Inhibition of aerobic glycolysis alleviates sepsis‑induced acute kidney injury by promoting lactate/Sirtuin 3/AMPK‑regulated autophagy

Abstract

Metabolism reprogramming influences the severity of organ dysfunction, progression to fibrosis, and development of disease in acute kidney injury (AKI). Previously we showed that inhibition of aerobic glycolysis improved survival rates and protected septic mice from kidney injury. However, the underlying mechanisms remain unclear. In the present study, it was revealed that sepsis or lipopolysaccharide (LPS) enhanced aerobic glycolysis as evidenced by increased lactate production and upregulated mRNA expression of glycolysis‑related genes in kidney tissues and human renal tubular epithelial (HK‑2) cells. The aerobic glycolysis inhibitor 2‑deoxy‑D‑glucose (2‑DG) downregulated glycolysis, and improved kidney injury induced by sepsis. 2‑DG treatments increased the expression of sirtuin 3 (SIRT3) and phosphorylation‑AMP‑activated protein kinase (p‑AMPK), following promoted autophagy and attenuated apoptosis of tubular epithelial cells in septic mice and in LPS‑treated HK‑2 cells. However, the glycolysis metabolite lactate downregulated SIRT3 and p‑AMPK expression, inhibited autophagy and enhanced apoptosis in LPS‑treated HK‑2 cells. Furthermore, pharmacological blockade of autophagy with 3‑methyladenine (3‑MA) partially abolished the protective effect of 2‑DG in sepsis‑induced AKI. These findings indicated that inhibition of aerobic glycolysis protected against sepsis‑induced AKI by promoting autophagy via the lactate/SIRT3/AMPK pathway.

Keywords: aerobic glycolysis; 2‑deoxy‑D‑glucose; autophagy; acute kidney injury; sepsis.

Figure 1

Assessment of glycolysis in kidney tissues of CLP-induced septic mice. Sepsis was induced in BALB/c mice by CLP. BALB/c mice were injected i.p. with either 2-DG (2 g/kg) or PBS in equivalent volumes 3 h before CLP. Sham-operated animals served as negative controls. At 24 h of CLP or sham operation, serum and kidney were harvested from mice. (A-C) The mRNA expression levels of glycolysis-related genes (PDK1, PKM2 and LDHA) were analyzed by real-time PCR in the kidney tissues of mice. n=6 mice per group. (D) The serum levels of lactate were measured using commercial kits. n=6 mice per group. Data are presented as the means ± SEM. ^*P<0.05 vs. the vehicle-treated sham. ^#P<0.05 vs. the CLP+2-DG group. CLP, cecal ligation and puncture; i.p., intraperitoneally; 2-DG, 2-deoxy-D-glucose; PBS, phosphate-buffered saline; PDK1, pyruvate dehydrogenase kinase 1; PKM2, pyruvate kinase M2; LDHA, lactate dehydrogenase A.

Figure 2

Glycolysis inhibitor 2-DG alleviates sepsis-induced AKI and apoptosis. Sepsis was induced in BALB/c mice by CLP. BALB/c mice were injected i.p. with either 2-DG (2 g/kg) or PBS in equivalent volumes 3 h before CLP. Sham-operated animals served as negative controls. At 24 h of CLP or sham operation, serum and kidneys were harvested from mice. (A-C) The levels of BUN, SCr and KIM-1 in mouse sera were analyzed using commercial kits. n=4-6 mice per group. (D) The effect of 2-DG on the morphological changes of kidney tissues in CLP or sham mice (×40). Data are presented as the means ± SEM. ^*P<0.05 vs. the vehicle-treated sham. ^#P<0.05 vs. CLP+2-DG group. 2-DG, 2-deoxy-D-glucose; AKI, acute kidney injury; CLP, cecal ligation and puncture; i.p., intraperitoneally; PBS, phosphate-buffered saline; BUN, blood urea nitrogen; Scr, serum creatinine; KIM-1, kidney injury molecule-1.

Figure 3

Glycolysis inhibitor 2-DG induces autophagy in kidney tissues of septic mice. Sepsis was induced in BALB/c mice by CLP. BALB/c mice were injected i.p. with either 2-DG (2 g/kg) or PBS in equivalent volumes 3 h before CLP. Sham-operated animals served as negative controls. At 24 h of CLP or sham operation, kidney tissues were harvested from mice. (A and B) Western blotting of LC3-II/I and p62 in the whole kidney of septic mice. n=4 experiments. (C) The apoptosis of renal tubular cells of mice was measured by TUNEL assay and quantified. Scale bars, 100 µm. n=4 mice per group. Data are presented as the means ± SEM. ^*P<0.05 vs. the vehicle-treated sham. ^#P<0.05 vs. the CLP+2-DG group. 2-DG, 2-deoxy-D-glucose; CLP, cecal ligation and puncture; i.p., intraperitoneally; PBS, phosphate-buffered saline; TUNEL, terminal UTP nick end labeling.

