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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Mar 5;177(9):1933–1948. doi: 10.1111/bph.14957

Protective effect of cilastatin against diclofenac‐induced nephrotoxicity through interaction with diclofenac acyl glucuronide via organic anion transporters

Xiaokui Huo 1,2,3, Qiang Meng 1,2,3, Changyuan Wang 1,2,3, Jingjing Wu 1,2,3, Chong Wang 1,2,3, Yanna Zhu 1,2,3, Xiaodong Ma 1, Huijun Sun 1,2,3, Kexin Liu 1,2,3,✉
PMCID: PMC7161545  PMID: 32000294

Abstract

Background and Purpose

Diclofenac is a widely used nonsteroidal anti‐inflammatory drug. However, adverse effects in the kidney limit its clinical application. The present study was aimed to evaluate the potential effect of cilastatin on diclofenac‐induced acute kidney injury and to clarify the potential roles of renal organic anion transporters (OATs) in the drug‐drug interaction between cilastatin and diclofenac.

Experimental Approach

The effect of cilastatin was evaluated in diclofenac‐induced acute kidney injury in mice. Human OAT1/3‐transfected HEK293 cells and renal primary proximal tubule cells (RPTCs) were used to investigate OAT1/3‐mediated transport and the cytotoxicity of diclofenac.

Key Results

Cilastatin treatment decreased the pathological changes, renal dysfunction and elevated renal levels of oxidation products, cytokine production and apoptosis induced by diclofenac in mice. Moreover, cilastatin increased the plasma concentration and decreased the renal distribution of diclofenac and its glucuronide metabolite, diclofenac acyl glucuronide (DLF‐AG). Similarly, cilastatin inhibited cytotoxicity and mitochondrial damage in RPTCs but did not change the intracellular accumulation of diclofenac. DLF‐AG but not diclofenac exhibited OAT‐dependent cytotoxicity and was identified as an OAT1/3 substrate. Cilastatin inhibited the intracellular accumulation and decreased the cytotoxicity of DLF‐AG in RPTCs.

Conclusion and Implications

Cilastatin alleviated diclofenac‐induced acute kidney injury in mice by restoring the redox balance, suppressing inflammation, and reducing apoptosis. Cilastatin inhibited OATs and decreased the renal distribution of diclofenac and DLF‐AG, which further ameliorated the diclofenac‐induced nephrotoxicity in mice. Cilastatin can be potentially used in the clinic as a therapeutic agent to alleviate the adverse renal reaction to diclofenac.

Abbreviations

AUC

area under concentration‐time curve

CLp

plasma clearance

Cmax

maximum plasma drug concentration

DDI

drug‐drug interaction

DLF‐AG

diclofenac acyl glucuronide

NSAID

nonsteroidal anti‐inflammatory drug

OATs

organic anion transporters

RPTCs

renal primary proximal tubule cells.

t1/2

half lifet

Tmax

time to reach Cmax

What is already known

  • Cilastatin is a promising agent with renal protective activity against nephrotoxic drugs.

What this study adds

  • Cilastatin inhibited renal transport and sequentially decreased nephrotoxicity of diclofenac.

What is the clinical significance

  • Cilastatin can potentially be used in clinics to alleviate the adverse renal reaction to diclofenac.

1. INTRODUCTION

https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=2714 is a nonsteroidal anti‐inflammatory drug (NSAID) widely used for the treatment of rheumatoid arthritis, osteoarthritis and acute injury pain (Altman, Bosch, Brune, Patrignani, & Young, 2015). Owing to its anti‐inflammatory, analgesic and antipyretic activities, diclofenac is the most widely prescribed NSAID worldwide (McGettigan & Henry, 2013). Unfortunately, diclofenac is associated with serious dose‐dependent gastrointestinal, cardiovascular and renal adverse effects (Coxib, traditional NTC, et al., 2013; McGettigan & Henry, 2013; Douros et al., 2018). While diclofenac‐induced enteropathy and cardiovascular risk are well studied, diclofenac‐induced acute kidney injury is less well investigated. It was reported that diclofenac was one of the most common causes of drug‐induced kidney injury and seemed to induce nephrotoxicity even in patients with normal baseline kidney function (Douros et al., 2018). The mechanisms underlying the nephrotoxicity of diclofenac include but are not limited to non‐selective COX inhibition and direct renal tubular cytotoxicity (Bao et al., 2019; Kovic, Vujovic, Srebro, Medic, & Ilic‐Mostic, 2016). Both induction of COX‐2 expression and co‐administration with agents possessing anti‐inflammatory, anti‐oxidative, and anti‐apoptotic activities were demonstrated to reduce diclofenac‐induced acute kidney injury (Bao et al., 2019; Fattori et al., 2017). In parallel, modifications of pharmacokinetic properties through the use of pharmaceutical approaches and chemical structural modification have been used to develop new drug products based on diclofenac with improved efficacy, safety and increased clinical utility (Altman et al., 2015). Therefore, it is necessary to clarify the mechanism of nephrotoxicity induced by diclofenac from a pharmacokinetic perspective.

The kidney is the main organ responsible for the elimination of diclofenac in humans. More than half of a radiolabelled dose was recovered from urine as unchanged diclofenac or its acyl glucuronide, diclofenac acyl glucuronide (DLF‐AG; Lagas, Sparidans, Wagenaar, Beijnen, & Schinkel, 2010), which resulted in high exposure in the kidney and consequently induced nephrotoxicity. Reducing the renal level of diclofenac will definitely decrease nephrotoxicity. It is worth noting, although controversially, that both diclofenac and diclofenac acyl glucuronide can interact with renal https://www.guidetopharmacology.org/GRAC/FamilyDisplayForward?familyId=198 (OATs) (Khamdang et al., 2002; Nieskens et al., 2016; Nieskens et al., 2018; Zhang et al., 2016). OATs are located on the basolateral membrane of renal proximal tubule cells and are gates for substrate entry into tubule cells (Huo & Liu, 2018; Yin & Wang, 2016). The substrate spectrum of OATs is broad and many toxic metabolites (uric acids, indoxyl‐sulfate and anionic uraemic toxins) and drugs (methotrexate and aristolochic acid) are substrates of OATs (Huo & Liu, 2018; Yin & Wang, 2016), the excessive accumulation of which can induce nephrotoxicity and lead to unexpected clinical outcomes. Recently, a clinical study reported that a population with the single nucleotide polymorphism (SNP) (−475T > T/G), which induces https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1025 expression, had a higher frequency of chronic kidney disease (Sun et al., 2018). In contrast, Oat1 and https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1027 gene knockout mice seemed to show increased tolerance to aristolochic acid, a natural product with renal toxicity (Dickman, Sweet, Bonala, Ray, & Wu, 2011). Therefore, OATs are the determinant of the renal accumulation of toxic substances and the initiation of nephrotoxicity.

https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5166 was developed to improve the stability of the antibiotic imipenem by inhibiting renal https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1393 (Balfour, Bryson, & Brogden, 1996). Recently, cilastatin was identified as a promising agent with renal protective activity against nephrotoxicity induced by vancomycin, cisplatin, colistin and aminoglycosides (Hori et al., 2017; Humanes et al., 2012). In addition to its anti‐inflammatory, anti‐oxidative and anti‐apoptotic activities, an important mechanism of renoprotection by cilastatin is blockade of the renal uptake of toxic drugs (Hori et al., 2017; Humanes et al., 2012). However, the effect of cilastatin on the nephrotoxicity induced by diclofenac has never been studied. In our previous studies, cilastatin was found to decrease the nephrotoxicity of imipenem partially through OATs inhibition (Huo et al., 2019; Zhu et al., 2018). These findings encouraged us to investigate whether OATs played a potential role in the drug‐drug interaction (DDI) between diclofenac and cilastatin.

