Indoxyl sulphate induces oxidative stress and the expression of osteoblast-specific proteins in vascular smooth muscle cells

Nephrol Dial Transplant. 2009 Jul;24(7):2051-8. doi: 10.1093/ndt/gfn757. Epub 2009 Jan 22.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alkaline Phosphatase / biosynthesis*
  • Cells, Cultured
  • Core Binding Factor Alpha 1 Subunit / biosynthesis*
  • Humans
  • Indican / pharmacology*
  • Muscle, Smooth, Vascular / cytology
  • Muscle, Smooth, Vascular / drug effects*
  • Muscle, Smooth, Vascular / physiology*
  • Osteopontin / biosynthesis*
  • Oxidative Stress / drug effects*

Substances

  • Core Binding Factor Alpha 1 Subunit
  • Osteopontin
  • Alkaline Phosphatase
  • Indican