In vitro toxicity of α-amanitin in human kidney cells and evaluation of protective effect of polymyxin B

Abstract

α-Amanitin intoxications have been associated with acute kidney injury and renal failure, besides its well-known hepatotoxic effects. Currently, no effective antidote against α-amanitin toxicity exists. Recent in vivo studies have shown that polymyxin B (PolB) decreases αamanitin toxicity and that the associated renal damage is largely decreased by this antibiotic. This work aimed to characterize α-amanitin cytotoxicity in HK-2 cells and evaluate PolB’s putative antidotal effectiveness in this in vitro system. HK-2 cells were exposed to α-amanitin (0.01-10 μM) at different time-points and cytotoxicity evaluated by the MTT reduction and neutral red uptake assays. To assess PolB putative protective effects, two paradigms were used: (i) 30 min pre-incubation with PolB followed by 48h incubation with α-amanitin (0.5 and 1 μM) or (ii) PolB co-incubation with α-amanitin (5 and10 μM) for 2h followed by a 48h drug/toxin-free period. α-Amanitin led to cytotoxicity effects on kidney cells at clinical relevant concentrations. The effectiveness of a previously described antidote, PolB, was not verified in vitro, which highlights the importance of further investigation on this antidotal strategy and its mechanisms.