The AMPK Activator ATX-304 Reprograms Tubular Cell Metabolism to Protect Against Cisplatin-Induced AKI

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Background: Acute kidney injury (AKI) is characterized by widespread disruption of energy metabolism pathways. Targeting the metabolic regulator AMP-activated protein kinase (AMPK) is a potential strategy for addressing the problem of AKI. ATX-304 is a novel PAN-AMPK activator with reported benefits in models of diabetes, obesity and aging. Methods: C57Bl/6 mice were fed chow containing ATX-304 (1mg/gm) versus control chow for 7-days prior to cisplatin-induced AKI (CI-AKI). Primary cultures of tubular epithelial cells (TECs) were pre-treated with ATX-304 (20 μM, 4-hrs), prior to exposure to cisplatin (20 μM, 23-hours). Results: ATX-304 protected against CI-AKI as measured by serum creatinine at 48-hours (control 0.05±0.03 mM vs ATX-304 0.02+0.01 mM, P=0.004), western blot for neutrophil gelatinase-associated lipocalin (NGAL) (control 3.3±1.8-fold vs ATX-304 1.2±0.55-fold, P=0.002), RT-PCR for NGAL (control 99.6±22.8-fold vs ATX-304 35.2±5.9-fold, P=0.006), monocyte chemoattractant protein-1 (control 18.7±5.1-fold vs ATX-304 10.4±1.3-fold, P=0.048) and receptor-interating protein kinase 3 (CI-AKI control 6.9±2.4-fold vs CI-AKI ATX-304 4.9±1.3-fold, P=0.02), and histological injury score (control 3.5±0.59 vs ATX-304 2.7±0.74, P=0.03). In TECs, pre-treatment with ATX-304 protected from cisplatin mediated cell death, measured by lactate dehydrogenase release assay (control 27.1±4.2% lysis vs ATX304 -0.6±2.5% lysis, p<0.0001), and maintained cell viability in the presence of cisplatin, as measured by MTS assay. ATX-304 protection against cisplatin was lost in AMPK-null murine embryonic fibroblasts. In TECs metabolomic analysis revealed that ATX-304 (20 μM, 4-hrs) altered the level of 66/126 detected metabolites, including changes in fatty acids, tricarboxylic acid cycle metabolites, and amino acids. Energy metabolism studies of live TECs using the XFe96 Seahorse analyser found ATX-304 increased basal oxygen consumption rate by 38%, whereas maximal respiration was unchanged. Conclusions: The mechanism of ATX-304 protection against cisplatin injury is AMPK- dependent and involves widespread metabolic reprogramming. AMPK activation by ATX-304 is a promising therapeutic strategy for AKI. Funding: Commercial Support - Amplifier Therapeutics (Betagenon), Government Support - Non-U.S.

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