Desflurane Preconditioning Protects Against Renal Ischemia-Reperfusion Injury and Inhibits Inflammation and Oxidative Stress in Rats Through Regulating the Nrf2-Keap1-ARE Signaling Pathway

Abstract

Objective: Kidney is sensitive to ischemia-reperfusion (I/R) injury because of its special structure and function. In this study, we aimed to explore the mechanism of desflurane (DFE) preconditioning effecting on renal I/R injury in rats.

Methods: Renal I/R injury rats model was constructed, and the expressions of serum renal function parameters (blood urea nitrogen (BUN) and serum creatinine (SCr)) and lipid peroxidation-related factors were detected using corresponding commercial kits to assess the degrees of renal functional damage and oxidative stress. Hematoxylin--eosin (HE) staining and Masson trichrome staining were applied to measure the renal histologic damage. The expressions of inflammation-related factors were determined by ELISA assay. The cell apoptosis was analyzed using TUNEL, Western blot and immunohistochemistry (IHC). IHC was also used to detect the number of myeloperoxidase (MPO)-positive cells. The expressions of proteins associated with the Nrf2-Keap1-ARE pathway were assessed by Western blot and IHC.

Results: DFE preconditioning inhibited I/R injury-induced BUN and SCr increase and renal histologic injury in rats. Also, DFE suppressed the inflammation, apoptosis and oxidative stress caused by renal I/R injury in vivo. In addition, DFE preconditioning repressed peroxide-related factors (MDA, MPO and NO) expressions and promoted antioxidant-related factors (GSH, SOD, GPx and CAT) expressions. In addition, DFE promoted Nrf2-Keap1-ARE-related proteins including Nrf2, NQO1, HO-1, γ-GCS, GSR and GCLc expressions.

Conclusion: DFE preconditioning protected the kidney as well as inhibited the inflammation, cell apoptosis and oxidative stress in renal I/R injury rats by activating the Nrf2-Keap1-ARE signaling pathway.

Keywords: Nrf2-Keap1-ARE signaling pathway; animal model; desflurane; ischemia–reperfusion; kidney.

Figure 1

DFE preconditioning decreased BUN and Scr contents in the serum of renal I/R injury rat models. The rats were divided into four groups (n=8) according to different treatments, including sham group, sham + DFE group, I/R group and I/R + DFE group. (A) Urea Assay Kits were used to detect the BUN index in the serum of four groups of rats. (B) Creatinine Assay Kit was used to detect the Scr index in the serum of four groups of rats. Data were shown as mean ± SD. **P < 0.01 vs sham group, ^#P < 0.05 vs I/R group.

Figure 2

DFE preconditioning alleviated I/R-induced renal histological injury. (A) The renal histological injury of four groups was measured by HE staining and ATN valuing. (B) The renal fibrosis of four groups was determined by Masson trichrome staining. Data were shown as mean ± SD. **P < 0.01 vs sham group, ^#P < 0.05 vs I/R group.

Figure 3

DFE preconditioning reduced I/R induced renal inflammation. (A–D) The protein expression of inflammation-related proteins TNF-α, IL-1β, IL-6 and IL-10, were detected using ELISA assay. Data were shown as mean ± SD. **P < 0.01 vs sham group, ^#P < 0.05, ^##P < 0.01 vs I/R group.

Figure 4

DFE preconditioning inhibited I/R-induced cell apoptosis. (A) The numbers of apoptotic cells in the kidney of four groups were detected using TUNEL assay. (B) The expression levels of cell apoptosis-related proteins, including Bax, caspase 3, cleaved-caspase 3, caspase 9, cleaved-caspase 9 and Bcl-2, were measured using Western blot. (C) The expression levels of cleaved-caspase 3 in four groups were assessed using IHC. Data were shown as mean ± SD. **P < 0.01 vs sham group, ^#P< 0.05, ^##P < 0.01 vs I/R group.

Figure 5

DFE preconditioning suppressed I/R induced oxidative stress and lipid peroxidation. (A) The contents of MDA, MPO, GSH, SOD, GPx, CAT and NO in the renal cells lipid offour groups were detected by commercial kits. (B) The numbers of MPO-positive cells in four groups were measured using IHC. Data were shown as mean ± SD. **P < 0.01, ^#P < 0.05, ^##P < 0.01 vs sham group.

Figure 6

DFE preconditioning regulated the Nrf2-Keap1-ARE signaling pathway after renal I/R injury. (A) The expressions of Nrf2 in cell nucleus and cytoplasm and the expression of Keap1 in whole cell were detected using Western blot. (B) The expressions of Nrf2 of four groups were assessed using IHC. (C) The expression levels of HO-1, NQO-1, GSR, GCLc and γ-GCS were measured using Western blot. Data were shown as mean ± SD. **P < 0.01 vs sham group, ^##P < 0.01 vs I/R group.