Apigenin activates AKT1 to coordinate MTOR-mediated anti-apoptosis and FOXO3-driven antioxidant defense in calcium oxalate nephropathy

Background: Kidney stone disease, primarily composed of calcium oxalate (CaOx) crystals, represents a significant global health burden with high recurrence rates. Current therapeutic strategies fail to adequately address the oxidative stress and apoptosis triggered by CaOx crystals in renal tubular cells. While the natural flavonoid apigenin (API) shows promise, its precise mechanism in CaOx nephropathy remains unclear. Methods: We employed an integrated strategy combining network pharmacology and machine learning to identify potential therapeutic targets of apigenin in kidney stone disease. Experimental validation was conducted using both in vivo and in vitro models: a mouse model of CaOx nephropathy induced by glyoxylate and HK-2 cells exposed to calcium oxalate monohydrate crystals. The interaction between apigenin and AKT1 was investigated through surface plasmon resonance, molecular docking, cellular thermal shift assays, and drug affinity responsive target stability assays. Downstream signaling effects were analyzed using quantitative PCR, Western blotting, reactive oxygen species measurement, and apoptosis assessment. The functional role of AKT1 was further examined using the specific inhibitor MK-2206, siRNA-mediated knockdown, and FOXO3 overexpression experiments. Results: Apigenin treatment significantly reduced CaOx crystal deposition, improved renal function markers, and attenuated tubular damage in mice in a dose-dependent manner. Bioinformatic analysis identified AKT1 as the core target, and experimental validation confirmed that apigenin directly binds to AKT1 with high affinity, leading to enhanced phosphorylation. This activation modulated two critical downstream pathways. The mTOR pathway suppressed apoptosis through downregulation of BAX and upregulation of BCL-2; Phosphorylation of FOXO3 decreased its transcriptional activity on Keap1, resulting in reduced Keap1 expression, subsequent Nrf2 stabilization, nuclear translocation, and upregulation of the antioxidant enzyme HO-1. The essential role of AKT1 was further supported by the finding that MK-2206 and si-AKT1 treatment abolished apigenin’s protective effects. Additionally, FOXO3 overexpression reversed apigenin-induced Nrf2 activation, confirming the involvement of the AKT1-FOXO3-Keap1-Nrf2 axis. Conclusion: Apigenin alleviates CaOx-induced renal injury through direct activation of AKT1, which coordinates dual protective mechanisms. Anti-apoptosis via the mTOR pathway and antioxidant defense through the FOXO3-Keap1-Nrf2 axis. This study provides comprehensive evidence of apigenin’s AKT1-centered mechanism of action in CaOx nephropathy, supporting its potential as a novel therapeutic agent for kidney stone disease.