Cilastatin protects against imipenem-induced nephrotoxicity via inhibition of renal organic anion transporters (OATs)

Abstract

Imipenem is a carbapenem antibiotic. However, Imipenem could not be marketed owing to its instability and nephrotoxicity until cilastatin, an inhibitor of renal dehydropeptidase-I (DHP-I), was developed. In present study, the potential roles of renal organic anion transporters (OATs) in alleviating the nephrotoxicity of imipenem by cilastatin were investigated in vitro and in rabbits. Our results indicated that imipenem and cilastatin were substrates of hOAT1 and hOAT3. Cilastatin inhibited hOAT1/3-mediated transport of imipenem with IC₅₀ values comparable to the clinical concentration, suggesting the potential to cause a clinical drug-drug interaction (DDI). Moreover, imipenem exhibited hOAT1/3-dependent cytotoxicity, which was alleviated by cilastatin and probenecid. Furthermore, cilastatin and probenecid ameliorated imipenem-induced rabbit acute kidney injury, and reduced the renal secretion of imipenem. Cilastatin and probenecid inhibited intracellular accumulation of imipenem and sequentially decreased the nephrocyte toxicity in rabbit primary proximal tubule cells. Renal OATs, besides DHP-I, was also the target of interaction between imipenem and cilastatin, and contributed to the nephrotoxicity of imipenem. This therefore gives in part the explanation about the mechanism by which cilastatin protected against imipenem-induced nephrotoxicity. Thus, OATs can potentially be used as a therapeutic target to avoid the renal adverse reaction of imipenem in clinic.

Keywords: BUN, blood urea nitrogen; CKD, chronic kidney disease; CLp, plasma clearance; CLr, renal clearance; CRE, creatinine; Cil, cilastatin; Cilastatin; DDIs, drug-drug interactions; DHP-I, renal dehydropeptidase-I; ES, estrone-3-sulfate; GSH, glutathione; Imipenem; Imp, imipenem; MDA, malonaldehyde; Nephrotoxicity; OATs; OATs, renal organic anion transporters; PAH, p-aminophenol acid; Prb, probenecid; Probenecid; SNP, single nucleotide polymorphism; hOAT, human OAT; hOAT1; hOAT3; rOAT, rat OAT; rPTCs, rabbit primary proximal tubule cells; raOAT, rabbit OAT; t1/2, half life.

Graphical abstract

Figure 1

hOAT1/3-induced interaction between Imp and Cil in Mock-, hOAT1-, and hOAT3-HEK293 cells. (A) Uptake of Imp (100 μmol/L) with or without Prb (200 μmol/L) for 10 min. (B) Uptake of Cil (100 μmol/L) with or without Prb (200 μmol/L) for 10 min. ^*P < 0.05 vs Mock cells; ^#P < 0.05 vs control group. (C) PAH (10 μmol/L) and ES (10 μmol/L) uptake with or without Prb (200 μmol/L), Imp (100 μmol/L) and Cil (200 μmol/L) for 10 min. (D) Concentration-dependent inhibition of Cil (0–800 μmol/L) on Imp (100 μmol/L) uptake ^*P < 0.05 vs PAH or ES alone group. Data are expressed as mean±SEM, n = 3.

Figure 2

Effects of Cil and Prb on the cytotoxicity of Imp on Mock-, hOAT1/3-HEK293 cells. (A) Cytotoxicity of Imp (0–1 mmol/L) in Mock-HEK293 cells in the absence or presence of Prb (200 μmol/L) and Cil (200 μmol/L). (B) Cytotoxicity of Imp (0–1 mmol/L) in hOAT1-HEK293 cells in the absence or presence of Prb (200 μmol/L) and Cil (200 μmol/L). (C) Cytotoxicity of Imp (0–1 mmol/L) in hOAT3-HEK293 cells in the absence or presence of Prb (200 μmol/L) and Cil (200 μmol/L). ^*P < 0.05, Imp+Prb group vs Imp alone group. ^#P < 0.05, Imp+Cil group vs Mock cells. Data are expressed as mean±SEM, n = 3.

Figure 3

Protective effect of Cil and Prb on renal histology in Imp-treated rabbits. Imp (200 mg/kg) was injected intravenously (i.v.) through the ear vein to rabbits with or without Cil (200 mg/kg) or Prb (50 mg/kg). Kidney was collected at 48 h after administration of Imp for Gross observation (A), HE staining (B) and PAS staining (C). Renal tubular epithelial cell swelling (black arrows), tubule dilatation (red arrows) and necrosis and shedding of cell (green arrows) were indicated in representative images of HE staining. Glomerular atrophy and matrix increase were indicated by black arrows in PAS staining. Bars = 100 μm at 200×; bars = 50 μm at 400×. Data are expressed as mean±SEM, n = 3.

Figure 4

Effects of Cil and Prb on Imp-induced nephrotoxicity in rabbits. Imp (200 mg/kg) was injected intravenously (i.v.) through the ear vein to rabbits with or without Cil (200 mg/kg) or Prb (50 mg/kg). Urine and blood were collected at 0, 24 and 48 h after administration of Imp for the determination of urine protein, CRE and BUN. ^*P < 0.05 vs control group. ^#P < 0.05 vs Imp group. Data are expressed as mean±SEM, n = 3.

Figure 5

Effects of Cil and Prb on the plasma concentration and urinary excretion of Imp in rabbits. Imp (200 mg/kg) was injected intravenously (i.v.) through the ear vein to rabbits with or without Cil (200 mg/kg) or Prb (50 mg/kg). Plasma and urine were collected for the determination of Imp and Imp-M by LC–MS/MS. ^*P < 0.05, Imp+Cil vs Imp group; ^#P < 0.05, Imp+Prb vs Imp group. Data are expressed as mean±SEM, n = 3.

Figure 6

Effects of Cil and Prb on the Intracellular accumulation of Imp in rPTCs. (A) PAH (10 μmol/L) and ES (10 μmol/L) uptake by rPTCs at 37 or 4 °C with or without Prb (200 μmol/L) and Cil (200 μmol/L) for 10 min. (B) Imp (100 μmol/L) uptake by rPTCs at 37 or 4 °C with or without Prb (200 μmol/L) and Cil (200 μmol/L) for 10 min. (C) PAH (10 μmol/L) and ES (10 μmol/L) uptake by rPTCs transfected with siRNA of DHP-I (si-DHP-I) at 37 or 4 °C with or without Prb (200 μmol/L) and Cil (200 μmol/L) for 10 min. (D) Imp (100 μmol/L) uptake by rPTCs transfected with si-DHP-I at 37 or 4 °C with or without Prb (200 μmol/L) and Cil (200 μmol/L) for 10 min. (E) Concentration-dependent inhibition of Cil (0–800 μmol/L) on Imp (100 μmol/L) uptake by rPTCs transfected with si-DHP-I at 37 °C for 10 min. IC₅₀ was determined after the deduction of uptake at 4 °C. ^*P < 0.05 vs control or si-DHP-I group. Data are expressed as mean±SEM, n = 3.

Figure 7

Effects of Cil and Prb on the cytotoxicity of Imp in rPTCs. (A) Cytotoxicity of Imp in rPTCs in the absence or presence of Prb (200 μmol/L) and Cil (200 μmol/L). ^*P < 0.05 vs control. (B) Cytotoxicity of Imp in rPTCs transfected with si-DHP-I in the absence or presence of Prb (200 μmol/L) and Cil (200 μmol/L). ^*P < 0.05 vs si-DHP-I group. Data are expressed as mean±SEM, n = 3.