Increased plasma phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression
J. Jankowski, M. van der Giet, V. Jankowski, S. Schmidt, M. Hemeier, B. Mahn, G. Giebing, M. Tölle, H. Luftmann, H. Schlüter, W. Zidek, M. Tepel
J. Jankowski, M. van der Giet, V. Jankowski, S. Schmidt, M. Hemeier, B. Mahn, G. Giebing, M. Tölle, H. Luftmann, H. Schlüter, W. Zidek, M. Tepel
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Article Endocrinology

Increased plasma phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression

Abstract

NO prevents atherogenesis and inflammation in vessel walls by inhibition of cell proliferation and cytokine-induced endothelial expression of adhesion molecules and proinflammatory cytokines. Reduced NO production due to inhibition of either eNOS or iNOS may therefore reinforce atherosclerosis. Patients with end-stage renal failure show markedly increased mortality due to atherosclerosis. In the present study we tested the hypothesis that uremic toxins are responsible for reduced iNOS expression. LPS-induced iNOS expression in mononuclear leukocytes was studied using real-time PCR. The iNOS expression was blocked by addition of plasma from patients with end-stage renal failure, whereas plasma from healthy controls had no effect. Hemofiltrate obtained from patients with end-stage renal failure was fractionated by chromatographic methods. The chromatographic procedures revealed a homogenous fraction that inhibits iNOS expression. Using gas chromatography/mass spectrometry, this inhibitor was identified as phenylacetic acid. Authentic phenylacetic acid inhibited iNOS expression in a dose-dependent manner. In healthy control subjects, plasma concentrations were below the detection level, whereas patients with end-stage renal failure had a phenylacetic acid concentration of 3.49 ± 0.33 mmol/l (n = 41). It is concluded that accumulation of phenylacetic acid in patients with end-stage renal failure inhibits iNOS expression. That mechanism may contribute to increased atherosclerosis and cardiovascular morbidity in patients with end-stage renal failure.

Authors

J. Jankowski, M. van der Giet, V. Jankowski, S. Schmidt, M. Hemeier, B. Mahn, G. Giebing, M. Tölle, H. Luftmann, H. Schlüter, W. Zidek, M. Tepel

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Expression of iNOS measured by real-time PCR, protein blotting of iNOS, ...
Expression of iNOS measured by real-time PCR, protein blotting of iNOS, or nitrite formation in RAW 264.7 cells. (a) The iNOS expression was measured by real-time PCR after stimulation by LPS (1 μg/ml) (control) and in the presence of various concentrations of PAA (0.1 mmol/l, 0.5 mmol/l, 1.0 mmol/l, and 5.0 mmol/l). Basal, unstimulated iNOS expression was set to 100% (n = 6). *P < 0.05 compared with control. (b) Representative protein blotting of iNOS and β-actin after 12 hours of stimulation (+) or without stimulation (–) of RAW 264.7 cells with LPS (1 μg/ml) and in the presence of PAA (0.1 mmol/l, 0.5 mmol/l, 1.0 mmol/l, and 5.0 mmol/l). The iNOS protein was detected as a band with a molecular mass of approximately 125 kDa. (c) Signals of iNOS were quantified and normalized to those of β-actin using a bioimaging analyzer. Data represent means of triplicate determinations from each of three protein preparations (n = 3). *P < 0.05 compared with control. (d) Effect of various concentrations of PAA (0.1 mmol/l, 0.5 mmol/l, 1.0 mmol/l, and 5.0 mmol/l) on LPS-induced nitrite production in RAW 264.7 cells. Data are means ± SEM (n = 6). *P < 0.05 compared with control (+).