Anti-inflammatory and renoprotective effects of difelikefalin, a kappa opioid receptor agonist, in a rat model of renal ischemia-reperfusion-induced acute kidney injury

Abstract

Background: This study evaluated the effects of difelikefalin, a kappa opioid receptor (KOR) agonist, on the inflammatory response and renal dysfunction in a rat model of acute kidney injury induced by renal ischemia-reperfusion injury, comparing it with other KOR agonists.

Methods: One week after right nephrectomy, rats received one of three drugs (difelikefalin, nalfurafine hydrochloride, or U-50488H). Thirty minutes later, ischemia was induced in the left kidney by clamping its artery and vein for 45 min. Rats were then transferred to metabolic cages for 24-h urine collection, and blood was taken at the 24-h time point. Renal function (blood urea nitrogen [BUN], serum creatinine [SCr], and creatinine clearance [CCr]) and 23 serum cytokines were assessed, along with histopathological evaluation of renal tubular damage.

Results: Compared with the sham group, the vehicle group showed increased BUN and SCr levels, reduced CCr, and renal tubular damage. Difelikefalin improved BUN, SCr, and CCr levels and mitigated renal tubular damage. Similar effects were observed with nalfurafine hydrochloride and U-50488H. Thirteen serum cytokines were elevated in the vehicle group. Difelikefalin suppressed all elevated cytokines, U-50488H showed moderate efficacy, and nalfurafine hydrochloride exhibited little inhibitory effect.

Conclusion: These findings indicate that difelikefalin has anti-inflammatory and renoprotective effects in a rat acute kidney injury model, with varying anti-inflammatory efficacy among KOR agonists.

Keywords: Cytokine; Difelikefalin; Kappa opioid receptor; Nalfurafine hydrochloride; Renal ischemia–reperfusion injury.

Fig. 1

Scheme of the experiment. I/R: ischemia–reperfusion, nor-BNI: nor-binaltorphimine, i.v.: intravenously, p.o.: per os, i.p.: intraperitoneally

Fig. 2

Effects of difelikefalin, nalfurafine hydrochloride, and U-50488H on biochemical parameters. A BUN, B SCr, and C CCr were measured or calculated 24 h after surgical establishment of renal I/R injury. In addition to the effects of KOR agonists, the inhibitory effect of the KOR antagonist nor-BNI on the effect of difelikefalin was evaluated. nor-BNI (3 mg/kg, i.p.) was administered 30 min before difelikefalin (0.01 mg/kg, i.v.). Each column and bar represents the mean ± standard error of the mean in 12 rats (nor-BNI + difelikefalin group: 9 rats). ^###P < 0.001 compared with sham group (Student’s t-test or Aspin–Welch t-test). *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle group (Dunnett’s test or Steel’s test). ^$P < 0.05, ^$$P < 0.01 compared with difelikefalin group (0.01 mg/kg) (Student’s t-test or Aspin–Welch t-test). BUN: blood urea nitrogen, SCr: serum creatinine, CCr: creatinine clearance, nor-BNI: nor-binaltorphimine

Fig. 3

Representative images of renal tissues with periodic acid–Schiff staining. Left kidneys extracted 1 day after the I/R procedure in rats were microscopically observed. A Sham group: normal tubular epithelial cells. B Vehicle group: severe necrosis and loss of the brush border of tubular epithelial cells with moderate luminal obstruction (black arrows). C Difelikefalin (0.01 mg/kg, i.v.) group: slight necrosis and loss of the brush border of the tubular epithelial cells with moderate luminal obstruction (red arrows). D Nalfurafine hydrochloride (1 mg/kg, p.o.) group: severe necrosis and loss of the brush border of tubular epithelial cells with slight luminal obstruction (blue arrows). E U-50488H (1 mg/kg, i.v.) group: slight necrosis and loss of the brush border of tubular epithelial cells with moderate luminal obstruction (green arrows). Scale bar = 50 μm

Fig. 4

Effects of difelikefalin, nalfurafine hydrochloride, and U-50488H on renal tubular injury. Histopathological scoring of renal tubular injury of kidneys extracted 1 day after I/R procedure. See Methods for scoring criteria. Each column and bar represents the mean ± standard error of the mean in 12 rats. ^###P < 0.001 compared with sham group (Aspin–Welch t-test). *P < 0.05, **P < 0.01, ***P < 0.001 compared with vehicle group (Steel’s test)

Fig. 5

Effects of difelikefalin, nalfurafine hydrochloride, and U-50488H on serum cytokines. Cytokines in rat serum collected 24 h after surgical establishment of renal I/R injury were measured. A TNF-α, B IL-6, C IL-1α, D IFN-γ, E IL-2, F IL-4, G IL-5, H IL-10, I IL-13, J IL-17, K G-CSF, L M-CSF, and M MIP-3α were higher in the serum of the vehicle group than sham group. In each bar chart, the concentrations of serum cytokines in the drug treatment group were compared with those in the vehicle group. Each column and bar represents the mean ± standard error of the mean in 12 rats. S: sham, V: vehicle, Nal: nalfurafine hydrochloride, U: U-50488H. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 compared with sham group (Student’s t-test). *P < 0.05, **P < 0.01 compared with vehicle group (Dunnett’s test or Steel’s test). TNF: tumor necrosis factor, IL: interleukin, IFN: interferon, G-CSF: granulocyte colony-stimulating factor, M-CSF: macrophage colony-stimulating factor, MIP: macrophage inflammatory protein