To elucidate the primary mechanism of high glucose cytotoxicity, the cytoprotective properties of antioxidants against metabolical disorders were assessed in human mesangial cell (HMC) cultures. An 8-day incubation of HMC with high glucose concentration (30 mM) resulted in an extracellular accumulation of the matrixprotein fibronectin (FN), owing to both an expansion of the matrix-associated pericellular FN and a 60% increase of the soluble molecule in the culture medium. The high glucose-induced FN alterations were not due to osmotical effects, as assessed by an iso-osmotic mannitol control. Rather, they are mediated by oxygen-free radicals because the combined treatment of HMC with high glucose and either the antioxidative flavonoid silibinin (given as the water soluble derivative silibinin-C-2,3-dihydrogensuccinate disodium salt) or a radical scavenger cocktail totally prevented the extracellular FN accumulation. This is corroborated further by the determination of malondialdehyde, a product of lipid peroxidation. Incubation of HMC with high glucose resulted in an increase of malondialdehyde in cell homogenates which was completely counteracted by either silibinin or a radical scavenger cocktail. Silibinin alone had no effects on protein synthesis and culture growth. The data presented are compatible with oxidative stress induced by high glucose concentration in HMC cultures. The study further substantiates the proposed role of silibinin in the amelioration of glucose cytotoxicity in renal cells.