Peroxynitrite, the reaction product of superoxide (O-2(center dot)-) and nitric oxide (NO), nitrates tyrosine residues, unsaturated fatty acids, cyclic guanosine monophosphate and other phenolics. We report herein that indoxyl sulfate (IS) is also nitrated by peroxynitrite in vitro and forms 2-nitro-IS, as determined from spectral characteristics and H-1-NMR. IS is one of the very important uremic toxins that accelerate the progression of chronic kidney disease via various mechanisms. However, cell viability experiments with human proximal tubular cells show that the cytotoxicity of 2-nitro-IS is several-fold higher than that of IS. The explanation for this finding seems to be that 2-nitro-IS induces a much more pronounced generation of intracellular reactive oxygen species (ROS) than IS. Results with inhibitors revealed that an organic anion transporter, several intracellular enzymes and nonprotein-bound iron ions are reasons for this finding. Most importantly, however, as detected by immunofluorescence and Western blotting, 2-nitro-IS induces the expression of heme oxygenase-1 and thereby the formation of ROS; most probably through the Fenton reaction. The final result of the increased amounts of ROS is death of the kidney cells. Thus, nitration of uremic toxins by peroxynitrite may help us to understand the initiation and progress of chronic kidney diseases. (C) 2015 Elsevier Inc. All rights reserved.