IS induces oxidative stress in VSMCs 2051 31. Van Den Born J, Van Den Heuvel LP, Bakker MA et al. A monoclonal antibody against GBM heparan sulfate induces an acute selective proteinuria in rats. Kidney Int 1992; 41: 115-123 32. Bertolatus JA, Klinzman D. Macromolecular sieving by glomerular basement membrane in vitro: effect of polycation or biochemical modifications. Microvasc Res 1991; 41: 311-327 33. Kanwar YS, Farquhar MG. Isolation of glycosaminoglycans (heparan sulfate) from glomerular

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... sement membranes. Proc Natl Acad Sci U S A 1979; 76: 4493-4497 34. Nayak BR, Spiro RG. Localization and structure of the asparaginelinked oligosaccharides of type IV collagen from glomerular basement membrane and lens capsule. J Biol Chem 1991; 266: 13978-13987 35. Van Den Hoven MJ, Wijnhoven TJ, Li JP et al. Reduction of anionic sites in the glomerular basement membrane by heparanase does not lead to proteinuria. Kidney Int 2008; 73: 278-287 36. Chen S, Wassenhove-McCarthy DJ, Yamaguchi Y et al. Loss of heparan sulfate glycosaminoglycan assembly in podocytes does not lead to proteinuria. Kidney Int 2008; 74: 289-299 37. Dawes J, Pepper DS. Human vascular endothelial cells catabolise exogenous glycosaminoglycans by a novel route. Thromb Haemost 1992; 67: 468-472 38. Comper WD, Tay M, Wells X et al. Desulphation of dextran sulphate during kidney ultrafiltration. Biochem J 1994; 297(Pt 1): 31-34 39. Tay M, Comper WD, Singh AK. Charge selectivity in kidney ultrafiltration is associated with glomerular uptake of transport probes. Am J Physiol 1991; 260: F549-F554 40. Takeda T, McQuistan T, Orlando RA et al. Loss of glomerular foot processes is associated with uncoupling of podocalyxin from the actin cytoskeleton. 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 ISinduced 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.