In the present study, we hypothesized that the combination of diclofenac and cilastatin induced OATs‐mediated DDI, which might contribute to the renal protective effect of cilastatin against diclofenac‐induced nephrotoxicity. To test this hypothesis, the effect of cilastatin on acute kidney injury was evaluated in diclofenac‐treated mice in vivo and in primary renal proximal tubule cells (RPTCs) in vitro. The intracellular accumulation and cytotoxicity of diclofenac and diclofenac acyl glucuronide was investigated using human OAT1/3‐overexpressing cells and RPTCs in vitro with or without cilastatin. For the first time, the effect of cilastatin on diclofenac nephrotoxicity was studied and the potential role of OATs was explored to clarify the nephrotoxicity mechanism of diclofenac from a pharmacokinetic perspective.

2. METHODS

2.1. Materials

Diclofenac and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4357 were purchased from Dalian Meilun Biology Technology Co., Ltd. (Dalian, People's Republic of China). Cilastatin were purchased from Topscience Co., Ltd. (Shanghai, People's Republic of China). Diclofenac acyl glucuronide (DLF‐AG) was purchased from Toronto Research Chemicals (Toronto, Canada). Kits for the determination of blood urea nitrogen (BUN), creatine (CRE), glutathione (GSH), superoxide dismutase (SOD), glutathione (https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6737) and malondialdehyde (MDA) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). ELISA kits of kidney injury molecule‐1 (Kim‐1), https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4974, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4983, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4998 and https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5074 were purchased from Elabscience Biotechnology Co., Ltd. (Wuhan, China). The following primary antibodies were used: anti‐caspase‐3/cleaved caspase‐3 (Cell Signaling Technology, Beverly, USA; Cat# 9662, RRID:AB_331439), anti‐caspase‐9/cleaved caspase‐9 (Cell Signaling Technology, Beverly, USA; Cat# 9508, RRID:AB_2068620), anti‐BAX (Proteintech, Wuhan, China; Cat# 50599‐2‐Ig, RRID:AB_2061561), anti‐BCL2 (Proteintech, Wuhan, China; Cat# 26593‐1‐AP, RRID:AB_2818996) and anti‐β‐actin (Proteintech, Wuhan, China; Cat# 66009‐1‐Ig, RRID:AB_2687938). All other chemicals were of analytical grade and were available from commercial sources.

HEK293 cells (RRID:CVCL_0045) stably transfected with mock vector (mock‐HEK293), full length hOA1 cDNA (OAT1‐HEK293), and hOAT3 cDNA (OAT3‐HEK293) were established previously (Deguchi et al., 2004) and kindly provided by Prof. Yuichi Sugiyama (Sugiyama Laboratory, RIKEN, Japan).

2.2. Animals

Kunming male mice (18–22 g) were obtained from the Experimental Animal Center of Dalian Medical University (Dalian, China; permit number SYXK 2018‐0007). All animal experiments were performed according to local institutional guidelines for the care and use of laboratory animals. Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology. All animal studies complied with the principle for replacement, refinement or reduction (the 3Rs).

2.3. Diclofenac‐induced acute kidney injury in mice

Mice were divided randomly into six groups: (a) vehicle group, (b) cilastatin (100 mg·kg−1, i.p., single dose) group, (c) diclofenac (200 mg·kg−1, p.o., single dose) group, (d) diclofenac (200 mg·kg−1, p.o., single dose) + cilastatin (25 mg·kg−1, i.p., single dose) group, (e) diclofenac (200 mg·kg−1, p.o., single dose) + cilastatin (50 mg·kg−1, i.p., single dose) group, and (f) diclofenac (200 mg·kg−1, p.o., single dose) + cilastatin (100 mg·kg−1, i.p., single dose) group. The doses used in the in vivo studies were set according to previous research (Bao et al., 2019; Fattori et al., 2017; Hori et al., 2017). Cilastatin and diclofenac were dissolved in saline. Cilastatin was given 0.5 hr before diclofenac injury. Mice were killed by cervical dislocation after systemic anaesthesia with isoflurane at 24 hr after diclofenac administration. The blood was collected to obtain plasma immediately. The kidneys were removed and one was fixed in neutral 10% buffered formalin, while the other one was used for biochemical analysis. Plasma and kidney specimens were kept at −80°C until analysis. The levels of BUN and CRE in plasma were determined according to the manufacturer's instructions. The kidneys were homogenized (IKA‐T10 homogenizer; IKA, Staufen, Germany) in ice‐cold saline and the contents of GSH, SOD, MDA, Kim‐1, IL‐1β, IL‐18, IL‐6, and TNF‐α were determined using commercial kits following the manufacturer's protocol.

2.4. Kidney histology

The kidneys fixed in neutral 10% buffered formalin were sectioned at a thickness of 5 μm in paraffin and stained with haematoxylin & eosin (HE) or DeadEnd™ Fluorometric TUNEL System (Promega, Madison, WI, USA) for histological examination and apoptosis assay (Shu et al., 2019).

2.5. Western blot assay

Proteins from kidney tissue lysates were electrophoretically separated on an SDS‐PAGE (8–15%) gel and then transferred onto a PVDF membrane. Western blots were probed with specific primary antibodies and incubated with the corresponding secondary antibodies. The proteins were visualized using enhanced chemiluminescence‐plus reagents (ECL) and Chemidoc (Bio‐Rad, Hercules, CA, USA). β‐Actin acted as a standard protein. The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.

2.6. Pharmacokinetic study

Mice were divided randomly into two groups: (a) diclofenac (200 mg·kg−1, p.o., single dose) group and (b) diclofenac (200 mg·kg−1, p.o., single dose) + cilastatin (100 mg·kg−1, i.p., single dose) group. Cilastatin was given 0.5 hr before diclofenac administration. At 0, 5, 15, 30, 60, 120, 180, 240, 360, 480, 600 and 720 min after administration of diclofenac, mice were killed, and blood samples were collected to obtain plasma immediately. The kidney, liver, and intestine were removed simultaneously. Plasma and tissue samples were stored at −80°C until determination of diclofenac. For the determination of diclofenac acyl glucuronide, samples were acidified with citric acid (final concentration at 40 mM) immediately after collection to stabilize the diclofenac acyl glucuronide (Zhang et al., 2016). The maximum plasma drug concentration (C max) and the time to reach C max (T max) were obtained directly from the experimental data. The area under concentration‐time curve (AUC), plasma clearance (CLp), and half‐life (t 1/2) were calculated by DAS 2.0 pharmacokinetic program.

2.7. Cell culture and mouse renal primary proximal tubule cell (RPTC) isolation

Mock‐, hOAT1‐, and hOAT3‐HEK293 cells were routinely maintained in DMEM (Invitrogen, USA) supplemented with 10% FBS (heat inactivated), 1% non‐essential amino acid solution, 100 U·ml−1 penicillin and 0.1 mg·ml−1 streptomycin and G418 sulfate (400 μg·ml−1). Renal primary proximal tubule cells were isolated from mice according to a previous method with modifications (Huo et al., 2019; Li et al., 2016). Briefly, mice were killed and the kidneys were removed and minced in a sterile cell culture dish on ice. The minced tissue was isolated mechanically by sequential filtration through 80‐mesh and 100‐mesh cell strainers. The residue on the 100‐mesh cell strainer was washed repeatedly in HBSS solution containing 1% penicillin/streptomycin and digested in HBSS solution containing collagenase IV (1 mg·ml−1) by shaking for 20 min at 37°C. The isolated fragments were resuspended in DMEM/F12 supplemented with 10% FBS, 1% penicillin/streptomycin and 1% insulin‐transferrin‐selenium and then seeded into collagen I coated 96‐ or 24‐well culture plates (Costar Corning Inc., Corning, NY, USA). The cells were kept at 37°C in a 95% relative humidity atmosphere containing 5% CO2.

