Phenylacetic acid stimulates reactive oxygen species generation and tumor necrosis factor-α secretion in vascular...

Tumor necrosis factor (TNF)-α and oxidative stress are considered to play crucial roles in atherosclerosis and vascular calcification. "Uremic toxins" detected in patients with chronic kidney disease (CKD) could cause impaired signal transduction and dysfunction in many organs. Since phenylacetic acid (PAA), identified as one of the uremic toxins, has an inhibiting property of monocytes as well as osteoblastic cells, we examined the effects of PAA on TNF-α secretion and oxidative stress in vascular endothelial cells. In human aortic endothelial cells, TNF-α secretion was assessed after treatment with PAA using an ELISA kit and following the manufacturer's instructions. For determination of reactive oxygen species (ROS), 8-hydroxydeoxyguanosine (8-OHdG) in the culture medium was measured in the presence or absence of PAA. Treatment with PAA in aortic endothelial cells for 24 h significantly stimulated TNF-α secretion in a dose-dependent manner ranging between 0.5 and 5 mM. On the other hand, the 8-OHdG level in the culture medium was significantly increased in the cells incubated with 1 mM PAA for 12 h. To determine if PAA-induced TNF-α secretion is mediated by ROS production, the effect of free radical scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL) was examined. It was found that PAA-induced TNF-α secretion was significantly inhibited by TEMPOL. Our findings indicate that PAA stimulates TNF-α secretion at least in part through ROS production in aortic endothelial cells. The plasma PAA level was reported to be approximately 3.5 mM in end-stage CKD patients, whereas it was <5 µM in healthy subjects; thus, PAA could be involved in the pathological changes of the vasculature in CKD.