NADPH oxidase–associated oxidative stress and ferroptosis-related injury in calcium oxalate-exposed renal tubular epithelial cells: pharmacological evidence for PI3K/AKT involvement

Background/Objectives Calcium oxalate (CaOx) crystals contribute to renal tubular injury in nephrolithiasis, but the relationship among NADPH oxidase (NOX), oxidative stress, ferroptosis-related injury, and PI3K/AKT signaling remains incompletely defined. We examined CaOx-induced injury in NRK-52E renal tubular epithelial cells and tested the effects of pharmacological NOX and PI3K inhibition on oxidative and ferroptosis-related readouts.Methods NRK-52E cells were exposed to CaOx (0–10 mM) and assessed by CCK-8, DCFH-DA flow cytometry, NOX activity assay, NADPH and Fe2+ assays, GSH and malondialdehyde (MDA) assays, quantitative real-time PCR (qRT-PCR), Western blotting, and transcriptome sequencing. Ferrostatin-1 (Fer-1, 2 μM), Erastin (10 μM), the NOX inhibitor GKT137831 (10 μM), and the PI3K inhibitor LY294002 (10 μM) were used as pharmacological probes.Results CaOx reduced cell viability and increased intracellular ROS in a concentration-dependent manner. At 1 mM, CaOx was associated with lower NADPH and GSH, higher Fe2+, MDA, and ROS, increased NOX activity and NOX4 mRNA and protein expression, reduced GPX4, and increased ACSL4. Fer-1 ameliorated, whereas Erastin aggravated, several ferroptosis-associated readouts. CaOx also increased the p-PI3K/PI3K and p-AKT/AKT ratios. GKT137831 and LY294002 each attenuated CaOx-associated oxidative stress and ferroptosis-related phenotypes in this cell model.Conclusions The findings support a contribution of NOX-associated oxidative stress and ferroptosis-related injury to CaOx-exposed NRK-52E cells. Pharmacological experiments indicate that NOX and PI3K/AKT signaling participate in this phenotype. These findings are limited to an in vitro cell model and require validation in animal and human-relevant systems.