Figure 4

Assessment of glycolysis in HK-2 cells stimulated with LPS. A total of 1×10⁶ HK-2 cells were treated with the doses of LPS (1 µg/ml), 2-DG (2 mM), and LPS+2-DG for 12 h at 37°C in 5% CO₂. (A-C) The mRNA expression of glycolysis-related genes (PDK1, PKM2 and LDHA) in HK-2 cells was analyzed by real-time PCR. n=6 samples per group. (D) The lactate levels in the supernatant of HK-2 cells were measured using commercial kits. n=6 samples per group. Data are presented as the means ± SEM. ^*P<0.05 vs. the control group. ^#P<0.05 vs. the LPS+2-DG group. LPS, lipopolysaccharides; 2-DG, 2-deoxy-D-glucose; PDK1, pyruvate dehydrogenase kinase 1; PKM2, pyruvate kinase M2; LDHA, lactate dehydrogenase A.

Figure 5

Glycolysis inhibitor 2-DG induces autophagy in HK-2 cells stimulated with LPS. A total of 1×10⁶ HK-2 cells were treated with the doses of LPS (1 µg/ml), 2-DG (2 mM), and LPS+2-DG for 12 h at 37°C in 5% CO₂. (A) Western blotting of LC3-I/II and p62 in HK-2 cells. n=4 experiments. (B) Immunofluorescence staining of LC3 (green) expression in HK-2 cells. Scale bars, 20 µm. (C) The apoptosis of HK-2 cells was detected by flow cytometry. n=4 samples per group. Data are presented as the means ± SEM. ^*P<0.05 vs. the control group. ^#P<0.05 vs. the LPS+2-DG group. 2-DG, 2-deoxy-D-glucose; LPS, lipopolysaccharides.

Figure 6

Glycolysis inhibitor 2-DG induces autophagy by regulating the SIRT3/AMPK pathway. Sepsis was induced in BALB/c mice by CLP. BALB/c mice were injected i.p. with either 2-DG (2 g/kg) or PBS in equivalent volumes 3 h before CLP. Sham-operated animals served as negative controls. At 24 h of CLP or sham operation, kidneys were harvested from mice. (A and B) Immunoblot analysis and quantification of SIRT3 and p-AMPK/AMPK in the kidney tissues of septic mice in the presence/absence of 2-DG (2 g/kg). n=4 experiments. (C and D) Immunoblot analysis and quantification of SIRT3 and p-AMPK/AMPK expression in HK-2 cells stimulated with LPS (1 µg/ml) for 12 h in the presence/absence of 2-DG (2 mM). n=4 experiments. Data are presented as the means ± SEM. ^*P<0.05 vs. the vehicle-treated sham group or control group. ^#P<0.05 vs. CLP+2-DG or LPS+2-DG group. 2-DG, 2-deoxy-D-glucose; SIRT3, sirtuin 3; AMPK, AMP-activated protein kinase; CLP, cecal ligation and puncture; i.p., intraperitoneally; PBS, phosphate-buffered saline; p-, phosphorylated; LPS, lipopolysaccharides.

Figure 7

Lactate decreases autophagy via the SIRT3/AMPK pathway in HK-2 cells. A total of 1×10⁶ HK-2 cells were treated with the indicated doses of LPS (1 µg/ml), LPS+2-DG (2 mM), LPS+2-DG+lactate (25 µM) for 12 h at 37°C in 5% CO₂. (A and B) Immunoblot analysis and quantification of LC3-I/II and p62 in HK-2 cells stimulated with LPS (1 µg/ml) in the presence/absence of lactate (25 µM) or 2-DG (2 mM, before 3 h). n=3 mice per group. (C and D) Immunoblot analysis and quantification of SIRT3 and p-AMPK/AMPK in HK-2 cells stimulated with LPS in the presence/absence of lactate (25 µM) or 2-DG (2 mM, before 3 h). n=4 experiments. (E) Apoptosis of HK-2 cells stimulated with LPS in the presence/absence of lactate (25 µM) or 2-DG (2 mM, before 3 h). n=3 experiments. The data are presented as the means ± SEM. ^*P<0.05, vs. the indicated groups. SIRT3, sirtuin 3; AMPK, AMP-activated protein kinase; LPS, lipopolysaccharides; 2-DG, 2-deoxy-D-glucose; p-, phosphorylated.

Figure 8

2-DG-induced protective effects against sepsis-induced AKI depend on regulating autophagy. Mice were injected i.p. with either 3-MA (30 mg/kg, CLP+3-MA) or PBS in equivalent volumes 1 h before CLP in the presence/absence of 2-DG (2 g/kg, before 3 h). Kidney tissues and blood samples were collected at 24 h after CLP. (A) Immunoblot analysis and quantification of LC3-I/II and in kidney tissue of septic mice. n=3 mice per group. (B-D) BUN and Scr and KIM-1 were analyzed using commercial kits. n=7 mice per group. (E) Apoptosis of renal tubular cells of mice was measured by TUNEL assay and quantified. Scale bars, 100 µm. n=4 mice per group. Data are presented as the means ± SEM. ^*P<0.05, ^**P<0.01 and ^***P<0.001 vs. the indicated groups. 2-DG, 2-deoxy-D-glucose; AKI, acute kidney injury; i.p., intraperitoneally; 3-MA, 3-methyladenine; CLP, cecal ligation and puncture; PBS, phosphate-buffered saline; BUN, blood urea nitrogen; Scr, serum creatinine; KIM-1, kidney injury molecule-1; TUNEL, terminal UTP nick end labeling.