Loss of von Hippel‐Lindau protein in renal collecting duct cells is associated with massive changes in gene expression and induces a mesenchymal phenotype

Clear‐cell renal cell carcinoma (ccRCC) is the most common form of kidney cancer. It is characterized by a loss of function mutation of the von‐Hippel‐Lindau tumor suppressor (VHL). The proximal tubule is traditionally the source of ccRCC but there is also evidence that a subset con also originate from collecting duct. Alongside the renal nephron, cells are exposed to increasing concentrations of NaCl and urea. In the collecting duct, osmolality can reach up to 1200 mosmol/kg, generating a hyperosmolar environment. It has previously been shown, that VHL deficient mice express a phenotype of polyuria and reduced aquaporin‐2 expression, which is predicted to be caused by a hypoosmolar environment in the nephron. This implicates a physiological function of VHL in the collecting duct segment. Most research however, that discusses the effects of VHL deletion has been conducted with renal cancer cell lines such as 786‐0, Caki or RENCA, which harbor various other specific mutations. Within this study we analyzed the impact of VHL deletion, using “healthy” renal collecting duct cells. Materials and methods We created a VHL deficient mpkCCD cell line by using CRISPR/Cas9. Single clones with functional VHL deletion were selected. The knockout was confirmed by western blotting. Cell morphology studies were performed, using the immune fluorescence method. The gene expression profiling was performed by Next Generation Sequencing analysis and results were validated with real time PCR. Proliferation and migration assays were performed for functional characterization of the cells. Results We successfully generated single clones harboring dysfunctional VHL. This was associated with stable Hif1a expression. VHL deficient mpkCCD cells undergo drastic morphological and functional changes. We observed the partial loss of tight and adherent junctions. Deletion of VHL is also associated with higher migration and lower proliferation potential compared to wildtype cells. The expression of Aqp2, a specific marker of the collection duct epithelial cells, was massively reduced. RNA‐seq analysis showed massive changes in global gene expression pattern and cancer associated pathways were affected by VHL deletion. Correlation with the Humans Pathology Atlas showed that VHL loss induces an unfavorable gene expression pattern. Conclusions Since the mpkCCD cells only lack active VHL, we are able to conclude a direct link between the loss of function of VHL and the cancerous transformation of epithelial cells. Therefore, our results might be useful for further research and novel treatment strategies in the future. Support or Funding Information German Research Fundation