title: Differential effects of indoxyl sulfate and inorganic phosphate in a murine cerebral endothelial cell line (bEnd.3).
abstract: Endothelial dysfunction plays a key role in stroke in chronic kidney disease patients. To explore the underlying mechanisms, we evaluated the effects of two uremic toxins on cerebral endothelium function. bEnd.3 cells were exposed to indoxyl sulfate (IS) and inorganic phosphate (Pi). Nitric oxide (NO), reactive oxygen species (ROS) and O2•⁻ were measured using specific fluorophores. Peroxynitrite and eNOS uncoupling were evaluated using ebselen, a peroxide scavenger, and tetrahydrobiopterin (BH₄), respectively. Cell viability decreased after IS or Pi treatment (p < 0.01). Both toxins reduced NO production (IS, p < 0.05; Pi, p < 0.001) and induced ROS production (p < 0.001). IS and 2 mM Pi reduced O2•⁻ production (p < 0.001). Antioxidant pretreatment reduced ROS levels in both IS- and Pi-treated cells, but a more marked reduction of O2•⁻ production was observed in Pi-treated cells (p < 0.001). Ebselen reduced the ROS production induced by the two toxins (p < 0.001); suggesting a role of peroxynitrite in this process. BH₄ addition significantly reduced O2•⁻ and increased NO production in Pi-treated cells (p < 0.001), suggesting eNOS uncoupling, but had no effect in IS-treated cells. This study shows, for the first time, that IS and Pi induce cerebral endothelial dysfunction by decreasing NO levels due to enhanced oxidative stress. However, Pi appears to be more deleterious, as it also induces eNOS uncoupling.
authors.affiliation: Inserm U1088, Department of Pharmacy, 1 rue des Louvels, Amiens F-80037 Cédex 1, France. andreastinghen@ufpr.br.
authors.forename: Andréa E M
authors.initials: AE
authors.lastname: Stinghen
authors.affiliation: Inserm U1088, Department of Pharmacy, 1 rue des Louvels, Amiens F-80037 Cédex 1, France. jean-marc.chillon@u-picardie.fr.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Chillon
authors.affiliation: Inserm U1088, Department of Pharmacy, 1 rue des Louvels, Amiens F-80037 Cédex 1, France. massy@u-picardie.fr.
authors.forename: Ziad A
authors.initials: ZA
authors.lastname: Massy
authors.affiliation: Inserm U1088, Department of Pharmacy, 1 rue des Louvels, Amiens F-80037 Cédex 1, France. agnes.boullier@u-picardie.fr.
authors.forename: Agnès
authors.initials: A
authors.lastname: Boullier
mesh_terms: D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D001393:Azoles / Q000494:pharmacology; D001708:Biopterins / Q000031:analogs & derivatives / Q000494:pharmacology; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D002540:Cerebral Cortex / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007200:Indican / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism*; D054833:Isoindoles; D051379:Mice; D018696:Neuroprotective Agents / Q000494:pharmacology; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000037:antagonists & inhibitors / Q000378:metabolism; D016566:Organoselenium Compounds / Q000494:pharmacology; D018384:Oxidative Stress / Q000187:drug effects; D010710:Phosphates / Q000037:antagonists & inhibitors / Q000097:blood / Q000378:metabolism*; D017382:Reactive Oxygen Species / Q000819:agonists / Q000378:metabolism; D013481:Superoxides / Q000037:antagonists & inhibitors / Q000378:metabolism; D015854:Up-Regulation / Q000187:drug effects; D014511:Uremia / Q000097:blood / Q000188:drug therapy / Q000378:metabolism* / Q000473:pathology
chemical_list: D000975:Antioxidants; D001393:Azoles; D004791:Enzyme Inhibitors; D054833:Isoindoles; D018696:Neuroprotective Agents; D016566:Organoselenium Compounds; D010710:Phosphates; D017382:Reactive Oxygen Species; D013481:Superoxides; D001708:Biopterins; D009569:Nitric Oxide; C042986:ebselen; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse; C003402:sapropterin; D007200:Indican