Chapter 3 56 Discussion Here, we investigated whether HS-GAGs play an essential role in glomerular permeability using homozygous dak/ext2 mutant zebrafish in which HS-GAGs are lacking in the entire embryo. We found that homozygous dak/ext2 mutants develop an overall phenotype similar to previous reports.(34, 36) PEI staining of the GBM revealed significantly fewer anionic sites in homozygous mutants compared to WT embryos; however, despite having fewer negative sites in the GBM, homozygous dak/ext2 mutants had normal glomerular permeability. Furthermore, our ultrastructural analysis revealed no difference between homozygous dak/ext2 mutants and WT embryos with respect to podocyte foot width. The classic paradigm based on the results of Kanwar et al.(8, 42) suggests that HSGAGs are essential for normal glomerular filtration. In their study, the authors found that glomerular permeability increased after perfusing the kidney with heparinase III, an enzyme that cleaves HS-GAG chains. However, several other studies showed that podocyte-specific knockout of HS-GAGs did not result in proteinuria, suggesting that HS-GAGs may not necessarily be required for normal glomerular filtration.(23-27) Consistent with this notion, our results show that even when HS-GAGs are deleted in the entire embryo (and not specifically in podocytes), glomerular filtration is essentially intact. It remains possible that the size- and charge-selective properties of the GFB are altered in HS-GAG deficiency. However, overall glomerular permeability remains intact despite a severe and global loss of HS-GAG, as was hypothesized. Previous in vivo models of HS-GAG deficiency used proteinuria as a readout of glomerular permeability; thus, albumin retrieval by proximal tubule epithelial cells may have masked changes in glomerular permeability, as suggested by Chen et al.(25) Therefore, we used tubular reabsorption of 70-kDa dextran particles as our readout of glomerular permeability; this method has previously been reported as providing a reliable measure of overall glomerular permeability.(40, 43) Although dextrans are known to move through a network as chains rather than as hydrodynamic spheres, dextrans have been validated as a sensitive marker for loss of glomerular permeability in various zebrafish models.(40, 4346) Tubular reabsorption mechanisms were found to be fully functional in homozygous dak/ext2 mutants, as a similar number of 3-kDa dextran droplets, which readily pass the GFB, was found between mutant and wild-type embryos. Pericardial edema has previously been used as an indicator of renal damage in zebrafish. (43) Interestingly, although homozygous dak/ext2 mutants lack HS-GAGs throughout the entire body and develop pericardial edema, they have essentially normal glomerular permeability. We therefore speculate that the pericardial edema observed in homozygous
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