2.8. Uptake studies in transporter‐transfected cells and renal primary proximal tubule cells (RPTCs)

hOAT1/3‐ and mock‐HEK293 cells were seeded in 24‐well culture plates for 48 hr before uptake studies (Huo et al., 2019). After washing three times with transport buffer (containing 118‐mM NaCl, 23.8‐mM NaHCO3, 4.8‐mM KCl, 1.0‐mM KH2PO4, 1.2‐mM MgSO4, 12.5‐mM HEPES, 5.0‐mM glucose, 1.5‐mM CaCl2, pH 7.4), monolayers were incubated in transport buffer for 15 min at 37°C. Uptake studies were initiated by the addition of 1 ml of buffer containing substrates. Diclofenac (10 μM) and diclofenac acyl glucuronide (10 μM) were added into transport buffer to initialize uptake. Following incubation for 10 min at 37°C with gentle shaking, the medium was removed, and the cells were washed three times with 1 ml of ice‐cold buffer to stop uptake. For inhibition assays, various concentrations of cilastatin (0‐800 μM) were added simultaneously to the buffer, and the uptake was examined after incubation. Cell monolayers were subsequently lysed by freeze thawing. Concentrations of substrates in cell lysates were determined using LC‐MS/MS.

2.9. Cytotoxicity assay

HEK293 and RPTC cells were seeded at 4 × 103 cells per well in 96‐well plates and cultured for 24 hr. Fresh medium containing diclofenac with or without cilastatin (100 μM) was subsequently added, and the cells were incubated for an additional 24 hr. Cell viability was determined by a CCK‐8 assay (Solarbio, China).

2.10. Tetraethylbenzimidazolycar bocyanine iodine (jc‐1) staining (mitochondrial membrane potential assay)

Renal primary proximal tubule cells were seeded at 4 × 103 cells per well in six‐well plates and cultured for 24 hr. Fresh medium containing diclofenac (400 μM) with or without cilastatin (100 μM) was subsequently added, and the cells were incubated for an additional 24 hr. Then, the cells were stained with JC‐1 (2 μM, Beyotime Institute of Biotechnology, Shanghai, China) at 37°C in a dark environment for 20 min. Monomeric JC‐1 green fluorescence emission and aggregate JC‐1 red fluorescence emission were photographed using a Leica DM 14000B microscope fitted with a digital camera to determine.

2.11. Liquid chromatography‐tandem mass spectrometry (lc–ms/ms) analysis

Preparation of various biological samples was conducted as previously described (Huo et al., 2019). Detailed methods were provided in the Supporting Information. The concentrations of diclofenac and diclofenac acyl glucuronide were determined by an AB QTRAP 5500 LC–MS/MS System (Foster City, CA, USA) as previously described (Huo et al., 2018; Zhang et al., 2016). Chromatographic separation was performed on a Hypersil BDS‐C18 column (150 × 4.6 mm, 5 μm; Elite Analytical Instruments Co. Ltd., Dalian, China) at ambient temperature. The mobile phase consisted of acetonitrile and water with 0.1% (v/v) formic acid (90:10) at a flow rate of 0.6 ml·min−1. The detection was performed by multiple reaction monitoring. The selected m/z transitions were 294.0 → 250.0 for diclofenac, 470.0 → 193.1 for diclofenac acyl glucuronide, and 283.1 → 268.0 for physcion (internal standard). Other parameters were listed in Table S1. Representative chromatograms of analytes were shown in Figure S1. Analyst 1.6.3 software (Applied Biosystems) was used to control the equipment and for data acquisition and analysis.

2.12. Data analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology 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 data are expressed as the mean ± SEM. GraphPad Prism 7.00 software (GraphPad Software Inc., San Diego, CA, USA; RRID:SCR_002798) was used to handle the statistical analyses. All results were obtained from at least five independent experiments. The difference between the groups was calculated by a one‐way ANOVA followed by Tukey's post hoc test when comparing multiple independent groups, and when comparing two different groups, the unpaired Student's t‐test was carried out. Post hoc tests were run only if F achieved P < .05, and there was no significant variance inhomogeneity. P < .05 was considered a significant difference.

2.13. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (RRID:SCR_013077) (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).

3. RESULTS

3.1. Protective effect of cilastatin on diclofenac‐induced acute kidney injury in mice

To evaluate the effect of cilastatin on diclofenac nephrotoxicity, acute kidney injury in mice was induced by a single oral dose of diclofenac (200 mg·kg−1, p.o.), and the changes in kidney micrographs and biomarkers of renal function were studied after co‐administration of cilastatin (25‐100 mg·kg−1, i.p.). Twenty‐four hours after diclofenac treatment, mouse kidneys exhibited obvious acute tubular necrosis, including vacuolization in the tubular epithelium, tubular dilation, sloughing of tubular cells into the lumen and loss of brush border (Figure 1a), while the kidneys of mice in the vehicle and cilastatin‐only groups exhibited normal renal tissue morphology after haematoxylin & eosin (HE) staining (Figure 1a). The pathological changes in the kidneys of mice induced by diclofenac were significantly reduced in a dose‐dependent manner after co‐administration of cilastatin (Figure 1a). Meanwhile, cilastatin decreased diclofenac‐induced elevated levels of creatine (CRE) and blood urea nitrogen (BUN) in the plasma and kidney injury molecule‐1 (Kim‐1) in the kidneys in a dose‐dependent manner (Figure 1b), indicating that cilastatin ameliorated diclofenac‐induced acute kidney injury. Additionally, diclofenac induced oxidative stress and an inflammatory response in mouse kidneys as evidenced by the depletion of the antioxidant defence molecules glutathione (GSH) and superoxide dismutase (SOD), increased malondialdehyde (MDA) production, and increased levels of inflammatory cytokines IL‐1β, IL‐18, IL‐6, and TNF‐α. These effects were significantly prevented by cilastatin treatment (Figure 1b). On the other hand, cilastatin alone did not induce any changes in the levels of the biochemical indicators mentioned above. The results suggested that cilastatin protected against diclofenac‐induced acute kidney injury and inhibited oxidative stress and the inflammatory response induced by diclofenac challenge.

Figure 1.

Figure 1

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Protective effect of cilastatin against diclofenac‐induced acute kidney injury. Mice received diclofenac (200 mg·kg−1) orally with or without cilastatin (25, 50, and 100 mg·kg−1), and the kidneys and blood were collected after 24 hr. (a) Representative micrographs of haematoxylin & eosin (HE) staining of kidney specimens. Renal tubular epithelial cell swelling, tubule dilatation and necrosis, and shedding of cells are indicated in representative images of HE staining. Bars = 100 μm at 200×; bars = 50 μm at 400×. (b) Plasma levels of blood urea nitrogen (BUN) and creatinine (CRE), and biochemical indicators for kidney injury, oxidative stress, and inflammation in kidney tissues were determined by commercial kits. *P < .05 versus control group. # P < .05 versus diclofenac group. Data are expressed as mean ± SEM, n = 6

To further explore the protective effect of cilastatin on diclofenac‐induced acute kidney injury, apoptosis in mouse kidneys induced by diclofenac was evaluated in the presence or absence of cilastatin. First, TUNEL staining was performed to visualize apoptosis in the kidneys. Twenty‐four hours after diclofenac treatment, scattered and bright TUNEL‐positive nuclei could be observed over the entire cortex but were gradually reduced with increasing doses of cilastatin (Figure 2a,b). TUNEL‐positive nuclei were rarely observed in the kidneys from the vehicle and cilastatin‐only groups (Figure 2a,b). The results suggested that diclofenac induced apoptosis in mouse kidneys and cilastatin inhibited diclofenac‐induced apoptosis. This conclusion was further confirmed by western blot analysis, in which expression levels of apoptosis‐related proteins (cleaved caspase‐3/9, BAX, and Bcl‐2) in the kidney were determined. Diclofenac significantly increased the protein levels of cleaved caspase‐3/9 and the ratio of Bax/Bcl‐2 in the kidney (Figure 2c), which were markedly decreased by cilastatin treatment (Figure 2c). These results indicated that cilastatin prevented diclofenac‐induced apoptosis in the kidney.

