Sirtuin 6 overexpression relieves sepsis-induced acute kidney injury by promoting autophagy

Abstract

Sirtuin 6 (SIRT6) has the function of regulating autophagy. The aim of this study was to investigate the mechanism through which SIRT6 relieved acute kidney injury (AKI) caused by sepsis. The AKI model was established with lipopolysaccharides (LPS) using mice. Hematoxylin-eosin (HE) staining and streptavidin-perosidase (SP) staining was used to observe kidney tissue and test SIRT6 and LC3B proteins in kidney. Enzyme-linked immunosorbent assay (ELISA) was performed to detected the tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) concentrations. Cell counting kit-8 (CCK-8) assay and flow cytometry were carried out to test the cell viability and apoptosis rate respectively. Protein and mRNA were determined by Western blot and quantitative real-time polymerase chain reaction (qRT-PCR). AKI induced by LPS had self-repairing ability. At 12 h after modeling, the expression levels of TNF-α, IL-6, SIRT6 and LC3B-II/LC3B-I were first significantly increased and were then significantly decreased at 48 h after modeling. LPS inhibited the growth of HK-2 cells and promoted the expressions of TNF-α, IL-6, SIRT6 and LC3B. Overexpression of SIRT6 down-regulated the secretion of TNF-α and IL-6 induced by LPS. SIRT6 overexpression inhibited apoptosis induced by LPS and promoted autophagy in HK-2 cells. Silencing of the SIRT6 gene not only promoted the secretion of TNF-α and IL-6 by HK-2 cells, but also promoted apoptosis and reduced autophagy. LPS up-regulated the expression of SIRT6 gene in HK-2 cells. Overexpression of the SIRT6 gene could inhibit apoptosis and induce autophagy, which might be involved in repairing kidney damage caused by LPS.

Keywords: Sirtuin 6; acute kidney injury; apoptosis; autophagy.

Figure 1.

Effects of LPS on mouse kidney. (a) HE staining was used to detect kidney tissue morphology. (b–d) Cr, BUN and Cys C in the serum of mice were measured. *P< 0.05,**P< 0.01, versus 1 h. (biological replicate = 12, technical replicate = 3).

Figure 2.

Effects of LPS on inflammatory factors, SIRT6 and autophagy-related proteins. (a–b) ELISA was used to test the concentration of TNF-α and IL-6 in serum. (c–g) QRT-PCR and Western blot were applied to tested mRNAs and proteins of SIRT6 and LC3B. *P< 0.05,**P< 0.01, versus control group. (biological replicate = 12, technical replicate = 3).

Figure 3.

Effects of LPS on autophagy-related proteins. (a–b) SP staining was performed to detect the LC3B and SIRT6 proteins in kidney. *P< 0.05,**P< 0.01, versus control group. (biological replicate = 12).

Figure 4.

Effects of LPS on HK-2 cells. (a) The CCK-8 assay was applied to detect cell viability. (b–c) ELISA was used to test the concentration of TNF-α and IL-6 in culture. (d–h) SIRT6 and LC3B mRNAs and proteins were detected by QRT-PCR and Western blot. *P< 0.05,**P< 0.01, versus 0 h. (biological replicate = 12, technical replicate = 3).

Figure 5.

Effects of SIRT6 overexpression on HK-2 cells. (a) The CCK-8 assay was applied to detect cell viability. (b–c) ELISA was used to test the concentration of TNF-α and IL-6 in culture. (d–e) Apoptosis rate was detected using flow cytometry. (f–j) SIRT6 and LC3B mRNAs and proteins were detected by QRT-PCR and Western blot. *P< 0.05,**P< 0.01. (biological replicate = 12, technical replicate = 3).

Figure 6.

Effect of SIRT6 silencing on HK-2 cells. (a) The CCK-8 assay was applied to detect cell viability. (b–c) ELISA was used to test the concentration of TNF-α and IL-6 in culture. (d–e) Apoptosis rate was detected using flow cytometry. (f–j) SIRT6 and LC3B mRNAs and proteins were detected by QRT-PCR and Western blot. *P< 0.05,**P< 0.01. (biological replicate = 12, technical replicate = 3).