Indoxyl sulphate induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells.
BACKGROUND
Previously, we demonstrated that indoxyl sulphate (IS), a uraemic toxin, induced aortic calcification in hypertensive rats. This study aimed to determine if IS induces the production of reactive oxygen species (ROS) and the expression of osteoblast-specific proteins in human aortic smooth muscle cells (HASMCs).


METHODS
In order to achieve these goals, HASMCs were incubated with IS. ROS were detected using probes with a fluorescence detector. The expression of alkaline phosphatase (ALP), osteopontin and organic anion transporters (OAT1, OAT3) was studied by western blotting. The expression of core binding factor 1 (Cbfa1), ALP, osteopontin and NADPH oxidases (Nox1, Nox2 and Nox4) was analysed by reverse transcription-polymerase chain reaction (RT-PCR). Knockdown of Nox4 was performed by RNA interference (RNAi).


RESULTS
IS induced ROS generation and the expression of Nox4, Cbfa1, ALP and osteopontin in HASMCs. A NADPH oxidase inhibitor and antioxidants inhibited IS-induced ROS production and mRNA expression of Cbfa1 and ALP. Knockdown of Nox4 using small interfering RNA (siRNA) inhibited IS-induced ROS production and mRNA expression of Cbfa1, ALP and osteopontin. OAT3 was expressed in HASMCs.


CONCLUSIONS
IS induces ROS generation by upregulating Nox4, and the expression of osteoblast-specific proteins such as Cbfa1, ALP and osteopontin in HASMCs.
Muteliefu
Gulinuer
G
Department of Clinical Preventive Medicine, Nagoya University Hospital, Nagoya, Japan.
Enomoto
Atsushi
A
Jiang
Ping
P
Takahashi
Masahide
M
Niwa
Toshimitsu
T
D000469:Alkaline Phosphatase / Q000096:biosynthesis*; D002478:Cells, Cultured; D050659:Core Binding Factor Alpha 1 Subunit / Q000096:biosynthesis*; D006801:Humans; D007200:Indican / Q000494:pharmacology*; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology*; D053495:Osteopontin / Q000096:biosynthesis*; D018384:Oxidative Stress / Q000187:drug effects*
D050659:Core Binding Factor Alpha 1 Subunit; D053495:Osteopontin; D000469:Alkaline Phosphatase; D007200:Indican