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

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.

Figure 1

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

Figure 2

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

Figure 3

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

Figure 4

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

Figure 5

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

Figure 6

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

Figure 7

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

Figure 8

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