Supplementary Material for: The <b><i>von Hippel-Lindau</i></b> Gene Is Required to Maintain Renal Proximal Tubule and Glomerulus Integrity in Zebrafish Larvae
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<b><i>Background:</i></b> <i>von Hippel-Lindau</i> (<i>VHL</i>) disease is characterized by the development of benign and malignant tumours in many organ systems, including renal cysts and clear cell renal cell carcinoma. It is not completely understood what underlies the development of renal pathology, and the use of murine <i>Vhl</i> models has been challenging due to limitations in disease conservation. We previously described a zebrafish model bearing inactivating mutations in the orthologue of the human <i>VHL</i> gene. <b><i>Methods:</i></b> We used histopathological and functional assays to investigate the pronephric and glomerular developmental defects in <i>vhl</i> mutant zebrafish, supported by human cell culture assays. <b><i>Results:</i></b> Here, we report that <i>vhl</i> is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos. <i>vhl</i> mutant glomeruli are enlarged, <i>cxcr4a+</i> capillary loops are dilated and the Bowman space is widened. While we did not observe pronephric cysts, the cells of the proximal convoluted and anterior proximal straight tubule are enlarged, periodic acid schiff (PAS) and Oil Red O positive, and display a clear cytoplasm after hematoxylin and eosine staining. Ultrastructural analysis showed the <i>vhl</i><sup>–/–</sup> tubule to accumulate large numbers of vesicles of variable size and electron density. Microinjection of the endocytic fluorescent marker AM1–43 in zebrafish embryos revealed an accumulation of endocytic vesicles in the <i>vhl</i> mutant pronephric tubule, which we can recapitulate in human cells lacking <i>VHL.</i> <b><i>Conclusions:</i></b> Our data indicates that <i>vhl</i> is required to maintain pronephric tubule and glomerulus integrity during zebrafish development, and suggests a role for VHL in endocytic vesicle trafficking.
