Zebrafish model of early stage human ccRCC

Von Hippel–Lindau (VHL) disease is caused by a germline mutation in the VHL gene that leads to the development of several tumors including clear cell renal cell carcinoma (ccRCC) (Maher and Kaelin, 1997). ‘Clear cell’ tumors are characterized by large, proliferating epithelial cells with clear cytoplasm, and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a (HIF1a and HIF2a) that leads to up-regulation of genes involved in cell proliferation, angiogenesis and erythropoiesis (Maxwell et al., 1999). In a new study by Noonan et al., 2016, a zebrafish ccRCC model with a homozygous knockout of the VHL gene (vhl−/−) recapitulates several aspects of the human disease. Authors characterized for the first time the epithelial abnormalities present in the kidney of the vhl−/− zebrafish larvae and showed that treatment of vhl−/− zebrafish embryos with a HIF2a inhibitor partially rescued the mutant phenotype.

To develop the model of ccRCC in zebrafish authors assessed vhl−/− zebrafish pronephric epithelial cells for features of ccRCC cells. The tubular epithelium of each nephron is divided into a proximal and a distal portion. Differences between the vhl−/− larvae and their wt siblings were most clearly observed in the proximal pronephros corroborating the idea that human ccRCC likely originates in the proximal tubule (Chen et al., 2016). Hematoxylin and eosin (H&E) staining of the proximal pronephric tubules revealed several differences between the vhl−/− zebrafish larvae and their wt siblings. The architecture of the vhl−/− pronephric tubules appeared to be distorted compared to their wt siblings and the proximal vhl−/− pronephric epithelium contained abundant cytoplasmic vesicles that were absent in the wt siblings. Resembling the clear cell histology of RCC, transmission electron microscopy (TEM) and staining with a lipophilic fluorescent dye showed that the renal epithelium of vhl−/− larvae contained significantly more lipids both in the proximal and distal pronephros than their wt siblings. In addition, the cilia coating the tubules, which were uniform in the wt siblings, were disorganized and in lower frequencies in the vhl−/− larvae. The presence of cellular glycogen is commonly associated with the clear cell histology in ccRCC (Shuch et al., 2015). PAS staining confirmed that the proximal vhl−/− pronephric tubule had increased glycogen compared to their wt siblings. To assess if the pronephros of vhl−/− zebrafish larvae recapitulates the highly proliferative characteristics of human ccRCC, authors stained larvae with the cell proliferation marker BrdU using a transgenic zebrafish line, Tg(ATPase1.a1A4:GFP) (Majumdar et al., 2000). Confocal microscopy showed that Vhl−/− larval pronephric epithelium displayed significantly higher cellular proliferation compared to wt siblings through development. In addition, Vhl−/− larval pronephric epithelium also displayed significantly higher cell death compared to wt siblings in early development.

Because HIF2a functions as an oncogene in VHL-associated and sporadic cases of ccRCC, authors tested if the expression of the zebrafish orthologs of human HIF2a are responsible for the phenotype reported. Larvae were treated with HIF2a inhibitor Compound 76 or DMSO vehicle control. Treatment of vhl−/− larvae with Compound 76 significantly improved the proximal pronephric tubule phenotype compared to vhl−/− larvae treated only with DMSO. Treated larvae had more organized nuclei, less lipid vesicles and a significantly larger and more structured lumen meaning that the abnormal structure of the proximal vhl−/−pronephric tubule was partially rescued by the HIF2a inhibitor treatment.

In summary, authors identified for the first time a zebrafish model of early stage human ccRCC. The results of the Compound 76 assay suggest that small-molecule HIF2a inhibitors show promise in the treatment of ccRCC. Given the similarities of hypoxia, angiogenesis and erythropoiesis pathways between humans and zebrafish (Kajimura et al., 2006Rojas et al., 2007Paffett-Lugassy et al., 2007), zebrafish vhl mutants provide an important and unique tool for further studies of VHL-related tumor biology, ccRCC development and as a model for developing effective new drug treatments (MacRae and Peterson, 2015).

  • Noonan HR, Metelo AM, Kamei CN, Peterson RT, Drummond IA, & Iliopoulos O (2016). Loss of vhl in the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma. Disease models & mechanisms, 9 (8), 873-84 PMID: 27491085
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