Figure 2.

Figure 2

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Protective effect of cilastatin against diclofenac‐induced apoptosis in vivo. Mice received diclofenac (200 mg·kg−1) orally with or without cilastatin (25, 50, and 100 mg·kg−1), and the kidneys were collected after 24 hr. (a) Representative micrographs of TUNEL staining of kidney specimens. (b) Quantitative analysis of TUNEL‐labelled cells among the groups. *P < .05 versus control group. # P < .05 versus diclofenac group. Data are expressed as mean ± SEM, n = 5. (c) Protein expression levels of caspase‐3, caspase‐9, Bax, and BCL‐2 in mouse kidneys were determined by western blot

3.2. Effect of cilastatin on the pharmacokinetics of diclofenac in mice

To preliminarily explore the protective mechanisms of cilastatin, the plasma concentration and tissue distribution of diclofenac were determined by LC‐MS/MS in the presence or absence of cilastatin. After oral administration, diclofenac was rapidly absorbed, and the plasma concentration of diclofenac was generally raised when cilastatin was co‐administered (Figure 3a). The AUC(0–12 hr) of diclofenac was increased by 35.4% in the presence of cilastatin (Table 1). Plasma clearance (CLp) of diclofenac was significantly reduced and the half‐life (t 1/2) was increased by cilastatin (Table 1). Diclofenac displayed a typical enterohepatic circulation profile with two plasma concentration peaks reaching the first plasma concentration peak at 0.90 ± 0.25 hr (Figure 3a; Table 1). Co‐administration of cilastatin had little effect on the peak time of diclofenac but significantly increased the maximum plasma concentration (C max1) by twofold (Figure 3a; Table 1). Additionally, the second peak time (T max2) of diclofenac was shifted from 4.40 ± 0.77 hr to 7.60 ± 0.00 hr after co‐administration of cilastatin, while C max2 was slightly reduced by 23.1% (Table 1). Unlike the plasma concentrations, the concentrations in both the kidneys and liver were significantly decreased by cilastatin, less than 6 hr after administration, but the effect of cilastatin was reversed to up‐regulation afterwards (Figure 3b,c). Concurrent use of cilastatin decreased the C max and AUC(0–12 hr) of diclofenac significantly and T max was markedly delayed (Table 1; Table S2). In the intestine, the concentrations of the two groups were similarly less than 6 hr after administration and a slight increase was observed in the presence of cilastatin at 8 and 10 hr (Figure 3d; Table S2). These findings suggested that cilastatin increased the plasma concentration of diclofenac and a possible mechanism was the inhibition of the tissue distribution of diclofenac to the kidney and liver. Reduced renal exposure of diclofenac via combination with cilastatin might consequently contribute to low renal toxicity.

Figure 3.

Figure 3

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Effects of cilastatin on the plasma concentration and tissue distribution of diclofenac in mice. Diclofenac (200 mg·kg−1) was orally administered to mice with or without cilastatin (100 mg·kg−1). The plasma (a), kidney (b), liver (c), and intestine (d) were collected for the determination of diclofenac by LC‐MS/MS. *P < .05 versus diclofenac group. Data are expressed as mean ± SEM, n = 5

Table 1.

Pharmacokinetic parameters of diclofenac after oral administration of diclofenac (200 mg·kg−1) with or without cilastatin (100 mg·kg−1) in mice

Parameter Unit Diclofenac Diclofenac + Cilastatin
Plasma
T max1 hr 0.90 ± 0.22 1.00 ± 0.37
C max1 μg·ml−1 55.0 ± 9.99 142 ± 32.3a
T max2 hr 4.40 ± 0.69 7.60 ± 0.73a
C max2 μg·ml−1 50.6 ± 11.6 36.8 ± 1.55
CLz/F L·hr−1·kg−1 0.769 ± 0.155 0.286 ± 0.056a
t 1/2z hr 1.78 ± 0.155 6.16 ± 0.889a
AUC(0–12 hr) μg·ml−1·hr−1 246 ± 47.6 333 ± 68.0
AUC(0–∞) μg·ml−1·hr−1 315 ± 71.2 603 ± 43.1a
Kidney
T max1 hr 0.25 ± 0.08 0.87 ± 0.41
C max1 μg·g−1 44.1 ± 5.39 20.3 ± 3.87a
T max2 hr 2.60 ± 0.30 9.20 ± 0.36a
C max2 μg·g−1 35.8 ± 4.20 14.1 ± 3.85a
AUC(0–12 hr) μg·g−1·hr−1 151 ± 93.2 101 ± 37.3a
AUC(0–∞) μg·g−1·hr−1 184 ± 57.8 222 ± 82.6
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a

P < .05 versus diclofenac‐only group. Data are expressed as mean ± SEM, n = 5.

3.3. Effect of cilastatin on the cytotoxicity of diclofenac in renal primary proximal tubule cells

To further reveal the molecular mechanism underlying the renal interaction between diclofenac and cilastatin, the relationship between cytotoxicity and the intracellular accumulation of diclofenac was evaluated in the absence or presence of cilastatin using renal primary proximal tubule cells. Effect of cilastatin (100 μM) on the cytotoxicity of diclofenac (0‐2,000 μM) was first evaluated by CCK‐8 assays. After incubation for 24 hr, diclofenac decreased the survival rates of renal primary proximal tubule cells in a concentration‐dependent manner with an IC50 value of 420 ± 143 μM (Figure 4a), suggesting direct cytotoxicity towards renal primary proximal tubule cells. In the presence of cilastatin, the survival rates of renal primary proximal tubule cells treated with cilastatin were significantly increased, and the IC50 value increased to 908 ± 143 μM (Figure 4a), indicating that cilastatin protected against diclofenac‐induced cytotoxicity. As mentioned above, diclofenac induced apoptosis via the mitochondria‐dependent pathway in mouse kidneys. The mitochondrial membrane potential of renal primary proximal tubule cells was then determined through JC‐1 staining assays after incubation with diclofenac (400 μM) in the absence or presence of cilastatin (100 μM) for 24 hr. Diclofenac treatment induced scattered bright green fluorescence after JC‐1 staining, which was significantly reduced by co‐incubation with cilastatin (Figure 4b,c). The results suggested that cilastatin reduced diclofenac‐induced mitochondrial damage and consequently protected against the cytotoxicity of diclofenac in renal primary proximal tubule cells. To clarify whether the protective effect of cilastatin was due to alteration of the intracellular exposure of diclofenac, the intracellular accumulation of diclofenac was determined by LC‐MS/MS after incubation with diclofenac for 10 min under different conditions. Disappointingly, the intracellular accumulation of diclofenac was similar regardless of whether the renal primary proximal tubule cells were incubated with diclofenac at 37°C or 4°C (Figure 4d), indicating that the transmembrane transport of diclofenac might depend mainly on passive diffusion rather than active transport mediated by a transporter. Indeed, neither cilastatin nor probenecid, inhibitors of OATs, changed the intracellular exposure of diclofenac in renal primary proximal tubule cells, which ruled out OATs‐mediated drug‐drug interactions between diclofenac and cilastatin. The findings suggested that cilastatin reduced diclofenac‐induced cytotoxicity but had no effect on the intracellular accumulation of diclofenac.

Figure 4.

