduced the LDH leakage from MDCK‐hOCT2 cells caused by NC (P < 0.05) (Figure 5B and 5C), which further confirmed the contribution of OCT2 to the toxicity of NC.

Figure 5.

Concentration‐dependent cytotoxicity induced by NC on MDCK‐hOCT2, MDCK‐hOCT2/hMATE1 cells and mock cells (A). The effects of 50 μM (+)‐THP and 50 μM cimetidine on the reduction in cell viability (B) and increased LDH release (C) induced by NC (0.5 μM) in MDCK‐hOCT2 cells. Cells were treated with NC at 37°C for 24 h in the absence or presence of (+)‐THP and cimetidine. Values are displayed as percentage of the vehicle group (% of control). n = 6. #P < 0.05 in comparison with the vehicle group; one‐way ANOVA with Bonferroni post hoc test; *P < 0.05 in comparison with the NC‐alone group; one‐way ANOVA with Dunnett's post hoc test.

OCT2 inhibitors reduced the accumulation and cytotoxicity of NC in rat PCPT cells

In this study, we could not measure the protein level of transporters, but based on the finding that the mRNA level of SLC22A2 (OCT2) was approximately 2.2‐foldthat of SLC47A1 (MATE1) in rPCPT cells, the capacity or intrinsic activity (Cl _int : V _max/K _M) of OCT2 for NC was much higher than MATE1. Therefore, rPCPT cells were utilized to evaluate the role of OCT2 in the renal disposition of NC. As shown in Figure 6A, the uptake of NC into rPCPT cells could be significantly inhibited by OCT2 inhibitors, (+)‐THP and cimetidine (P < 0.05), indicating that OCT2 plays an important role in the NC uptake into the proximal tubule cells.

Figure 6.

Cimetidine and (+)‐THP reduced the accumulation and cytotoxicity of NC in rPCPT cells. (A) Cells were incubated for 2 min in 2 μM NC with or without 50 μM (+)‐THP or 50 μM cimetidine. *P < 0.05 in comparison with the vehicle group (0.2% DMSO). (B) Cells were incubated with 0.1–50 μM NC for 24 h in the absence or presence of 50 μM (+)‐THP or 50 μM cimetidine. n = 5. *P < 0.05 in comparison with NC‐alone group. One‐way ANOVA with Dunnett's post hoc test.

To assess the cytotoxicity of NC in rPCPT cells, cell viability was also evaluated, and the results showed that after incubation of rPCPT cells with 0.1–50 μM of NC for 24 h, the viability of cells was concentration‐dependently decreased (87.0%–44.0% of control, respectively) (Figure 6B), whereas OCT2 inhibitors, (+)‐THP and cimetidine, attenuated the NC‐induced toxicity with the cell viability increased to 121.5%–90.3% and 110.0%–72.7% (P < 0.05) respectively. These results suggest that OCT2 inhibitors could protect the rPCPT cells from the cytotoxicity induced by NC; thus, we deduced that OCT2 might be involved in NC‐induced toxicity of proximal tubule cells in vivo.

Twenty day repeated toxicity study

Because OCT2 is highly expressed in the kidney, we assumed that the kidney might be a target for NC‐associated toxicity. Thus, the accumulation and toxicity of NC in rats’ kidneys were investigated to evaluate the crucial role of OCT2 in NC‐induced renal injury.

Macroscopic observations and body weights

All the rats tested survived after i.v. doses of 5 mg·kg⁻¹ day⁻¹ NC for 20 successive days. Sedation, relaxation and reduced locomotor activity were observed in the NC‐treated group after the first dose, followed by asthenia, a reduction in food and water consumption during the repeated‐dose treatment. Moreover, the body weights significantly decreased from day 13 to day 20, compared with the vehicle group (P < 0.05) (Figure 7).

Figure 7.

Body weight changes of rats induced by administration of 5 mg·kg^−1· day⁻¹ NC for 20 days. The control group received 10% HPC containing 0.5% DMSO solution (n = 6), and the NC‐treated group was successively administered NC (5 mg·kg⁻¹·day⁻¹) by the tail vein (i.v.) for 20 days (n = 12). *P < 0.05 in comparison with the vehicle group (control: 10% HPC containing 0.5% DMSO solution); Student's t‐test for unpaired samples.

