3 Glomerular permeability is not affected by heparan sulfate glycosaminoglycan deficiency in zebrafish embryos 57 dak/ext2 mutants is not caused by renal damage. For example, HS-GAG deficiency may have had an effect on the permeability of systemic capillaries because of an altered glycocalyx composition. Such a correlation between the endothelial glycocalyx of systemic capillaries and vascular permeability has been reported previously.(47) An important difference between Kanwar et al.’s study and our approach using a germline mutation is that Kanwar et al. investigated the acute loss of HS-GAG by enzymatic activity, whereas our approach deleted HS-GAGs for an extended period. This difference may account for the difference in results between the two studies. For example, with a germline mutation, the effects will be present throughout the animal’s life; therefore, other GAGs may have functionally replaced the deleted HS-GAGs. In the in situ set-up used by Kanwar et al., loss of HS-GAG is acute and there is not enough time for such a compensatory mechanism to take place. Another possible explanation for the different results is that the enzymes used by Kanwar et al. may have caused collateral damage to other components of the GFB, which could have led to their observed increase in glomerular permeability. The charge-selective nature of the GBM has long been attributed to the presence of HSGAG.(7) In contrast, Miner proposed that charge selectivity is not an essential factor in glomerular permeability, as several studies found that a decreased number of anionic sites does not necessarily coincide with proteinuria.(48) Consistent with this notion, we found that glomerular permeability was not affected in homozygous dak/ext2 mutants, despite the fact that these animals had significantly fewer PEI particles in the GBM. It is important to note that although negatively charged sites were significantly reduced in the GBM of homozygous dak/ext2 mutants, these sites were not completely absent. Thus, these residual anionic sites could reflect negatively charged particles other than HS-GAGs. For example, type IV collagen, which is one of the primary constituents of the GBM, contains negatively charged sialic acid residues.(49) Therefore, we cannot exclude the possibility that the residual negatively charged sites in the GBM are sufficient for conferring charge selectivity. Ourultrastructuralanalysisoftheglomeruliinzebrafishembryosrevealedthathomozygous dak/ext2 mutants have normal podocyte foot processes. Although similar results were reported in podocyte-specific agrin-deficient and perlecan-deficient mice, podocytespecific Ext1 knockout mice have foot process effacement.(23-25) The difference between our global ext2 deficient zebrafish model and the podocyte-specific Ext1 knockout mice may be due to differential effects of Ext1 and Ext2 on podocyte cytoskeletal organization. Both EXT1 and EXT2 have been reported to influence cytoskeletal organization in
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