Figure 4

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Effects of cilastatin on the cytotoxicity and intracellular accumulation of diclofenac in mouse renal proximal tubular cells (RPTCs). (a) RPTCs were incubated with diclofenac (0‐2,000 μM) in the absence or presence of cilastatin (100 μM) for 24 hr, and cell survival was determined by CCK‐8 assays. (b) RPTCs were incubated with diclofenac (400 μM) in the absence or presence of cilastatin (100 μM) for 24 hr, and mitochondrial membrane potential was evaluated by JC‐1 staining assays. (c) Quantitative analysis of J‐monomers fluorescence intensity among the groups. *P < .05 versus control group. # P < .05 versus diclofenac group. (d) Diclofenac (10 μM) uptake by RPTCs at 37°C or 4°C with or without probenecid (200 μM) and cilastatin (100 μM) for 10 min, and intracellular accumulation of diclofenac was determined by LC‐MS/MS. Data are expressed as mean ± SEM, n = 6

3.4. Roles of OAT1/3 in drug‐drug interactions between cilastatin and diclofenac in vitro

To further clarify the effect of OATs on the protective role of cilastatin against diclofenac cytotoxicity, cell viability was assessed by CCK‐8 assays again after incubation of diclofenac for 24 hr in the absence or presence of cilastatin using hOAT1/3‐transfected cells instead. When incubated with diclofenac alone, the survival rate of hOAT1/3‐HEK293 cells was slightly lower and the IC50 values of diclofenac in hOAT1‐HEK293 cells and hOAT3‐HEK293 cells were decreased by 13.2% and 16.4%, respectively, compared with that in mock‐HEK293 cells (Figure 5a‐c; Table S3). Meanwhile, co‐incubation with cilastatin slightly decreased diclofenac cytotoxicity and caused an 18.1‐23.2% increase in IC50 values (Figure 5a‐c; Table S3). Furthermore, mock cells and hOAT1/3‐transfected cells showed similar intracellular accumulation levels of diclofenac and OATs inhibitors cilastatin and probenecid did not change the intracellular exposure of diclofenac in hOAT1/3‐transfected cells (Figure 5d). The findings proved that diclofenac was not an OATs substrate and OATs had little effect on the protective role of cilastatin against diclofenac cytotoxicity in vitro.

Figure 5.

Figure 5

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Effects of OAT1/3 and cilastatin on the cytotoxicity and intracellular accumulation of diclofenac in HEK293 cells. Mock‐ (a), hOAT1‐ (b), and hOAT3‐HEK293 cells (c) were incubated with diclofenac (0‐1,000 μM) in the absence or presence of cilastatin (100 μM) for 24 hr and cell survival was determined by CCK‐8 assays. (d) Diclofenac (10 μM) uptake by mock‐, hOAT1‐ and hOAT3‐HEK293 cells at 37°C with or without probenecid (200 μM) and cilastatin (100 μM) for 10 min, and intracellular accumulation of diclofenac was determined by LC‐MS/MS. Data are expressed as mean ± SEM, n = 6

Considering the high exposure level and toxicity risk in humans, diclofenac acyl glucuronide, the major metabolite of diclofenac in vivo, was assessed to evaluate the potential drug‐drug interaction induced by OATs between cilastatin and diclofenac acyl glucuronide. Similarly, cytotoxicity and the intracellular accumulation of diclofenac acyl glucuronide were evaluated in the absence or presence of cilastatin using hOAT1/3‐transfected cells. After incubation of diclofenac acyl glucuronide for 24 hr, the survival rate of hOAT1/3‐HEK293 cells was significantly lower than that in mock‐HEK293 cells, and the IC50 values of diclofenac acyl glucuronide in hOAT1/3‐HEK293 cells were only approximately half of that in mock‐HEK293 cells (Figure 6a‐c). Meanwhile, co‐incubation with cilastatin markedly decreased diclofenac acyl glucuronide cytotoxicity, and the IC50 values of diclofenac acyl glucuronide in Mock‐, hOAT1‐, and hOAT3‐HEK293 cells were increased by 1.37‐, 1.82‐ and 2.78‐fold by cilastatin, respectively (Figure 6a‐c, Table S4). The results suggested that diclofenac acyl glucuronide exhibited OAT1/3‐dependent cytotoxicity and OAT1/3 inhibition reduced the cytotoxicity induced by diclofenac acyl glucuronide. Furthermore, hOAT1‐ and hOAT3‐HEK293 cells showed an elevated intracellular concentration of diclofenac acyl glucuronide relative to mock‐HEK293 cells (Figure 6d). Diclofenac acyl glucuronide uptake by hOAT1/3‐HEK293 cells was significantly inhibited in the presence of OATs inhibitors cilastatin and probenecid (Figure 6d). Indeed, diclofenac acyl glucuronide uptake by hOAT1/3‐HEK293 cells was inhibited by cilastatin in a concentration‐dependent manner with IC50 values comparable to the clinical plasma concentration of cilastatin (Figure S2). To clarify the inhibition modes of cilastatin, concentration‐dependent uptake of diclofenac acyl glucuronide by hOAT1/3‐HEK293 cells was examined, and Eadie‐Hofstee plot analysis was conducted. K M values for diclofenac acyl glucuronide uptake by hOAT1/3‐HEK293 cells were significantly increased in the presence of cilastatin while V max values were not changed (Figure 6d,e, Table S5), suggesting that cilastatin competitively inhibited diclofenac acyl glucuronide uptake by OAT1/3. The findings proved that diclofenac acyl glucuronide was an OAT1/3 substrate and cilastatin inhibited OAT1/3‐mediated intracellular accumulation of diclofenac acyl glucuronide and consequently protected against diclofenac acyl glucuronide cytotoxicity in vitro.

Figure 6.

Figure 6

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Effects of OAT1/3 and cilastatin on the cytotoxicity and intracellular accumulation of diclofenac acyl glucuronide (DLF‐AG) in HEK293 cells. Mock‐ (a), hOAT1‐ (b), and hOAT3‐HEK293 cells (c) were incubated with diclofenac acyl glucuronide (DLF‐AG; 0–1,000 μM) in the absence or presence of cilastatin (100 μM) for 24 hr and cell survival was determined by CCK‐8 assays. (d) DLF‐AG (10 μM) uptake by mock‐, hOAT1‐ and hOAT3‐HEK293 cells at 37°C with or without probenecid (200 μM) and cilastatin (100 μM) for 10 min, and intracellular accumulation of DLF‐AG was determined by LC–MS/MS. (e) Concentration‐dependent uptake of DLF‐AG (5–400 μM) by hOAT1‐HEK293 cells at 37°C for 10 min with or without cilastatin (50 μM). Insets: Eadie–Hofstee plots. (f) Concentration‐dependent uptake of DLF‐AG (10–400 μM) by hOAT1‐ and hOAT3‐HEK293 cells at 37°C for 10 min with or without cilastatin (20 μM). Insets: Eadie–Hofstee plots. Data are expressed as mean ± SEM, n = 6

3.5. Effects of cilastatin on the cytotoxicity and exposure of diclofenac acyl glucuronide in renal primary proximal tubule cells and in mice

To finally clarify the role of OATs in the effect of cilastatin on the nephrotoxicity and pharmacokinetics of diclofenac, the cytotoxicity and exposure of diclofenac acyl glucuronide in renal primary proximal tubule cells and in mice were evaluated in the presence or absence of cilastatin. First, cell viability of renal primary proximal tubule cells was assessed by CCK‐8 assays after incubation with diclofenac acyl glucuronide for 24 hr. Like diclofenac, diclofenac acyl glucuronide decreased the survival rates of renal primary proximal tubule cells in a concentration‐dependent manner with an IC50 value of 372 ± 52.9 μM, which was increased to 764 ± 90.7 μM by cilastatin (Figure 7a), indicating that cilastatin protected against diclofenac‐induced cytotoxicity. Intracellular accumulation of diclofenac acyl glucuronide was then determined to reveal the role of OATs in diclofenac acyl glucuronide cytotoxicity. Compared with the uptake under 37°C, diclofenac acyl glucuronide uptake by renal primary proximal tubule cells was significantly abolished when renal primary proximal tubule cells were incubated at 4°C (Figure 7b). The temperature‐dependent uptake suggested that an active transport system may facilitate the transmembrane transport of diclofenac acyl glucuronide into renal primary proximal tubule cells. Additionally, diclofenac acyl glucuronide uptake was inhibited by probenecid and cilastatin (Figure 7b), indicating that renal OATs were involved in the transport of diclofenac acyl glucuronide and cilastatin inhibited OATs and reduced renal exposure of diclofenac acyl glucuronide, which might contribute to its renal protection activity.

