N-acetylcysteine Ameliorates Vancomycin-induced Nephrotoxicity by Inhibiting Oxidative Stress and Apoptosis in the in vivo and in vitro Models

Abstract

Background: Oxidative stress-related apoptosis is considered as the key mechanism implicated in the pathophysiology of nephrotoxicity with vancomycin (VCM) therapy. We evaluated the possible effects of N-acetylcysteine (NAC) on VCM-induced nephrotoxicity and the underlying mechanism. Methods: VCM-induced nephrotoxicity was established using HK-2 cells and SD rats and observed by measuring cell survival, kidney histological changes, renal function and kidney injury related markers (KIM-1 and NGAL). Oxidative stress, renal cell apoptosis and the involved signaling pathways were also evaluated. Results: In model rats, NAC could protect against VCM-induced acute kidney injury with histological damage, renal dysfunction, and increased Cre and BUN levels. In HK-2 cells, VCM-induced decreased cell viability was restored by NAC. In addition, increased expression of caspase-3, KIM-1 and NGAL suffering from VCM was also reversed by NAC in vivo and in vitro. NAC inhibited ROS production, decreased cell apoptosis by decreasing the Bax/Bcl-2 ratio and caspase-3 expression in HK-2 cells and regulated oxidative stress indicators in the kidney by decreasing GSH, SOD and CAT activity and increasing MDA levels. Furthermore, NAC could effectively reverse VCM-associated increased P38 MAPK/JNK phosphorylation. Conclusions: The results demonstrated that NAC had a protective effect against nephrotoxicity from VCM by inhibiting oxidative stress and apoptosis via P38 MAPK/JNK.

Keywords: N-acetylcysteine; nephrotoxicity; oxidative stress; renal protection; vancomycin.

Figure 1

Effect of NAC on decreased HK-2 cell viability induced by VCM. (A) Morphological changes in HK-2 cells exposed to VCM. (B) HK-2 cell viability after exposure to different concentrations of VCM detected by CCK-8 assay. (C, D) The effects of NAC and vitamin C on the viability of HK-2 cells exposed to VCM detected by CCK-8 assay. Data was performed from three independent experiments and presented as mean ± SEM. ^**p<0.01, significantly lower than that in the control group. ^#p<0.05, significantly higher than that in the VCM group. Significance was analyzed by ANOVA followed by Tukey's test.

Figure 2

Effect of NAC on changes in renal histopathology and kidney function induced by VCM in rats. (A) Sections of kidneys from different groups stained using HE. (B, C) Serum levels of Cre and BUN. Data was presented as mean±SEM (n=5 rats). ^**p<0.01, significantly higher than that in the control group. ^#p<0.05, significantly lower than that in the VCM group. Significance was analyzed by ANOVA followed by Tukey's test.

Figure 3

Effect of NAC on VCM-induced increased expression of KIM-1 and NGAL in HK-2 cells and rat kidneys. The effects of NAC (A, B, C) and vitamin C (D, E, F) on the expression of KIM-1 and NGAL in HK-2 cells using western blot and semiquantified analysis. (G, H) KIM-1 expression and analysis in kidney tissue detected using immunohistochemistry. (I, J) NGAL expression and analysis in kidney tissue detected using an immunohistochemical technique. Data was performed from three independent experiments or 5 rats and presented as mean ± SEM. ^*p<0.05, significantly higher than that in the control group. ^#p<0.05, significantly lower than that in the VCM group. Significance was analyzed by ANOVA followed by Tukey's test.

Figure 4

Effect of NAC on VCM-induced cell apoptosis in HK-2 cells and rat kidneys. (A, B, C, D) The expression and analysis of Bax, Bcl-2 and caspase-3 detected and analyzed by western blot in HK-2 cells treated with VCM and NAC or vitamin C. (E) Caspase-3 expression and analysis using immunofluorescence staining in HK-2 cells treated with VCM and NAC or vitamin C. (F) Caspase-3 expression and analysis using immunofluorescence staining in rat kidneys treated with VCM and NAC or vitamin C. Data was performed from three independent experiments or 5 rats and presented as mean ± SEM. ^*p<0.05, ^**p<0.01, significantly higher than that in the control group. ^#p<0.05, significantly lower than that in the VCM group. Significance was analyzed by ANOVA followed by Tukey's test.

Figure 5

Effect of NAC on oxidative stress induced by VCM in HK-2 cells and rat kidneys. (A) ROS production in HK-2 cells detected by immunofluorescence. (B) Quantified analysis of ROS levels in HK-2 cells. (C) MDA contents in rat kidneys from different groups. (D) GSH contents in rat kidneys. (E) SOD activity in rat renal tissue. (F) CAT activity in rat renal tissue. Data was performed from three independent experiments or 5 rats and presented as mean ± SEM. ^*p<0.05, ^**p<0.01, significantly different compared with that in the control group. ^#p<0.05, significantly different compared with that in the VCM group. Significance was analyzed by ANOVA followed by Tukey's test.

Figure 6

Effect of NAC on the VCM-activated P38 MAPK/JNK signaling pathway. (A) Phosphorylation levels of P38 MAPK and JNK in HK-2 cells treated with VCM and NAC. (B) Phosphorylation levels of P38 MAPK and JNK in HK-2 cells treated with VCM and vitamin C. Data was performed from three independent experiments and presented as mean ± SEM. ^*p<0.05, ^**p<0.01, significantly higher than that in the control group. ^#p<0.05, significantly lower than that in the VCM group. Significance was analyzed by ANOVA followed by Tukey's test.

Figure 7

Mechanism of N-acetylcysteine against VCM-induced nephrotoxicity.