Luteolin alleviated calcium oxalate crystal induced kidney injury by inhibiting Nr4a1-mediated ferroptosis

Abstract

Background: The global incidence of calcium oxalate (CaOx) kidney stones is rising, and effective treatments remain limited. Luteolin (Lut), a naturally flavonoid present in several plants, is recognized for its anti-inflammatory, anti-injury, and neuroprotective effects. However, its effects on CaOx kidney stone formation and the associated kidney damage are still unknown.

Objective: Our study seeks to explore the therapeutic impact of Lut on kidney injury and renal fibrosis caused by CaOx crystal and to elucidate the underlying mechanisms.

Methods: CaOx stone models were established in mice via intraperitoneal injection of glyoxylate (Gly, 100 mg/kg) for 12 days. Lut (50 mg/kg or 100 mg/kg) was administered intraperitoneally 7 days before and with the period of Gly treatment. Kidney function and histopathology changes in renal tissues were assessed. RNA sequencing was used to explore potential mechanisms between the model and Lut treatment groups. Molecular docking simulations evaluated the interaction between Lut and the downstream target Nr4a1. Moreover, Nr4a1 knockout mice and knockdown plasmids were used to validate the mechanism of Lut in the treatment of CaOx crystal-induced kidney injury.

Results: Lut significantly mitigated kidney injury and renal fibrosis induced by CaOx crystal, as evidenced by improved kidney function, histopathology staining and Western blot analysis. Lut treatment also significantly inhibited lipid peroxidation and mitochondrial injury. In vitro experiments further demonstrated that Lut treatment alleviated injury and fibrosis in HK-2 cells. Mechanistically, RNA sequencing and molecular docking simulations indicated that Lut binds to Nr4a1 to regulate ferroptosis, thereby alleviating kidney injury induced by CaOx crystal. Overexpression of Nr4a1 negated Lut's beneficial effects, whereas Nr4a1 knockout exhibited a protective effect against kidney injury.

Conclusion: Lut exerts its protective effects by inhibiting ferroptosis via targeting Nr4a1-Slc7a11-GPX4 pathway, alleviating kidney injury and renal fibrosis caused by CaOx crystal deposition.

Keywords: Calcium oxalate stone; Ferroptosis; Kidney injury; Luteolin; Nr4a1.