Ethyl Vanillin Protects against Kidney Injury in Diabetic Nephropathy by Inhibiting Oxidative Stress and Apoptosis

Abstract

Diabetes-induced oxidative stress and apoptosis is regarded as a critical role in the pathogenesis of diabetic nephropathy (DN). Treating diabetes-induced kidney damage and renal dysfunction has been thought a promising therapeutic option to attenuate the development and progression of DN. In this study, we investigated the renoprotective effect of ethyl vanillin (EVA), an active analogue of vanillin isolated from vanilla beans, on streptozotocin- (STZ-) induced rat renal injury model and high glucose-induced NRK-52E cell model. The EVA treatment could strongly improve the deterioration of renal function and kidney cell apoptosis in vivo and in vitro. Moreover, treating with EVA significantly decreased the level of MDA and reactive oxygen species (ROS) and stabilized antioxidant enzyme system in response to oxidative stress by enhancing the activity of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) in vivo and in vitro. Furthermore, EVA also markedly suppressed cleaved caspase-3, Bax, and nuclear transcription factor erythroid 2-related factor (Nrf2) expression in STZ-induced rats. Therefore, these results of our investigation provided that EVA might protect against kidney injury in DN by inhibiting oxidative stress and cell apoptosis.

Figure 1

Chemical structure of ethyl vanillin.

Figure 2

Effect of EVA on the biochemical parameters in STZ-induced rats. EVA was administrated to STZ-induced rats for 8 weeks. (a) Level of BUN was detected by a standard method; (b) level of SCr was detected by a standard method. Results are presented with means ± SEM (n = 8). ^#P < 0.01 compared to control group; ^∗P < 0.05 compared to STZ-induced group; ^∗∗P < 0.01 compared to STZ-induced group.

Figure 3

Effect of EVA to protect DN-induced renal injury in vivo and in vitro. (a) Kidney sections stained with HE and TUNEL staining (×200). (b) Semiquantitative analysis of TUNEL staining in the rat kidney images was shown. Results are presented with means ± SEM (n = 8). ^#P < 0.01 compared to control group; ^∗P < 0.01 compared to STZ-induced group. (c) Annexin V/FITC-PI staining and flow cytometric analysis of apoptosis. (d) Cell viability was measured by the MTT assay. Results are presented with means ± SEM (n = 5). ^#P < 0.01 compared to control group; ^∗P < 0.05 compared to HG-induced group; ^∗∗P < 0.01 compared to HG-induced group. (e) Semiquantitative analysis of TUNEL staining in the NRK-52E cells was shown. Results are presented with means ± SEM (n = 5). ^#P < 0.01 compared to control group; ^∗P < 0.01 compared to HG-induced group. (f) Apoptotic rates of NRK-52E cells in Annexin V/FITC-PI staining were shown. Results are presented with means ± SEM (n = 5). ^#P < 0.01 compared to control group; ^∗P < 0.01 compared to HG-induced group. (g) The level of apoptosis in NRK-52E cells analyzed with TUNEL staining (×200).

Figure 4

Effect of EVA to protect against diabetes-induced oxidative stress in vivo and in vitro. (a) Level of ROS was observed by a fluorescence microscope (×400): (A) Control, (B) HG only, (C) HG+1 μM EVA, (D) HG+3 μM EVA, and (E) HG+10 μM EVA. (b) Semiquantitative image analysis of ROS level. Data are presented as the percentage of control group (mean ± SD; n = 5). ^#P < 0.01 compared to control group; ^∗∗P < 0.01 compared to HG-induced group. (c) Levels of MDA were detected by a standard method. Results are presented with means ± SEM (n = 8). ^#P < 0.01 compared to control group; ^∗P < 0.05 compared to STZ-induced group.

Figure 5

Effect of EVA on antioxidant enzyme activity in vivo and in vitro. After treatment of EVA on STZ-induced rats, (a) SOD activity was detected in the kidney of DN rats; (b) CAT activity was detected in the kidney of DN rats; (c) GSH-Px activity was detected in the kidney of DN rats. Results are presented with means ± SEM (n = 8). ^#P < 0.01 compared to control group, ^∗P < 0.05 compared to STZ-induced group, ^∗∗P < 0.01 compared to STZ-induced group. After treatment of EVA on HG-induced cells, (d) SOD activity was detected using a standard assay; (e) CAT activity was detected using a standard assay; (f) GSH-Px activity was detected using a standard assay. Data are shown as the percentage of the control group, and results are presented with means ± SEM (n = 5). ^#P < 0.01 compared to control group, ^∗P < 0.05 compared to HG-induced group, ^∗∗P < 0.01 compared to HG-induced group.

Figure 6

Effects of EVA on the expression of Nrf2 in STZ-induced rats and n-Nrf2 in HG-induced NRK-52E cells. (a) The expression of Nrf2 protein was observed by western blotting. (b) The quantitative analysis of Nrf2 protein expression. Results are presented with means ± SEM (n = 3). ^#P < 0.01 compared to control group, ^∗P < 0.05 compared to STZ-induced group. (c) The expression of n-Nrf2 protein was observed by western blotting. (d) The quantitative analysis of n-Nrf2 protein expression. Results are presented with means ± SEM (n = 3). ^#P < 0.05 compared to control group, ^∗P < 0.05 and ^∗∗P < 0.01 compared to STZ-induced group.

Figure 7

Inhibitory effect of EVA on the changes in Bax and activated caspase-3 in STZ-induced rats. (a) The expression of Bax and cleaved caspase-3 protein was observed by western blotting. (b, c) Semiquantitative analysis of cleaved caspase-3 and Bax protein expression. Results are presented with means ± SEM (n = 5). ^#P < 0.01 compared to control group, ^∗P < 0.05 compared to STZ-induced group, ^∗∗P < 0.01 compared to STZ-induced group.

Figure 8

Schematic flow diagram of the renoprotective effects of EVA on HG- and STZ-induced kidney injury and its underlying mechanism.