Loss ofvhlin the zebrafish pronephros recapitulates early stages of human clear cell renal cell carcinoma

Abstract

Patients with von Hippel–Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. ‘Clear cell’ tumors contain large, proliferating 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 (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl−/−) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl−/− zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl−/− zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl−/− pronephros is reminiscent of clear cell histology, indicating that the vhl−/− mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl−/− zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC.

The <b><i>von Hippel-Lindau</i></b> Gene Is Required to Maintain Renal Proximal Tubule and Glomerulus Integrity in Zebrafish Larvae

“…We have previously shown that zebrafish mutant in the VHL tumour suppressor develop key aspects of the human disease condition, including the activation of the HIF signalling pathway, the development of polycythemia and excessive neovascularization of the retina and brain, and recently, the loss of vhl was also shown to recapitulate ccRCC characteristics in zebrafish [29]. Here we report that vhl is required to maintain pronephric tubule and glomerulus integrity in zebrafish embryos.…”

Ciliary Genes in Renal Cystic Diseases

“…Renal cancer cells devoid of active pVHL are cilia deficient [156], while re-expression of pVHL in RCC cells restores cilia in a manner dependent on HIF-1α degradation [157]. The VHL-mediated control of cilia formation is evolutionary conserved as vhl −/− zebrafish mutants develop disorganized proximal pronephric tubules with disordered cilia [158]. Furthermore, pVHL protects microtubules from depolimerization in vivo [157] and pVHL-mediated stabilization of the axoneme microtubules is attenuated by GSK3β-mediated phosphorylation [159].…”

Renal Cell Carcinoma in von Hippel–Lindau Disease—From Tumor Genetics to Novel Therapeutic Strategies

“…Given the diversity of response, predictive biomarkers, such as HIF-2α and p53, may be useful for effective, targeted treatment. In zebrafish, treatment of vhl −/− embryos with a specific HIF2α inhibitor rescued pronephric abnormalities similar to human precancerous disease ( 38 ).…”

“…Likewise in zebrafish embryos, homozygous inactivation of VHL ( vhl −/− ) results in a kidney with enlarged proximal pronephric tubules, disorganized cilia, accumulated lipid and glycogen, cell proliferation, and apoptosis. This phenotype was rescued by a specific HIF2α inhibitor, showing that the zebrafish model system could be used to facilitate rapid screening of candidate drugs ( 38 ).…”