5 Increased dynamin expression precedes proteinuria in glomerular disease 87 Animal studies: zebrafish Wild-type (WT) AB/TL strain zebrafish (Danio rerio H) were maintained using previously defined standards.(81) Embryos were obtained through natural crossings and kept in E3 medium at 28.5 °C. In the 1-4-cell stage, the embryos were injected with 1 nl of a morpholino (Gene Tools, Philomath, OR) targeting the dnm2 gene (to knockdown mRNA translation) or a scrambled control morpholino.(82) Glomerular permeability and tubular reabsorption assay A previously validated tubular dextran reabsorption model was used to assess glomerular permeability in zebrafish embryos.(40, 43) At 4 days post-fertilization (dpf), a group of wildtype embryos were injected with 3 nl puromycin aminonucleoside (PAN, 25 mg/ml, SigmaAldrich, St. Louis, MO) to induce kidney damage as a positive control for proteinuria.(40) At 5 dpf, all groups were injected with 3 nl of a mixture of TRITC-labeled 3 kDa (100 mg/ ml; Invitrogen) and FITC-labeled 70-kDa (25 mg/ml; Invitrogen) dextran. One hour after injection, the animals were fixed in 10 % formalin for 24 hours. After subsequent storage in 70 % ethanol, the embryos were embedded in paraffin, sectioned at 3 µm thickness, and examined using fluorescence microscopy for the presence of reabsorption droplets in the proximal tubule epithelial cells by an investigator who was blinded with respect to the treatment conditions. The number of reabsorption droplets in the proximal tubule cells was counted and compared between groups. The 3 kDa dextran tracer freely passes the GFB and is subsequently reabsorbed by the proximal tubule epithelial cells. Therefore, reabsorption of this tracer is used as an indicator of sufficient tubular reabsorption function. In contrast, the 70 kDa tracer does not readily pass the GFB under normal conditions and is therefore used as an indicator of glomerular permeability.(43) The PAN and dextran injections were performed under anesthesia with 4 % tricaine methanesulfonate. All zebrafish experiments were performed prior to the free-feeding embryo stage and are therefore not considered animal experiments in accordance with the EU Animal Protection Directive 2010/63/EU. Human materials Biopsies from 26 patients with a variety of proteinuric kidney diseases were obtained from the LUMC tissue archive. Eight biopsies were from patients with minimal change disease, three were from patients with focal segmental glomerulosclerosis, three were from patients with IgA nephropathy, six were from patients with lupus nephritis, and six were from patients with diabetic nephropathy. In addition, 16 samples were used as a control group; these samples consisted of biopsy material obtained from patients with no evidence of glomerular pathology whose kidneys were unsuitable for transplantation due
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