[AMP-activated protein kinase activation alleviates lipopolysaccharide-induced sepsis-associated acute kidney injury by inhibiting ferroptosis].

OBJECTIVE

To investigate the regulatory role of AMP-activated protein kinase (AMPK) activation in ferroptosis during lipopolysaccharide (LPS)-induced sepsis-associated acute kidney injury (SA-AKI) and its molecular mechanisms.

METHODS

Thirty SPF male C57BL/6 mice were randomly assigned to the control group, LPS group, and LPS+AMPK activator (AICAR) group using a random number table method, with 10 mice per group. Mice in the LPS+AICAR group received an intraperitoneal injection of AICAR at a dose of 500 mg/kg for intervention 30 minutes prior to model establishment, while those in the control group and LPS group were intraperitoneally injected with an equal volume of normal saline. The SA-AKI model was induced via intraperitoneal injection of LPS at 10 mg/kg, and the control group was intraperitoneally administered an equal volume of normal saline. Enzyme-linked immunosorbent assay (ELISA) was performed to measure the serum concentrations of inflammatory factors [including interleukins (IL-1β, IL-6), and tumor necrosis factor-α (TNF-α)]. Serum renal function indicators [including serum creatinine (SCr) and blood urea nitrogen (BUN)], as well as the levels of key ferroptosis-related factors [including Fe2+, malondialdehyde (MDA), and glutathione (GSH)] in renal tissues were measured using biochemical assays. Hematoxylin-eosin (HE) staining was conducted to observe histopathological changes in renal tissues under a light microscope, and transmission electron microscopy was used to examine the ultrastructural alterations of mitochondria in renal tissues. Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting were employed to determine the mRNA and protein expression levels of AMPK, key ferroptosis-related enzymes [long-chain acyl-CoA synthetase 4 (ACSL4), solute carrier family 7 member 11 (SLC7A11), and glutathione peroxidase 4 (GPX4)], as well as the nuclear factor E2-related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling pathway-related molecules in renal tissues.

RESULTS

Compared with the control group, mice in the LPS group exhibited renal tubular structural disorder, with vacuolar degeneration, necrosis, and shedding of epithelial cells, interstitial edema, and inflammatory cell infiltration. Mitochondria in renal tissues showed typical ferroptosis characteristics. Moreover, the levels of serum IL-1β, IL-6, TNF-α, SCr, and BUN were significantly elevated. In renal tissues, the levels of Fe2+ and MDA, along with the mRNA and protein expression of ACSL4, were increased, whereas the GSH level, protein expression of AMPK, and mRNA and protein expression of SLC7A11, GPX4, Nrf2, and HO-1 were markedly decreased (all P<0.05). In contrast to the LPS group, mice in the LPS+AICAR group displayed significant alleviation of renal tubular structural damage and vacuolar degeneration, as well as reduced interstitial edema and inflammatory infiltration. The ferroptosis-related ultrastructural changes in renal mitochondria were also notably ameliorated. The serum concentrations of IL-1β, IL-6, TNF-α, SCr, and BUN were significantly lower in the LPS+AICAR group than in the LPS group [IL-1β (ng/L): 68.93±9.37 vs. 91.25±10.55, IL-6 (ng/L): 80.03±7.86 vs. 93.10±6.16, TNF-α (ng/L): 484.77±51.00 vs. 558.59±48.54, SCr (μmol/L): 31.75±5.08 vs. 57.63±6.37, BUN (mmol/L): 23.93±4.44 vs. 36.35±5.47, all P<0.05]. Additionally, the levels of Fe2+ and MDA in renal tissues were significantly reduced in the LPS+AICAR group compared with the LPS group [Fe2+ (μmol/g): 4.02±0.74 vs. 6.84±1.41, MDA (μmol/g): 1.44±0.62 vs. 2.62±0.62, both P<0.05], accompanied by downregulated mRNA and protein expression of ACSL4 (ACSL4 mRNA: 1.98±0.50 vs. 3.67±0.80, ACSL4/GAPDH: 0.49±0.08 vs. 0.94±0.12, both P<0.05). Furthermore, the renal GSH level was higher in the LPS+AICAR group than in the LPS group (μmol/kg: 4.35±0.50 vs. 3.49±0.94), along with significantly upregulated mRNA and protein expression of AMPK, SLC7A11, GPX4, Nrf2, and HO-1 [mRNA expression: AMPK mRNA (2-ΔΔCt): 2.68±0.27 vs. 1.22±0.24, SLC7A11 mRNA (2-ΔΔCt): 0.04±0.01 vs. 0.01±0.00, GPX4 mRNA (2-ΔΔCt): 5.15±1.21 vs. 3.40±0.45, Nrf2 mRNA (2-ΔΔCt): 0.57±0.29 vs. 0.22±0.23, HO-1 mRNA (2-ΔΔCt): 0.12±0.03 vs. 0.02±0.01; protein expression: p-AMPK/AMPK ratio: 1.19±0.14 vs. 0.50±0.15, SLC7A11/GAPDH: 0.55±0.07 vs. 0.34±0.10, GPX4/GAPDH: 0.56±0.11 vs. 0.18±0.08, Nrf2/GAPDH: 0.89±0.20 vs. 0.36±0.08, HO-1/GAPDH: 0.75±0.07 vs. 0.54±0.06, all P<0.05].

CONCLUSIONS

AMPK may exert protective effects against LPS-induced SA-AKI in mice by inhibiting ferroptosis through the Nrf2/HO-1 signaling pathway.