Publication Type:Journal Article
Source:Kidney Int, Volume 86, Issue 1, p.58-66 (2014)
Keywords:Adolescent, Adult, alpha-Galactosidase, Animals, asb, Biomarkers, Case-Control Studies, Cell Line, Disease Models, Animal, Endothelial Cells, Fabry Disease, Human Umbilical Vein Endothelial Cells, Humans, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Middle Aged, Nitric Oxide Synthase Type III, RNA, Small Interfering, Tyrosine, Vascular Diseases, Young Adult
The endothelial dysfunction of Fabry disease results from α-galactosidase A deficiency leading to the accumulation of globotriaosylceramide. Vasculopathy in the α-galactosidase A null mouse is manifested as oxidant-induced thrombosis, accelerated atherogenesis, and impaired arterial reactivity. To better understand the pathogenesis of Fabry disease in humans, we generated a human cell model by using RNA interference. Hybrid endothelial cells were transiently transfected with small interfering RNA (siRNA) specifically directed against α-galactosidase A. Knockdown of α-galactosidase A was confirmed using immunoblotting and globotriaosylceramide accumulation. Endothelial nitric oxide synthase (eNOS) activity was correspondingly decreased by >60%. Levels of 3-nitrotyrosine (3NT), a specific marker for reactive nitrogen species and quantified using mass spectrometry, increased by 40- to 120-fold without corresponding changes in other oxidized amino acids, consistent with eNOS-derived reactive nitrogen species as the source of the reactive oxygen species. eNOS uncoupling was confirmed by the observed increase in free plasma and protein-bound aortic 3NT levels in the α-galactosidase A knockout mice. Finally, 3NT levels, assayed in biobanked plasma samples from patients with classical Fabry disease, were over sixfold elevated compared with age- and gender-matched controls. Thus, 3NT may serve as a biomarker for the vascular involvement in Fabry disease.