2 Cystinosis (ctns) zebrafish mutant shows pronephric glomerular and tubular dysfunction 29 both genotypes. (f) Transmission EM image of the glomerulus of a 6 dpf wt larva showing normal foot processes (bar=2 µm). A magnified EM image (right) of podocytes of 6 dpf wt larva showing preserved podocytes slit diaphragms (bar=1 µm). (g) Transmission EM image of the glomerulus of a 6 dpf ctns‒/‒ larva showing partial foot process effacement (black arrows) (bar=2 µm). A magnified EM image (right) of podocytes of 6 dpf ctns‒/‒ larva showing narrowed podocyte slit diaphragmatic spaces (white arrows) (bar=1 µm). (h) Quantitation of podocyte foot process width (FPW) in cut sections at the level of the glomerulus in both genotypes. bb, brush border; bs, Bowman’s space; g, glomerulus; n, nucleus; pt, proximal tubule. * P<0.05, *** P<0.001 between the 2 genotypes using student's t test. ctns‒/‒ zebrafish pronephros shows signs of glomerular disease defective glomerular permselectivity Since many aspects of human and zebrafish cystinosis were similar, we investigated the functional consequences of the disruption of ctns gene in zebrafish larvae. One of the functional tests for the zebrafish kidney is measuring the time required for dextran clearance from the pronephros. In case of a glomerular defect, high molecular weight(HMW)dextran is expected to be lost more rapidly from the vasculature due to impaired glomerular filtration barrier (GFB). Thus, we injected fluorescent labelled 70kDa dextran into the vascular system of 72 hpf larvae (N=20 of each genotype). After 24 hours we monitored the fluorescence intensity of each larva over the retinal vascular bed16. The fluorescence intensity in ctns‒/‒ larvae was significantly lower compared to wt larvae, P<0.001 (Fig. 6a-c). Furthermore, the number of 70-kDa dextran droplets visualized passing through the proximal tubular wall of 72 hpf ctns‒/‒ larvae fixed in 4% paraformaldehyde (PF) 1h after injection was significantly higher when compared to wt larvae denoting also the increased passage of the 70-kDa dextran in the glomerular filtrate (N=10 of each genotype), P=0.031 (Fig. 6). decreased glomerular filtration rate (GFR) Human cystinosis patients develop a slow and gradual decrease in GFR usually starting during childhood. Hence we evaluated the GFR of mutant ctns‒/‒ larvae compared to that of the wt by injecting FITC-inulin into the vascular system of larvae at 96 hpf36. Inulin is freely passing through the glomerular membrane, not reabsorbed and not excreted from the tubular cells, thus is widely used for the assessment of GFR. We monitored the percent of fluorescence intensity decline over 3 fixed anatomical positions in the caudal artery of each larva after 4 hours of injection (Supplementary Fig. S4 online). The percentage of decline of fluorescent intensity in ctns‒/‒ larvae (65.3±5.1%, N=43) was slightly but significantly reduced compared to wt larvae (68.7±4.2%, N=45), P<0.001 denoting the early affection of GFR in ctns‒/‒ zebrafish larvae. The difference was significant in two Figure 5. Continued
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