Exploring the Multi-target Potential of Genistein in Urolithiasis: Network Pharmacology, Molecular Simulation, and In Vitro Studies

Abstract

Urolithiasis is a complex renal disorder characterized by recurrent stone formation driven by oxidative stress, epithelial injury, inflammation, and dysregulated ion transport. High recurrence rates and limitations of existing therapies highlight the need for safer, multi-targeted treatment strategies. Genistein, a naturally occurring isoflavone with antioxidant and anti-inflammatory properties, has emerged as a potential candidate for renal protection. The present preclinical and exploratory study investigates the antiurolithiatic potential of genistein using an integrated in silico and in vitro approach. Genistein-associated targets predicted using SwissTargetPrediction were intersected with urolithiasis-related genes from GeneCards to identify common targets. Protein–protein interaction and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed key pathways involved in lithogenesis. Molecular docking showed favorable binding of genistein to matrix metalloproteinase-9 (MMP-9) (−10.1 kcal/mol) and epidermal growth factor receptor (−6.8 kcal/mol). Further, molecular dynamics simulation of the genistein–MMP-9 complex exhibited stable interactions throughout 100 ns. In vitro nucleation and aggregation assays showed concentration-dependent inhibition of calcium oxalate crystallization. Genistein exhibited 78.49% ± 1.94% inhibition in nucleation at 100 µg/mL. In contrast, lower inhibition in aggregation (61.19% ± 2.19%) showed statistically significant differences at higher concentrations (50–100 µg/mL), with significance levels ranging from p < .05 to p < .001 compared to the standard. These findings suggest that genistein may act as a potential multi-target agent in urolithiasis, with MMP-9 emerging as a potentially relevant target, warranting further validation.