Azilsartan Modulates HMGB1/NF-κB/p38/ERK1/2/JNK and Apoptosis Pathways during Renal Ischemia Reperfusion Injury

Abstract

Renal ischemia/reperfusion (IR) injury is characterized by an unexpected impairment of blood flow to the kidney. Azilsartan is an angiotensin receptor blocker that is approved for the management of hypertension. The present study aimed to investigate, on molecular basics, the nephroprotective activity of azilsartan on renal IR injury in rats. Rats were assigned into four groups: (1) Sham group, (2) Azilsartan group, (3) IR group, and (4) IR/Azilsartan-treated group. Histological examination and renal function were evaluated. Levels of KIM-1, HMGB1, caspase 3, GPX, SOD, NF-κB, and p53 proteins were investigated using ELISA. mRNA levels of IL-1β, IL6, IL10, TNF-α, NF-κB, p53, and bax were assessed by qRT-PCR. Expression of p38, JNK, and ERK1/2 proteins was investigated by Western blotting. IR injury resulted in tissue damage, elevation of creatinine, BUN, KIM-1, HMGB1, caspase 3, NF-κB, and p53 levels, decreasing GPX and SOD activities, and up-regulation of NF-κB, IL-1β, IL6, TNF-α, p53, and bax genes. Furthermore, it up-regulated the expression of phosphorylated/total ratio of p38, ERK1/2, and JNK proteins. Interestingly, treatment of the injured rats with azilsartan significantly alleviated IR injury-induced histopathological and biochemical changes. It reduced the creatinine, BUN, KIM-1, HMGB1, caspase-3, NF-κB, and p53 levels, elevated GPX and SOD activities, down-regulated the expression of NF-κB, IL-1β, IL6, TNF-α, p53, and bax genes, and up-regulated IL10 gene expression. Furthermore, it decreased the phosphorylated/total ratio of p38, ERK1/2, and JNK proteins. Azilsartan exhibited nephroprotective activity in IR-injured rats via its antioxidant effect, suppression of inflammation, attenuation of apoptosis, and inhibition of HMGB1/NF-κB/p38/ERK1/2/JNK signaling pathway.

Keywords: ERK1/2; JNK; NF-κB; apoptosis; azilsartan; renal ischemia/reperfusion injury.

Figure 1

Creatinine (A) and BUN (B) serum levels. Bars represent mean ± SD. Followed by post hoc Dunnett test, significant difference was analyzed by one-way ANOVA test, where * p < 0.001, compared to the sham group, and # p < 0.001, compared to the IR group. BUN; blood urea nitrogen, SD; standard deviation, IR; ischemia reperfusion.

Figure 2

Levels of KIM-1 (A), HMGB-1 (B), caspase 3 (C), GPX (D), SOD (E), NF-κB (F) and p53 (G). Bars represent mean ± SD. Followed by post hoc Dunnett test, significant difference was analyzed by one-way ANOVA test, where * p < 0.001, compared to the sham group, and # p < 0.001, compared to the IR group. KIM-1; kidney injury molecule-1, GPX; glutathione peroxidase, SOD; superoxide dismutase, NF-κB; nuclear factor-κB, SD; standard deviation, IR; ischemia reperfusion.

Figure 2

Levels of KIM-1 (A), HMGB-1 (B), caspase 3 (C), GPX (D), SOD (E), NF-κB (F) and p53 (G). Bars represent mean ± SD. Followed by post hoc Dunnett test, significant difference was analyzed by one-way ANOVA test, where * p < 0.001, compared to the sham group, and # p < 0.001, compared to the IR group. KIM-1; kidney injury molecule-1, GPX; glutathione peroxidase, SOD; superoxide dismutase, NF-κB; nuclear factor-κB, SD; standard deviation, IR; ischemia reperfusion.

Figure 3

The renal expression of IL-1β, IL6, IL10, TNF-α, NF-κB, p53, and bax genes. Quantitative RT-PCR was used to investigate the gene expression of different groups. Expression was represented relative to the untreated sham group and normalized to the corresponding GAPDH gene expression. Bars represent mean ± SD. Followed by post hoc Dunnett test, significant difference was analyzed by two-way ANOVA test, where **; p < 0.01, ***; p < 0.001, compared to the sham group, and #, p < 0.05; ###, p < 0.001, compared to the IR group. IL; interleukin, TNF-α; tumor necrosis factor-α, NF-κB; nuclear factor-κB, SD; standard deviation, IR; ischemia reperfusion.

Figure 4

Effect of azilsartan on the expression of p38 MAPK, ERK1/2, and JNK proteins. (A) Representative Western blots of phosphorylated and total p38 MAPK, ERK1/2, JNK, and β-actin proteins for different groups. (B) Expressions of phosphorylated/total ratio of proteins were densitometrically expressed as fold change, using bands in (A), relative to that of sham control rats, after normalization to the corresponding β-actin. Bars represent mean ± SD. Followed by post hoc Dunnett test, significant difference was analyzed by two-way ANOVA test, where ***; p < 0.001, compared to the sham group, and ### p < 0.001, compared to the IR group. p38 MAPK; Mitogen Activated Protein Kinase, ERK1/2; extracellular signal-regulated protein kinase, c-JNK; Jun N terminal kinase, SD; standard deviation, IR; ischemia reperfusion.

Figure 5

Representative photomicrographs of rat kidney tissues of different groups. Hematoxylin-eosin was used to stain kidney tissues. (A) The sham group (magnification; ×200), (B) the azilsartan-treated group (magnification; ×100), (C,D) the IR group (magnification; ×200 & ×100, respectively), and (E) the IR/azilsartan-treated group (magnification; ×100). (F) Histological score of kidney damage. Bars represent mean ± SD. Followed by post hoc Dunnett test, significant difference was analyzed by one-way ANOVA test, where * p < 0.001, compared to the sham group, and # p < 0.01, compared to the IR group. SD; standard deviation, IR; ischemia reperfusion.