Figure 7.

Figure 7

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Effects of cilastatin on the cytotoxicity and exposure of diclofenac acyl glucuronide (DLF‐AG) in renal primary proximal tubule cells (RPTCs) and in mice. (a) RPTCs were incubated with DLF‐AG (0–1,000 μM) in the absence or presence of cilastatin (100 μM) for 24 hr, and cell survival was determined by CCK‐8 assays. (b) DLF‐AG (10 μM) uptake by RPTCs at 37°C or 4°C with or without probenecid (200 μM) and cilastatin (100 μM) for 10 min, and intracellular accumulation of DLF‐AG was determined by LC–MS/MS. (c, d) Diclofenac (200 mg·kg−1) was orally administered to mice with or without cilastatin (100 mg·kg−1). The plasma (c) and kidneys (d) were collected for the determination of DLF‐AG by LC–MS/MS. *P < .05 versus diclofenac group. Data are expressed as mean ± SEM, n = 5

Furthermore, the effect of cilastatin on diclofenac acyl glucuronide pharmacokinetics was evaluated in vitro and in mice. Cilastatin (100–1,000 μM) did not change the formation of diclofenac acyl glucuronide in vitro (Figure S3), while cilastatin increased the plasma concentration and decreased the renal distribution of diclofenac acyl glucuronide in mice (Figure 7c,d). The AUC(0–12 hr) of diclofenac acyl glucuronide in plasma was increased by 46.7%, while the AUC(0–12 hr) in the kidney was slightly decreased by 18.0% in the presence of cilastatin (Table 2). These findings suggested that cilastatin inhibited the renal accumulation of diclofenac acyl glucuronide and consequently reduced the kidney injury induced by diclofenac acyl glucuronide.

Table 2.

Pharmacokinetic parameters of diclofenac acyl glucuronide (DLF‐AG) after oral administration of diclofenac (200 mg·kg−1) with or without cilastatin (100 mg·kg−1) in mice

Parameter Unit Diclofenac Diclofenac + Cilastatin
Plasma
T max1 hr 0.75 ± 0.24 1.10 ± 0.36
C max1 μg·ml−1 60.4 ± 13.1 74.0 ± 16.6
T max2 hr 3.60 ± 0.90 3.00 ± 0.24
C max2 μg·ml−1 47.1 ± 7.35 66.0 ± 5.33a
CLz/F L·hr−1·kg−1 0.644 ± 0.111 0.310 ± 0.044a
t 1/2z hr 1.78 ± 0.330 4.35 ± 0.482a
AUC(0–12 hr) μg·ml−1·hr−1 259 ± 25.1 380 ± 29.6a
AUC(0–12 hr) μg·ml−1·hr−1 348 ± 55.8 691 ± 83.6a
Kidney
T max1 hr 0.17 ± 0.06 0.37 ± 0.06a
C max1 μg·g−1 45.0 ± 13.5 21.6 ± 5.70
T max2 hr 2.10 ± 0.41 7.40 ± 1.19a
C max2 μg·g−1 38.3 ± 4.12 22.9 ± 6.20a
AUC(0–12 hr) μg·g−1·hr−1 178 ± 61.7 146 ± 52.7
AUC(0–12 hr) μg·ml−1·hr−1 207 ± 66.8 364 ± 105
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a

P < .05 versus diclofenac‐only group. Data are expressed as mean ± SEM, n = 5.

4. DISCUSSION

Adverse drug events, including gastrointestinal, cardiovascular and renal complications are the main limitation of clinical use of diclofenac (Coxib, traditional NTC, et al., 2013; Douros et al., 2018; McGettigan & Henry, 2013). The kidney is the main organ responsible for the elimination of diclofenac in humans (Davies & Anderson, 1997). The present study focused on the nephrotoxicity of diclofenac and aimed to clarify the pharmacokinetic mechanism of cilastatin in diclofenac‐induced acute kidney injury in mice. We found that cilastatin alleviated kidney injury and tubular cytotoxicity induced by diclofenac in mice and in renal primary proximal tubule cells through inhibition of oxidative stress, inflammation, and apoptosis. Co‐administration of cilastatin changed the pharmacokinetic of diclofenac and decreased the renal accumulation of diclofenac and its main metabolite diclofenac acyl glucuronide, both of which exhibited cytotoxicity to renal primary proximal tubule cells. diclofenac acyl glucuronide, but not diclofenac, was a substrate of OATs. The intracellular accumulation and renal transport of diclofenac acyl glucuronide mediated by OATs contributed to its cytotoxicity and nephrotoxicity. Inhibition of OATs by cilastatin reduced renal exposure of diclofenac acyl glucuronide and consequently protected against diclofenac‐induced renal injury in vitro and in vivo. Cilastatin could be used as a potential therapeutic drug candidate to reduce the nephrotoxicity of diclofenac.

4.1. Protective effect of cilastatin against diclofenac‐induced kidney injury

Cilastatin is a promising agent for alleviating various kinds of drug‐induced nephrotoxicity. Originally, cilastatin was found to inhibit imipenem‐induced acute tubular necrosis in rabbits (Birnbaum, Kahan, Kropp, & MacDonald, 1985). Then, large clinical trials proved that cilastatin protected against cyclosporin A‐induced nephrotoxicity following clinical transplantation (Tejedor et al., 2007). Recently, the renoprotection effect of cilastatin was further proven in nephrotoxicity induced by vancomycin, cisplatin, colistin and aminoglycosides (Hori et al., 2017; Humanes et al., 2012). Cilastatin exhibited generally protective activity against various forms of drug‐induced nephrotoxicity, which reminded us that the co‐administration of cilastatin could have an impact on diclofenac‐induced kidney injury. Diclofenac is associated with an increased risk of serious dose‐dependent renal side effects (Altman et al., 2015; Douros et al., 2018). In the present study, a relatively high dose (approximately 10 times the human clinical dose) was used to induce acute kidney injury in mice as previously described (Bao et al., 2019; Fattori et al., 2017). Here, cilastatin was used at doses of 25–100 mg·kg−1, which was in line with the therapeutic doses commonly used in humans and calculated using a scaling factor of 12 on a mg·m−2 body surface area basis. Indeed, cilastatin treatment decreased pathological changes and renal dysfunction induced by diclofenac in mice (Figure 1). Moreover, diclofenac exerted direct cytotoxicity on tubular epithelial cells with an IC50 (Figure 4a) comparable to the C max of diclofenac in mice (Table 1). The kidney is the main organ responsible for the elimination of diclofenac. Approximately 65% of the dose of diclofenac is excreted in the urine as intact diclofenac and its metabolites (Altman et al., 2015). Although the IC50 was higher than the C max observed in humans, a higher local concentration in the kidney could be expected in the clinic due to the extensive excretion of diclofenac by the kidney. Additionally, cilastatin showed a protective effect against diclofenac cytotoxicity to renal primary proximal tubule cells in vitro (Figure 4a). These findings demonstrated that cilastatin also protected against the nephrotoxicity of diclofenac. In a previous study, cisplatin induced oxidative stress, inflammation and apoptosis, which could be inhibited by the co‐administration of cilastatin (Humanes et al., 2012; Humanes et al., 2017). In the present study, diclofenac also induced elevated levels of oxidation products, cytokine production and apoptosis in the kidney (Figures 1b and 2a), which was consistent with the results of a previous study (Fattori et al., 2017) and could be prevented by cilastatin treatment. In renal primary proximal tubule cells, cilastatin also decreased the cytotoxicity of diclofenac by ameliorating mitochondrial damage induced by diclofenac (Figure 4a). The results indicated that cilastatin alleviated diclofenac‐induced kidney injury by restoring the redox balance, suppressing inflammation and reducing apoptosis.

