NDT Advance Access originally published online on October 27, 2007
Nephrology Dialysis Transplantation 2008 23(1):65-71; doi:10.1093/ndt/gfm475
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The uraemic toxin phenylacetic acid increases the formation of reactive oxygen species in vascular smooth muscle cells
Medizinische Klinik IV – Nephrology, Charité Campus Benjamin Franklin, Berlin, Germany
Correspondence to: Dr Med. Sven Schmidt, Charité, Campus Benjamin Franklin, Medizinische Klinik IV, Nephrology, Hindenburgdamm 30, 12200 Berlin. Email: s.schmidt{at}charite.de
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Abstract |
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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-1β 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 (H2DCFDA) fluorescence assays. To evaluate the iNOS cofactor tetrahydrobiopterin (BH4), the expression of the key enzyme GTP cyclohydrolase (GTPCH) was determined by real-time PCR.
Results. PAA enhanced the IL-1β-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 BH4 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 BH4 synthesis leading to an uncoupled electron transfer. Thus, PAA might contribute to the oxidative stress in ESRD patients.
Keywords: atherosclerosis; end-stage renal disease; iNOS; peroxynitrite; phenylacetic acid; uraemic toxin
Received for publication: 12. 2.07
Accepted in revised form: 22. 6.07