3 Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos 49 Introduction Proteinuria is associated with a wide variety of renal diseases. Proteinuria is an independent risk factor for loss of renal function, renal failure, and cardiovascular mortality.(1, 18) Proteinuria occurs when proteins pass the glomerular filtration barrier (GFB) and are not fully reabsorbed by tubular epithelial cells; proteinuria can also develop when the reabsorption system is saturated. The GFB is comprised of fenestrated endothelial cells covered by a glycocalyx, the glomerular basement membrane (GBM), and podocyte foot processes.(19) The GFB filters molecules based on size and charge.(20) The GFB’s charge selectivity has long been attributed primarily to heparan sulfate proteoglycans (HSPGs) in the GBM.(8) HSPGs consist of a core protein to which heparan sulfate glycosaminoglycan (HS-GAG) chains are covalently attached. The HS-GAG chain is polymerized by enzymes encoded by the EXT1 and EXT2 genes. A defect in either is considered to be sufficient severely disrupt HS-GAG synthesis. Both during and after polymerization, the chain can be modified by several sulfotransferases and an epimerase, resulting in the addition of sulfate groups, which give the HS-GAGs and thus the whole HSPG a negative net charge.(9, 21) HS-GAGs have been considered to be essential for GFB permeability as far back as 1980, when Kanwar et al. showed that enzymatic removal of HS-GAGs resulted in increased GBM permeability.(7, 8) These early results were supported by the finding that HSPG expression is reduced in several proteinuric renal diseases.(22) However, recent findings in various mouse models have challenged the notion that HS-GAGs are essential for maintaining GFB permeability; these models include podocyte-specific knockouts of perlecan and agrin (two specific types of HSPGs),(23, 24) Ext1,(25) Extl3,(26) and Ndst1 (an enzyme involved in GAG sulfation).(27) . Although most of these models have a loss of anionic charge in the GBM, they do not develop significant proteinuria. However, HSPGs are also synthesized by mesangial cells and by glomerular endothelial cells.(28, 29) HSPGs produced by glomerular endothelial cells have been shown to be important for GFB function, as removal of HS-GAG from glomerular endothelial cells results in increased permeability to albumin in in vitro experiments.(29) The effects of an in vivo global HS-GAG deficiency on glomerular permeability remains unknown. Thus, the primary goal of this study was to investigate the effect of a global (in contrast to a podocyte-specific) HS-GAG deficiency on glomerular permeability. In addition, although various experimental models using podocyte-specific HS-GAG deletion do not develop proteinuria, they may have increased glomerular permeability. Indeed, Chen et al. hypothesized that a relatively moderate increase in tubular reabsorption may
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