4.2. Pharmacokinetic mechanism underlying the protection effect of cilastatin on diclofenac nephrotoxicity

The kidney is one of the major elimination organs. According to a statistical analysis, 32% of the top 200 prescribed drugs in 2010 are cleared by renal mechanisms (Morrissey, Stocker, Wittwer, Xu, & Giacomini, 2013), which makes drugs that are primarily eliminated by the kidney a common cause of kidney injury. Drug‐induced acute kidney injury was reported to account for approximately 19–26% of cases of acute kidney injury in hospitalized patients (Markowitz, Bomback, & Perazella, 2015; Mehta et al., 2004). Indeed, reducing the renal accumulation of toxic substances is an important mechanism underlying the protective effect of cilastatin. It was reported that cilastatin inhibited the transport of vancomycin, cyclosporin A, cisplatin and imipenem into the kidney and thereby limited the nephrotoxicity of these drugs (Camano et al., 2010; Huo et al., 2019; Perez et al., 2004). Our results also found that cilastatin reduced the renal distribution of diclofenac and diclofenac acyl glucuronide (Figures 3b and 7d). To maximize the possibility of demonstrating an interaction, cilastatin was used at 100 mg·kg−1, which was calculated using the maximum approved dose in human. It was reported that plasma concentrations and the AUC of cilastatin were linearly related to the dose over the normal therapeutic range (0.25–1 g) in healthy subjects (Clissold, Todd, & Campoli‐Richards, 1987), which explained, at least in part, the dose‐dependent protective effect of cilastatin against diclofenac‐induced kidney injury in mice (Figure 1). As the adverse effects of diclofenac were dose‐dependent, low renal exposure would consequently contribute to the low renal toxicity after coadministration of cilastatin.

In particular, both diclofenac and diclofenac acyl glucuronide exhibited cytotoxicity to renal primary proximal tubule cells and HEK293 cells (Figures 4a and 7a; Figures 5 and 6). In fact, diclofenac acyl glucuronide was more cytotoxic than diclofenac in the present study and in the previous studies (Oda et al., 2017; Scialis & Manautou, 2016). Transporters act as a key determinant of intracellular levels of substrates. In our previous studies, cilastatin was found to decrease the in vivo renal elimination and in vitro intracellular accumulation of OATs substrate imipenem via OATs inhibition and alleviated its nephrotoxicity (Huo et al., 2019; Zhu et al., 2018). Therefore, the potential role of OATs in the intracellular accumulation of diclofenac and diclofenac acyl glucuronide was investigated. Interestingly, diclofenac and diclofenac acyl glucuronide showed different responses towards co‐incubation with cilastatin, and only diclofenac acyl glucuronide but not diclofenac was further identified as an OAT1/3 substrate (Figures 5d and 6d). In previous studies, the interaction between OATs and diclofenac was controversial. Diclofenac is generally used as an OATs inhibitor (Yu et al., 2017), although some researchers consider it an OATs substrate (Nieskens et al., 2016; Nieskens et al., 2018). None of OAT1‐4, or only OAT2, recognized diclofenac as a substrate, while diclofenac acyl glucuronide was a substrate of OAT1/3 (Khamdang et al., 2002; Zhang et al., 2016), which was consistent with our results. In the present study, the liver distribution of diclofenac was not increased but decreased after the co‐administration of cilastatin (Figure 3c), which might contribute to the lower liver toxicity induced by diclofenac. OAT2 is mainly expressed in the liver (Giacomini et al., 2010). The possible reason of decreased liver distribution might be the inhibition of OAT2 by cilastatin. Besides, several efflux transporters, including multidrug resistance proteins (MRPs) and breast cancer resistance protein (BCRP), were suggested to mediate the transport of diclofenac and diclofenac acyl glucuronide (Scialis, Aleksunes, Csanaky, Klaassen, & Manautou, 2019; Zhang et al., 2016). However, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1024 and novobiocin were found to significantly inhibited https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=768‐ and BCRP‐mediated efflux transport of rhodamine 123 and Hoechst 33342 respectively, while cilastatin did not affect the efflux transport of probe substrates (Figure S4). Moreover, cilastatin reduced intracellular accumulation of cisplatin, an MRP substrate, in rat renal primary proximal tubule cell in vitro and in rat kidney in vivo (Camano et al., 2010; Humanes et al., 2012), suggesting that cilastatin could not inhibit https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=780. Therefore, cilastatin might not interact with diclofenac and diclofenac acyl glucuronide through P‐gp, MRP2 and BCRP.

Given that diclofenac was not an OAT substrate, why did cilastatin decrease the renal distribution of diclofenac? We speculated that this phenomenon resulted from a drug–drug interaction between cilastatin and diclofenac acyl glucuronide mediated by OATs. First, diclofenac acyl glucuronide is unstable in plasma and undergoes conversion to parent diclofenac (Zhang et al., 2016). Second, diclofenac acyl glucuronide is the main metabolite, and its plasma concentration was comparable to that of diclofenac according to our results and a previous study (Zhang et al., 2016). It is possible for metabolites to induce a drug–drug interaction when systemic exposure exceeds 25% of parent drug exposure (Zamek‐Gliszczynski, Chu, Polli, Paine, & Galetin, 2014). Third, the concentrations of diclofenac and diclofenac acyl glucuronide in uptake studies using OAT1/3 cells and renal primary proximal tubule cells in vitro were set near the C max in the clinic (approximately 1.25–22.6 μM; Davies & Anderson, 1997). Additionally, the maximum clinical plasma concentrations of cilastatin are documented as 88 μg·ml−1 (Balfour et al., 1996; Norrby et al., 1983). Taking into account plasma protein binding of approximately 40%, the peak level of free cilastatin in human was calculated as approximately 140 μM, which was high enough to induce an in vivo drug–drug interaction mediated by OATs based on FDA criteria (C max/IC50 > 0.1; Feng & Varma, 2016). This means that in vivo interactions between cilastatin and diclofenac need a third participant, diclofenac acyl glucuronide, the major metabolite of diclofenac in vivo. Taken together, cilastatin decreased renal transport of diclofenac acyl glucuronide via OATs inhibition, which reduced the conversion to parent diclofenac in parallel (Figure 8). Here, OATs are considered as mediators of renal toxicity through the transport of toxic substances into nephrocytes. Moreover, it should be noted that the expression levels of Oat1/3 might be altered during diclofenac‐induced kidney injury. In our previous studies, Oat1/3 expression was down‐regulated in drug‐induced acute kidney injury but was improved upon treatment by agents with renal protective activity (Guo et al., 2013; Liu et al., 2014; Wen et al., 2018). From this perspective, OATs expression represents kidney function status and is always used as an index of renal function (Huo & Liu, 2018). In the present study, cilastatin was given 0.5 hr before the administration of diclofenac, which blocked the transport mediated by OATs through competitive inhibition in advance and avoided kidney injury and impaired OATs expression induced by high local exposure to diclofenac and diclofenac acyl glucuronide.

Figure 8.

