1-Methylhydantoin Cytotoxicity on Renal Proximal Tubular Cells in Vitro

Abstract

1-Methylhydantoin is produced by bacterial creatinine deaminase in the intestinal tract of uremic patients and retaken up into the body. The present study was designed to explore the toxic effect of 1-methylhydantoin on renal proximal tubular cells in vitro. HK-2 (Human renal proximal tubular cell line) was used as the subject. The cell viability was assessed by MTT assay. The cytotoxicity of 1-methylhydantoin to HK-2 was determined by NAG release test. Apoptosis of cultured HK-2 was determined by flow cytometry (light scatter and propidium iodide/annexin V-FITC fluorescence) and by nuclear staining with Hoechst 33258. Cells were exposed to 1-methylhydantoin (0.25mMol/L, 0.5mMol/L, or 1mMol/L), or creatinine (1mMol/L) for 24 h. 1-methylhydantoin induced a significant (p < 0.01) dose-dependent loss of cell viability. 1-methylhydantoin–treated HK-2 displayed characteristic microscopic features of apoptosis: reduced cell size, nuclear disintegration, and membrane bleb formation. FACS analysis demonstrated that 1-methylhydantoin induced apoptosis as well as cell changes consistent with necrosis. The proportion of cells with nuclear changes of apoptosis, identified by flow cytometry, increased significantly (p < 0.01) after 1-methylhydantoin (0.5mMol/L ) for 24 h. The results of the present study clearly demonstrate that both 1-methylhydantoin and creatinine are toxic for proximal tubular cells but that the damage resulting from the 1-methylhydantoin is more severe.

Keywords:

• 1-methylhydantoin
• apoptosis
• HK-2
• cytotoxicity
• uremia