5 Increased dynamin expression precedes proteinuria in glomerular disease 93 (A and B) Summary of urinary albumin excretion (A) and systolic blood pressure (B). (C and D) Summary of glomerular Dnm2 mRNA(C) and Dnm1 mRNA (D). (E and F) Example images of glomeruli from an SHR rat (E) and Dahl rat (F) immunostained for dynamin. Similar to the pattern seen in human kidney sections, dynamin protein was present in podocytes and the tubular brush border. (G – I) Summary of the percent glomerular positive area for dynamin protein (G), the percent glomerular positive area for cathepsin L protein (H), and glomerular cathepsin L (Ctsl) mRNA (I) in Dahl and SHR rats at the indicated ages. No difference was seen in both dynamin and cathepsin L percent glomerular positive area. *p < 0.05 versus the SHR group. The images in E and F were taken at the same magnification; scale bar = 50 µm. Knocking down dynamin translation causes proteinuria Next, we knocked down dynamin expression in zebrafish embryos using morpholino injection followed by injection with a mixture of 3 kDa and 70 kDa dextran tracers (Figure 2A and B); as a positive control for inducing proteinuria, a separate group of embryos received an injection of puromycin aminonucleoside (PAN). We then quantified the reabsorption of dextran droplets. Our analysis revealed that the mean number of 3 kDa dextran droplets was similar between control zebrafish (which received an injection of a scrambled morpholino), dynamin-knockdown zebrafish, and PAN-injected zebrafish (p = 0.96, Figure 2C), indicating that a knockdown of dynamin in this model does not significantly affect tubular reabsorption. In contrast, the dynamin-knockdown zebrafish had significantly more reabsorption of 70 kDa droplets compared to control zebrafish (p < 0.0001, Figure 2D), indicating that loss of dynamin increases glomerular permeability, as also shown in the zebrafish model by Schiffer et al.(75) Figure 2. Blocking the translation of dnm mRNA in zebrafish embryos causes proteinuria Wild-type zebrafish embryos were injected with an anti-dnm morpholino or a scrambled control morpholino, followed by a mixture of 3 kDa and 70 kDa dextran molecules. (A and B) Figure 1. Continued
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