The uraemic toxin phenylacetic acid increases the formation of reactive oxygen species in vascular smooth muscle cells.
BACKGROUND
Cardiovascular events are the most common cause of death in end-stage renal disease (ESRD) patients. Traditional risk factors do not sufficiently explain the marked increment in cardiovascular morbidity and mortality as compared with the general population. The role of uraemic toxins in the genesis of atherosclerosis remains elusive. Reactive oxygen species (ROS) play a major role in the development of atherosclerosis. In the present study, we describe the effect of the uraemic toxin phenylacetic acid (PAA) on the inducible nitric oxide synthase (iNOS) and the consecutive production of ROS.


METHODS
Vascular smooth muscle cells (VSMC) were stimulated by IL-1beta in the absence and presence of different concentrations of PAA (0.1-5.0 mM). Inducible NOS (iNOS) mRNA was determined by real-time PCR, iNOS protein was examined by western blotting. The NO degradation product, nitrite, was measured by Griess-assay and peroxynitrite (ONOO(-)) was assessed by hydroethidium, 2',7'-dichlorodihydro-fluorescein diacetate (H(2)DCFDA) fluorescence assays. To evaluate the iNOS cofactor tetrahydrobiopterin (BH(4)), the expression of the key enzyme GTP cyclohydrolase (GTPCH) was determined by real-time PCR.


RESULTS
PAA enhanced the IL-1beta-mediated induction of iNOS expression regarding both mRNA and protein. Nitrite was significantly increased only with high concentrations of 5 mM PAA. ONOO(-), however, was enhanced in a dose-dependent manner. GTPCH, the key enzyme in BH(4) synthesis, was not enhanced by PAA.


CONCLUSIONS
The uraemic toxin, PAA, leads to an induction of iNOS expression, resulting in an increase of ONOO(-) production. The increased production of ONOO(-) might be explained by a lack of GTPCH-catalysed BH(4) synthesis leading to an uncoupled electron transfer. Thus, PAA might contribute to the oxidative stress in ESRD patients.
Schmidt
Sven
S
Medizinische Klinik IV--Nephrology, Charité Campus Benjamin Franklin, Berlin. s.schmidt@charite.de
Westhoff
Timm H
TH
Krauser
Phillip
P
Zidek
Walter
W
van der Giet
Markus
M
D000818:Animals; D002478:Cells, Cultured; D009131:Muscle, Smooth, Vascular / Q000166:cytology* / Q000378:metabolism*; D010648:Phenylacetates / Q000494:pharmacology*; D051381:Rats; D011921:Rats, Inbred WKY; D017382:Reactive Oxygen Species / Q000378:metabolism*
D010648:Phenylacetates; D017382:Reactive Oxygen Species; C025136:phenylacetic acid