Chapter 3 52 were also injected with puromycin aminonucleoside (PAN, Sigma-Aldrich, St. Louis, MO) at 4 dpf.(40) One hour after the dextrans were injected, the samples were fixed in 10% formalin for 24 hours and stored in 70% ethanol until further processing. The samples were then embedded in paraffin, sectioned at 4-µm thickness, and examined using immunofluorescence microscopy. The samples were analyzed in a blinded manner. Under physiological conditions, 3-kDa dextran can readily pass the GFB, whereas 70kDa dextran does not. If the integrity of the GFB is sufficiently compromised, the 70-kDa dextran can pass the GFB, after which it is reabsorbed by proximal tubule epithelial cells in endosomes. Therefore, we measured the number of 70-kDa dextran particles in the proximal tubule cells as a measure of glomerular permeability. To confirm functional tubular reabsorption, we also measured the number of 3-kDa dextran particles in the proximal tubule cells. Statistical analyses Statistical analyses were performed using SPSS version 20.0 (IBM Corp., Armonk, NY). The proportion of zebrafish embryos with pericardial edema was analyzed using Fisher’s exact test. Dextran tracer measurements of glomerular permeability, foot process width, and the presence of anionic sites were analyzed using the Student’s unpaired t-test. Differences with a p-value <0.05 were considered significant. Results The dackel mutant phenotype Homozygous dak/ext2to273b (referred to hereafter as simply “dak/ext2”) mutants develop a clear phenotype, as described before.15 Consistent with previous reports, compared to wild-type (WT) embryos, mutant embryos lacked pectoral fins and had a protruding jaw, a non-inflated swim bladder, and a more concave body shape.(34, 36) In addition, homozygous dak/ext2 mutants had a significantly higher prevalence of pericardial edema compared to WT embryos (31/34 versus 1/140, respectively; p<0.0001). An example of morphological changes and pericardial edema in a mutant embryo is shown in Figure 1. Loss of anionic sites in the GBM Because HS-GAGs are believed to be the primary contributor to the negative charge in the GBM(7), we expected that the loss of HS-GAGs in the homozygous dak/ext2 mutants would result in reduced numbers of negatively charged sites in the GBM. We therefore tested this hypothesis using polyethylenimine (PEI) staining;(41) PEI molecules bind to
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