Figure 8

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Schematic illustration of the protective effect of cilastatin against diclofenac‐induced acute kidney injury. Diclofenac is rapidly absorbed after oral administration and mainly present as diclofenac and diclofenac acyl glucuronide (DLF‐AG) in vivo. DLF‐AG is unstable in plasma and undergoes conversion to parent diclofenac. Both diclofenac and DLF‐AG contribute to nephrotoxicity by inducing inflammation, oxidative stress, and apoptosis. DLF‐AG is a substrate of OAT1/3. Cilastatin decreases the renal distribution of diclofenac and DLF‐AG by inhibiting OATs. Additionally, cilastatin alleviates diclofenac‐induced acute kidney injury in mice by restoring the redox balance, suppressing inflammation and reducing apoptosis

Of course, an OAT‐independent mechanism also underlies the protective effect of cilastatin. Recently, cilastatin was reported to interact with megalin to block the entry of toxic substrates into nephrocytes. Megalin is an endocytic receptor expressed at the apical membranes of proximal tubules, facilitating the transmembrane transport of vancomycin, cisplatin, colistin, and aminoglycosides and mediating their nephrotoxicity (Hori et al., 2017; Moestrup et al., 1995; Suzuki et al., 2013). Cilastatin can inhibit megalin‐mediated uptake of these toxic drugs, thereby limiting their nephrotoxicity (Hori et al., 2017). Moreover, intrinsic anti‐inflammatory, anti‐oxidative and anti‐apoptotic activities of cilastatin can explain its protective effect against diclofenac‐induced cytotoxicity in vitro (Figure 8). For diclofenac acyl glucuronide, multiple mechanisms can be involved in the protective effect of cilastatin. According to Figure 7, the IC50 value for mock‐HEK293 cells increased by 37% in the presence of cilastatin without OAT1/3, while IC50 values for OAT1/3‐HEK293 cells were increased by 78–117% compared with that of mock‐HEK293 cells, suggesting that OAT inhibition contributed more to the protective effect of cilastatin against the cytotoxicity of diclofenac acyl glucuronide. As a promising agent for renal protection, other mechanisms may be involved in the protective effect of cilastatin on diclofenac‐induced renal injury, which will be studied in future research.

5. CONCLUSION

Cilastatin alleviated diclofenac‐induced acute kidney injury in mice by restoring the redox balance, suppressing inflammation, and reducing apoptosis. Cilastatin inhibited renal OATs and decreased the renal distribution of diclofenac and diclofenac acyl glucuronide, which further ameliorated diclofenac‐induced nephrotoxicity in mice.

AUTHOR CONTRIBUTIONS

K.L. and X.H. designed the experiments. X.H. and Q.M. performed the experiments. X.H., C.W. (Changyuan Wang), C.W. (Chong Wang), and J.W. analysed the data. X.M. and H.S. prepared the figures. X.H. and K.L. wrote the main text. H.S. revised the manuscript. All authors reviewed the manuscript.

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 as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/abs/10.1111/bph.14206, and as recommended by funding agencies, publishers and other organisations engaged with supporting research.

Supporting information

Table S1. Parameters of mass spectrometry for analytes

Table S2. Pharmacokinetic parameters of diclofenac after oral administration of diclofenac (200 mg/kg) with or without cilastatin (100 mg/kg) in mice liver and intestine

Table S3. IC50 values of diclofenac in Mock‐, hOAT1‐, and hOAT3‐HEK293 with or without cilastatin (100 μM)

Table S4. IC50 values of DLF‐AG in Mock‐, hOAT1‐, and hOAT3‐HEK293 with or without cilastatin (100 μM)

Table S5. Km and Vmax values of DLF‐AG in hOAT1/hOAT3‐HEK293 cells in the presence and absence of cilastatin

Table S6. Pharmacokinetic parameters of DLF‐AG after oral administration of diclofenac (200 mg/kg) with or without cilastatin (100 mg/kg) in mice liver and intestine

Figure S1 Representative chromatograms of DLF‐AG (1), IS (2), and diclofenac (3)

Figure S2 Concentration‐dependent inhibition of cilastatin (0‐1000 μM) on DLF‐AG (10 μM) uptake by hOAT1‐ and hOAT3‐HEK293 cells at 37 oC for 10 min. Data are expressed as mean ± SEM, n = 6

Figure S3 Inhibition of diclofenac glucuronidation by PPt and cilastatin in HLM. Diclofenac (10 μM) was incubated in HLM (0.05 mg/mL) with UDPGA‐generating system at 37 oC for 30 min in the presence or absence of PPt (100 μM) and cilastatin (100‐1000 μM). The formation of DLF‐AG was determined by LC‐MS/MS. Data are expressed as mean ± SEM, n = 6. PPt, 20(S)‐protopanaxatriol. Cil, cilastatin

Figure S4 Effect of cilastatin on efflux transport by P‐gp and BCRP. MDR1‐ and BCRP‐MDCK cells were seeded on 24‐well transwell inserts and grown for 2‐3 days to from cell monolayers. For efflux assay, rhodamine 123 (10 μM) and Hoechst 33342 (100 μM), used as probe substrate for P‐gp and BCRP, respectively, were added to basolateral side of the monolayer with or without P‐gp inhibitor cyclosporin A (10 μM), BCRP inhibitor novobiocin (100 μM) and cilastatin (1000 μM). After indicated time, medium from apical side was sampled and fluorescence was measured at 350 nm for excitation and 465 nm for emission or at 485 nm for excitation and 535 nm for emission for Hoechst 33342 or rhodamine 123,respectively. Data are expressed as mean ± SEM, n = 6

Click here for additional data file. (339.4KB, pdf)

ACKNOWLEDGEMENTS

The work was supported by a grant from the National Natural Science Foundation of China (81874324, 81903706, and U1608283) and Dalian Science and Technology Innovation Fund, China (2018J12SN065). The authors thank Prof. Yuichi Sugiyama (Sugiyama Laboratory, RIKEN, Japan) for kindly providing Mock/hOAT1/3‐HEK293 cells.

Huo X, Meng Q, Wang C, et al. Protective effect of cilastatin against diclofenac‐induced nephrotoxicity through interaction with diclofenac acyl glucuronide via organic anion transporters. Br J Pharmacol. 2020;177:1933–1948. 10.1111/bph.14957

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Table S1. Parameters of mass spectrometry for analytes

Table S2. Pharmacokinetic parameters of diclofenac after oral administration of diclofenac (200 mg/kg) with or without cilastatin (100 mg/kg) in mice liver and intestine

Table S3. IC50 values of diclofenac in Mock‐, hOAT1‐, and hOAT3‐HEK293 with or without cilastatin (100 μM)

Table S4. IC50 values of DLF‐AG in Mock‐, hOAT1‐, and hOAT3‐HEK293 with or without cilastatin (100 μM)

Table S5. Km and Vmax values of DLF‐AG in hOAT1/hOAT3‐HEK293 cells in the presence and absence of cilastatin

Table S6. Pharmacokinetic parameters of DLF‐AG after oral administration of diclofenac (200 mg/kg) with or without cilastatin (100 mg/kg) in mice liver and intestine

Figure S1 Representative chromatograms of DLF‐AG (1), IS (2), and diclofenac (3)

Figure S2 Concentration‐dependent inhibition of cilastatin (0‐1000 μM) on DLF‐AG (10 μM) uptake by hOAT1‐ and hOAT3‐HEK293 cells at 37 oC for 10 min. Data are expressed as mean ± SEM, n = 6

Figure S3 Inhibition of diclofenac glucuronidation by PPt and cilastatin in HLM. Diclofenac (10 μM) was incubated in HLM (0.05 mg/mL) with UDPGA‐generating system at 37 oC for 30 min in the presence or absence of PPt (100 μM) and cilastatin (100‐1000 μM). The formation of DLF‐AG was determined by LC‐MS/MS. Data are expressed as mean ± SEM, n = 6. PPt, 20(S)‐protopanaxatriol. Cil, cilastatin

Figure S4 Effect of cilastatin on efflux transport by P‐gp and BCRP. MDR1‐ and BCRP‐MDCK cells were seeded on 24‐well transwell inserts and grown for 2‐3 days to from cell monolayers. For efflux assay, rhodamine 123 (10 μM) and Hoechst 33342 (100 μM), used as probe substrate for P‐gp and BCRP, respectively, were added to basolateral side of the monolayer with or without P‐gp inhibitor cyclosporin A (10 μM), BCRP inhibitor novobiocin (100 μM) and cilastatin (1000 μM). After indicated time, medium from apical side was sampled and fluorescence was measured at 350 nm for excitation and 465 nm for emission or at 485 nm for excitation and 535 nm for emission for Hoechst 33342 or rhodamine 123,respectively. Data are expressed as mean ± SEM, n = 6

Click here for additional data file. (339.4KB, pdf)

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