Serum biochemistry

Serum BUN, ALP, URA, CRE, LDH, ALT and AST of rats were determined at day 0, 5 and 20 after administration of NC or vehicle. As shown in Figure 8, compared with day 0, BUN and LDH were increased at day 20 (P < 0.05) in the NC‐treated group, while ALP was significantly reduced at day 5 and day 20 (P < 0.05), and no significant changes were found in other biochemical profiles (including URA, CRE, ALT and AST) during the experimental period. In the vehicle group, the serum biochemistry was not obviously altered (data not shown). The above results indicate that NC causes significant changes in serum concentrations of chemicals that indicate renal function.

Figure 8.

Biochemical indicators of kidney function in the serum after NC treatment. (A) BUN, LDH and ALP; (B) URA, CRE and the liver function indicators ALT and AST at day 0, 5 and 20 in the NC‐treated group (5 mg·kg⁻¹ day⁻¹) respectively. Day 0 (before treatment, n = 6), day 5 (n = 12) and day 20 (n = 12). Values are displayed as percentage of the vehicle group (% of control). *P < 0.05 in comparison with day 0; one‐way ANOVA with Dunnett's post hoc test.

Macroscopic findings and organ weights

An autopsy study was carried out in rats treated with NC (5 mg·kg⁻¹·day⁻¹) or vehicle successively for 20 days. The organs, including the liver and kidneys (right and left), were carefully examined. No noticeable pathological changes were observed by naked eye in all organs. Compared with the vehicle group, the absolute weight was obviously lower in the NC‐treated group (P < 0.05), while the relative weight of kidneys (ratio of kidney weight/body weight) became significantly higher (P < 0.05) (Table 1).

Table 1.

Kidney and liver weights of rats after the last dose of the 20 day NC‐treated

	NC‐treated	Vehicle
Body weight (g)	136.3 ± 13a	199.3 ± 20
Absolute (g)
Liver	4.18 ± 0.55a	7.72 ± 0.88
Kidney (right)	0.664 ± 0.054a	0.816 ± 0.082
Kidney (left)	0.631 ± 0.050a	0.793 ± 0.081
Relative (g/g b.w.)
Liver	0.0307 ± 0.0029a	0.0387 ± 0.0021
Kidney (right)	0.00489 ± 0.00027a	0.00410 ± 0.00024
Kidney (left)	0.00465 ± 0.00031a	0.00398 ± 0.00019

Data represent mean ± SD (n = 6–12).

^a P < 0.05 in comparison with the vehicle group; Student's t‐test for unpaired samples.

Histopathology

After the 20 day NC treatment, histopathological observations were carried out. In the NC‐treated group, mild swelling in the renal tubular epithelial cells and mild vacuoles degeneration were found in nine of 12 rats. In addition, mucous membrane inflammation and neutrophil infiltration in the renal pelvis, as well as renal tubule degeneration, were noticed in rats treated with NC (Figure 9), whereas no pathological changes were found in the livers of NC‐treated rats (data not shown) or in any organ of the vehicle group.

Figure 9.

Slices of kidney were stained with haematoxylin and eosin for histopathological analysis. Representative histomicrographs of kidney sections of the vehicle group (A) and NC‐treated group (B–D). Magnification 100×. The arrow indicated mild ballooning degeneration (B), neutrophil (C) and occasional renal tubule degeneration (D).

Tissue distribution of NC in rats

To elucidate whether NC accumulated in the kidney and liver, the concentration of NC in the kidney, liver and plasma of rats at 0.25, 0.5 and 2 h after a single dose (5 mg·kg⁻¹) and 2 h after the last dose of a 20 day repeated treatment (5 mg·kg⁻¹ day⁻¹) was determined. The concentration of NC in the kidney and liver was much higher than that in plasma at an identical time after dosing. The concentration of NC in the kidney reached the maximum (44.6 μg·g⁻¹) at 0.5 h and remained at a high level (40.4 μg·g⁻¹) at 2 h after a single i.v. dose, which was 532‐ and 775‐fold higher than that in plasma respectively. NC reached the highest concentration (15.1 μg·g⁻¹) in liver at 0.25 h, but decreased to a very low level (1.3 μg·g⁻¹) at 2 h after a single dose, which was 182‐ and 25‐fold of that in plasma. Additionally, the concentrations of NC in the kidney and liver at 2 h after the last administration of 20 day repeated doses were 55.7 and 9.5 μg·g⁻¹, which were 1.4‐ and 7.3‐fold higher than that after a single dose respectively (Figure 10).

