Dexmedetomidine Preconditioning Protects Rats from Renal Ischemia-Reperfusion Injury Accompanied with Biphasic Changes of Nuclear Factor-Kappa B Signaling

Abstract

Acute kidney injury (AKI) is one of the most common and troublesome perioperative complications. Dexmedetomidine (DEX) is a potent α2-adrenoceptor (α2-AR) agonist with anti-inflammatory and renoprotective effects. In this study, a rat renal ischemia-reperfusion injury (IRI) model was induced. At 24 h after reperfusion, the IRI-induced damage and the renoprotection of DEX preconditioning were confirmed both biochemically and histologically. Changes in nuclear factor-kappa B (NF-κB), as well as its downstream anti-inflammatory factor A20 and proinflammatory factor tumor necrosis factor-α (TNF-α), were detected. Atipamezole, a nonselective antagonist, was then added 5 min before the administration of DEX to further analyze DEX's effects on NF-κB, and another anti-inflammatory medicine, methylprednisolone, was used in comparison with DEX, to further analyze DEX's effects on NF-κB. Different concentrations of DEX (0 nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, and 10 μM) were applied to preincubated human renal tubular epithelial cell line (HK-2) cells in vitro. After anoxia and reoxygenation, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium assay and enzyme-linked immunosorbent assay (ELISA) were performed to evaluate the levels of NF-κB downstream anti-inflammatory cytokines. The results showed that, unlike methylprednisolone, DEX preconditioning led to a time-dependent biphasic change (first activation then inhibition) of NF-κB in the rat renal IRI models that were given 25 μg/kg i.p. It was accompanied by a similarly biphasic change of TNF-α and an early and persistent upregulation of A20. In vitro, DEX's cellular protection showed a concentration-dependent biphasic change which was protective within the range of 0 to 100 nM but became opposite when concentrations are greater than 1 μM. The changes in the A20 and NF-κB messenger RNA (mRNA) levels were consistent with the renoprotective ability of DEX. In other words, DEX preconditioning protected the rats from renal IRI via regulation biphasic change of NF-κB signaling.

Figure 1

Experimental groups and protocol. Schematic diagram of the six groups of rats exposed to the experimental treatments.

Figure 2

Histological tests showed that dexmedetomidine (DEX) preconditioning provided renoprotective effects. The rats underwent left renal resection and right renal artery clamping for 45 min (renal ischemia–reperfusion injury (IRI)) or just dissection of the bilateral renal pedicles without occlusion (sham). Either DEX or methylprednisolone was given 30 min before ischemia. Representative microphotographs were taken from the following groups: (a) sham, (b) IRI, (c) DEX (25 μ/kg)+IRI, (d) atipamezole (250 μ/kg)+DEX+IRI, (e) DEX alone, and (f) methylprednisolone (30 mg/kg)+IRI. (g) Quantification of the histological scoring following IRI in rats preconditioned as above. The histological damage was indicated by red arrows. Bar = 50 μm. Data are mean ± standard deviation (SD; n = 5). ^∗p < 0.05, ^∗∗p < 0.01 vs. S; ^#p < 0.05, ^##p < 0.01 vs. I.

Figure 3

(a) Nuclear factor-kappa B (NF-κB) signaling changes after kidney IRI and DEX preconditioning. (b) DEX preconditioning led to a time-dependent biphasic change of NF-κB of the early activation (within 3 h of IRI) and later inhibition (6 h after IRI) of NF-κB signaling in the rat renal IRI models that were given 25 μg/kg i.p. (c) It was accompanied by the early (<3 h) upregulation and later (>6 h) significant inhibition of TNF-α and (d) the persistent upregulation of A20. The data are the mean ± standard deviation (SD; n = 5). ^∗p < 0.05, ^∗∗p < 0.01 vs. S; ^#p < 0.05, ^##p < 0.01 vs. I.

Figure 4

(a) Comparison of nuclear factor-kappa B (NF-κB) signaling in the groups after 1 hour of reperfusion. Dexmedetomidine (DEX) preconditioning caused the significant activation of (b) NF-κB and the upregulation of (c) tumor necrosis factor-α (TNF-α) and (d) A20 expression. This was completely reversed by atipamezole. Only DEX without IRI also increased the nuclear translocation of NF-κB and mildly increased A20. The data are the mean ± standard deviation (SD; n = 5). ^∗p < 0.05, ^∗∗p < 0.01 vs. S; ^#p < 0.05, ^##p < 0.01 vs. I.

Figure 5

Dexmedetomidine (DEX) preconditioning activated nuclear factor-kappa B (NF-κB) 1 hour after reperfusion. NF-κB activation was assessed immunohistochemically. (a) The nuclear translocation of NF-κB in Group S. (b) The nuclear translocation of NF-κB was observed in the renal tubules of Group I. (c) DEX preconditioning (D+I group) significantly increased NF-κB nuclear translocation. (d) These effects were obviously attenuated by atipamezole (A+D+I group). (e) DEX without IRI also induced a certain amount of NF-κB activation. (f) Methylprednisolone, as an anti-inflammatory agent, obviously inhibited NF-κB, thereby indicating that the underlying mechanism was different from that in DEX.

Figure 6

Dexmedetomidine (DEX) preconditioning upregulated A20 at 1 hour after reperfusion. A20 activation was assessed immunohistochemically. (a) The A20 expression in Group S. (b) The upregulation of A20 was observed in the renal tubules of Group I. (c) DEX preconditioning (D+I group) further increased the expression of A20. (d) Atipamezole (A+D+I group) reversed this effect. (e) DEX produced a slight increase in A20 expression over that observed in Group S (A). (f) Methylprednisolone obviously inhibited A20.

Figure 7

Effects of dexmedetomidine (DEX) concentrations on the survival rate and NF-κB and A20 mRNA levels of human renal proximal tubular epithelial cells after hypoxia and reoxygenation. (a) Call survival rate. (b) NF-κB mRNA. (c) A20 mRNA. The data are the mean ± standard deviation (SD). ^∗p < 0.05, ^∗∗p < 0.01 vs. C; ^#p < 0.05, ^##p < 0.01 vs. 0 nM DEX.