The role of 6-0-sulphated heparan sulphate in chronic renal fibrosis

Abstract

Heparan sulphate (HS) plays crucial roles during the genesis and resolution of inflammation by sequestration, stabilization and presentation of proinflammatory cytokines and growth factors. The interaction between many of these factors and HS is critically dependent on the variable distribution of anionic 6-O-sulphated glucosamine residues within the structure of HS. The pattern of 6-O-sulphation is generated during HS biosynthesis by HS-6-O-sulphotransferases (HS6STs) but can be modified later by cell-surface HS-6-O-endosulphatases (SULFs). This study was designed to examine the potential contribution of these enzyme families to renal fibrosis following chronic inflammation. Initial experiments showed that the fibrogenic cytokine TGF-β induced SULF2 expression by renal tubular epithelial cells. Immortalized renal tubular epithelial cells were transfected to constitutively overexpress either HS6ST1 or SULF2 in order to examine the effect of these enzymes on cytokine function. Cells which overexpressed HS6ST1 showed increased binding of FGF2 compared to mock transfected control cells; this FGF2 binding correlated with increased pERK expression and enhanced cell proliferation. The requirement for HS for these processes was validated by inhibition of FGF2 binding with soluble HS, heparin or heparitinase III, whilst the importance of HS sulphation for increased binding was demonstrated after treatment of the cells with chlorate. Structural analysis of 35S-labelled HS from HS6ST1 overexpressing cells demonstrated an increase in mono-6-O-sulphated disaccharides accompanied by a decrease in 2-O-sulphated iduronic acid. By contrast, SULF2 transfectants showed reduced FGF2 binding, ERK activation and proliferation. Structural analysis of 35S-labelled HS from these cells showed a 50% reduction in 6-O-sulphation with a parallel increase in mono-2-O-sulfated iduronic acid. The significance of the in vitro study for renal fibrosis was then examined using a murine unilateral ureteric obstruction (UUO) model. Immunochemical analysis of UUO kidney sections showed a significant increase in expression of epitopes containing N- and 6-O sulphated HS around the renal tubules. This change was accompanied by a 5-fold increase in expression of the SULF1 gene. In summary, this study suggests that modulation of the expression of sulphate at the 6-O position in HS plays a significant role in the progression of chronic renal fibrosis by alteration of the biological activity of fibrogenic growth factors.