Figure 10.

Mean concentration of NC in kidney, liver and plasma after single (n = 6) or repeated (n = 12) administration of NC in rats. *P < 0.05 in comparison with single dose at 2 h; Student's t‐test for unpaired samples.

Discussion

The results of the present study demonstrate that NC is a high‐affinity substrate of OCT2, and the LC₅₀ of NC‐induced toxicity in the MDCK‐hOCT2 cells was significantly lower than that in mock cells (0.47 ± 0.12 μM vs. 29.1 ± 7.1 μM). Our previous study (Li et al., 2014) proved that NC was a substrate of MATE1 located at brush border membranes of the renal proximal tubules, which implied that MATE1 could mediate the efflux of NC from proximal tubule cells. However, the transport capacity of OCT2 was much greater than that of MATE1 (the Cl _int of 297.0 μL·mg⁻¹ protein·min⁻¹ vs. 20.6 μL·mg⁻¹·protein·min⁻¹); the cellular accumulation of NC in MDCK‐hOCT2/pcDNA3.1 cells was slightly higher than that of MDCK‐hOCT2/hMATE1 cells (Figure 4B); concomitantly, the NC‐induced toxicity on MDCK‐hOCT2/hMATE1 cells was only a little lower when compared with MDCK‐hOCT2 cells (the LC₅₀ of 1.60 ± 0.03 μM vs. 0.47 ± 0.12 μM). It appears that OCT2 plays a crucial role in the transport and toxicity of NC, while MATE1 mediates its slight cellular efflux.

As CYPs, such as CYP3A4 and CYP2D6, which mediate the metabolism of NC in liver, are expressed at a very low level in the kidney, and the conjugation metabolism of NC was not found (data not shown), it is clear that OCT2 plays a vital role in the accumulation of NC in the kidney. To further confirm the role of OCT2 in the renal disposition of NC, primary‐cultured proximal tubular cells, which are recognized as an appropriate model for evaluating transport‐mediated drug disposition in kidney (Elwi et al., 2009; Fisel et al., 2014; Li et al., 2015), were used. In agreement with the uptake and accumulation of NC in transfected cells, the cellular accumulation of NC in rPCPT cells was also substantially high, and the OCT2 inhibitors obviously reduced NC accumulation, indicating that OCT2 was indeed responsible for NC uptake in proximal tubule cells. As a result, NC markedly decreased the cell viability of rPCPT cells in a dose‐dependent manner, and this increased cell death was significantly prevented by the OCT2 inhibitors, (+)‐THP and cimetidine (Figure 6). Thus, we deduced that OCT2‐mediated uptake was likely to be the predominant factor in NC‐induced nephrotoxicity in vivo. (+)‐THP (50 μM) or cimetidine (50 μM) reduced the NC uptake in MDCK‐hOCT2 and significantly increased the cell viability (up to 74.3% or 47.8% vs. 39.7%, respectively) (Figure 5), which also indicates that OCT2 ha a critical role in NC‐induced cytotoxicity.

Our in vivo study showed that body weight gain was markedly decreased from day 13 during NC treatment, and the absolute weights of the liver and kidney were reduced after the last dose of the 20 day NC treatment (5 mg·kg⁻¹·day⁻¹). The time‐dependent reductions in body weight gain might be attributed to NC‐induced kidney damage, which was indicated by the increase in serum BUN and LDH, decrease in ALP and obvious histopathological changes after the i.v. injections of NC for 20 successive days (Figure 8). Our study also revealed that a high concentration of NC accumulated in the kidney, for instance, the concentration of NC in kidney reached 55.7 μg·g⁻¹ (1185‐fold of that in plasma) at 2 h after the last dose (Figure 9). Additionally, this high concentration of NC in the kidney was maintained for a long period of time (44.6 to 40.4 μg·g⁻¹ within 0.25–2 h) after he rats were given a single i.v. dose of 5 mg·kg⁻¹ NC. These data suggest that the observed renal injury probably results from or correlates with NC accumulation in the proximal tubule cells. Based on the differential transport of NC by OCT2 and MATE1, we reasoned that NC accumulation in the kidney is probably the result of its significant uptake mediated by OCT2 and weak tubular secretion by MATE1.

