Chapter 3 58 chondrocytes obtained from patients with EXT1 or EXT2 mutations,(50) in which the specific mutation seemed to determine the effect on cytoskeletal organization. Whether EXT1 and/or EXT2 directly affect cytoskeletal organization in podocytes, independently of HS-GAG side chains, remains an open question. This study has several limitations. First, a direct local decrease in HS-GAGs could not be shown by immunohistochemistry. However, dak/ext2 mutants had significantly less anionic sites in the GBM than WT animals in our PEI labelling experiment. This is indirect proof of a decrease in HS-GAGs. Tus, the degree of HS-GAG deficiency in the glomerulus is at least 63%, as this is the relative difference in PEI particles. These particles could reflect HS-GAGs, other sulfated GAGs or other negatively charged areas. Also, the total decrease of HS-GAGs in dak/ext2 zebrafish embryos has previously been reported to be over 80%.(21, 30) Although a relative and significant decrease in PEI-particles was observed, the absolute extent of PEI-labeling is less than in mammalian systems. A possible explanation is that this is due to a methodological difference, as we did not perform an in vivo labelling.(25) Also, it could be a reflection of interspecies variation in anionic site distribution in the GBM. Furthermore, the dak/ext2 model does not have a complete loss of HS-GAG. Theoretically, a double knock out of ext1 and ext2 would result in even more pronounced loss of HS-GAG. As zebrafish have three ext1 genes (ext1a, ext1b and ext1c) and up to date, characterized mutants lines are not available for any of these semi-orthologues, this could not be confirmed experimentally. To the best of our knowledge, this is the first study to examine the effect of a global HS-GAG deficiency on glomerular permeability. Globally deleting HS-GAGs in most vertebrates causes extremely early lethality;(51) Tus, the homozygous dak/ext2 mutant zebrafish is currently the only viable model for global loss of HS-GAGs that survives gastrulation when both copies of the ext2 gene are impaired and that gives unique opportunity to study glomerular filtration that cannot be rescued by HS-GAGs produced by other type of cells. Although homozygous dak/ext2 mutants do not survive to adulthood, Lee et al. hypothesized that these mutants survive the first part of the embryonic phase due to the maternal contribution of Ext2;(30) by 5 dpf, maternal contribution of Ext2 decreases to non-detectable levels.(30) In conclusion, we provide the first report that glomerular permeability is not affected by a global HS-GAG deficiency. These results support the growing body of evidence that HSGAGs do not play an essential role in mediating glomerular permeability.
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