Although the concentration of NC in the liver was also higher than that in plasma, it was much lower than in the kidney. Both the absolute and relative weight of the liver were also significantly decrease in the NC‐treated group, but no histopathological changes were observed (data not shown). Moreover, the concentration of NC in the liver 2 h after the last dose was 7.3‐fold higher than that 2 h after a single dose, suggesting NC also accumulates in the liver. Thus, liver damage would be unavoidable during a long‐term treatment with NC. Additionally, OCT3 is expressed in the lung (Koepsell et al., 2007), and NC has been found to be a substrate of lung OCT3 (Li et al., 2014), which means that NC is likely to accumulate in the lung. Indeed, this hypothesis has been corroborated in our present study as the concentration of NC in the lung was dramatically high. However, unexpectedly, no noticeable lung injury was present after the 20 day successive treatment schedule (data not shown).

It was reported that NC can insert into DNA and inhibit topoisomerases I and II; thus, NC has tumour‐selective cytotoxicity (Gatto et al., 1996; Holden et al., 1999). Because OCT2 is primarily expressed in the human kidney, and NC is transported by OCT2 and accumulates in the kidney, it is reasonable to assume that NC might have potential as a treatment of renal tumours. A marked decrease in the expression of OCTs has been reported in tumour tissue, which is thought to be caused by epigenetics in cancer (Zolk and Fromm, 2012; Yang et al., 2013), thus normal proximal tubule cells might be more sensitive to NC‐induced toxicity than cancer cells. Because OCT2 expression in the human kidney is very abundant and much higher than that of MATE1 (Morrissey et al., 2013; Motohashi et al., 2013), the NC‐induced damage to the kidney in humans might be very severe; therefore, it is clear that NC is not a promising drug candidate for development. Although natural or traditional Chinese medicines containing NC, such as Liangmianzhen tablets and extracts of Toddalia asiatica (Gakunju et al., 1995), have been widely used in China at high doses, no severe kidney damage has been reported clinically. This might be due to its low oral bioavailability. However, it seems unlikely that the kidney damage can be avoided because the minimal effective i.v. dose was 5 mg·kg⁻¹ in cancer therapy studies on nude mice (Liu et al., 2009). In the present study, we did not evaluate the effect of OCT2 inhibitors, such as (+)‐THP and cimetidine, on the accumulation and toxicity of NC in the kidney. Therefore, the attenuation of NC‐induced toxicity by OCT2 inhibitors in vivo needs further studies.

Conclusion

In conclusion, in the present study it was demonstrated that NC is a high‐affinity substrate of OCT2, which mediates the uptake of NC in proximal tubule cells, causes its accumulation in the kidney, and subsequently induces kidney damage. Whereas MATE1 only plays a minor role in the efflux of NC from cells, and it slightly attenuates NC‐induced toxicity. Our results for the first time highlight the vital role of OCT2 in the renal disposition and toxicity of NC. The results give us more information about the possible mechanisms and risk factors associated with the renal cytotoxicity of NC and provide a useful reference for the application and development of NC and NC‐containing herbal medicines.

Author contributions

L.P.L., H.Z. and H.D.J. designed the research. L.P.L., X.Y., F.F.S., H.M.L. and J.M. performed the research. L.P.L., X.Y., Y.Y.W., J.M. and H.Z. analysed data. L.P.L., K.W. and H.D.J. wrote the paper.

Conflict of interest

The authors declare no conflicts of interest.

Declaration of transparency and scientific rigour

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.

Li, L. P. , Song, F. F. , Weng, Y. Y. , Yang, X. , Wang, K. , Lei, H. M. , Ma, J. , Zhou, H. , and Jiang, H. D. (2016) Role of OCT2 and MATE1 in renal disposition and toxicity of nitidine chloride. British Journal of Pharmacology, 173: 2543–2554. doi: 10.1111/bph.13537.