Human Induced Pluripotent Stem Cell (iPSC)-Derived Kidney Tubuloid System to Study the Earliest Cellular Consequences of VHL Loss

Background: The cellular consequences of genetic loss of the VHL (von Hippel–Lindau) and the emergence of clear cell renal cell carcinoma (ccRCC) from proximal tubules of the kidney are still poorly understood. Mouse studies have faced multiple hurdles to comprehensively address this unmet need. Thus, we focus on an iPSC-derived organoid/tubuloid/2D culture system to recapitulate fundamental kidney functions and address the earliest effects of removing VHL expression from proximal tubular cells. Methods: Using genomic engineering we generated iPSC lines to conditionally inactivate VHL in a heterozygous or homozygous state. To study VHL in human proximal tubules, iPSCs were differentiated into kidney organoids, followed by kidney tubuloids and finally seeded on permeable support. These cells closely resemble a matured renal epithelium based on immunofluorescence and qRT-PCR for markers of the different nephron cell lineages. Upon conditional inactivation of VHL, Western blotting and q-RTPCR were used to confirm VHL loss, and analyze effects of hypoxia, DNA damage and proliferation. Results: The differentiation protocol resulted in the successful generation of human iPSC-derived kidney tubuloids expressing marker proteins such as HNF4A, HNF1B, AQP1, SGLT1, and SGLT2. Moreover, the 2D culture exhibited a postmitotic phenotype resembling the native nephron epithelium. Conditional ablation of VHL resulted in the upregulation of hypoxia markers such as HIF2 and other VHL-target genes such as Aurora A kinase. Moreover, VHL-deficient cells also demonstrated genomic instability based on marker genes such phospho-Histone H2AX protein. Together, in our iPSC-derived kidney epithelial model the loss of VHL activates hypoxia signaling pathways and cell cycle dysregulation suggesting a shift of a specific population of cells toward a pre-neoplastic state. Conclusion: We have established a conditional allele of VHL in iPSCs and a differentiation protocol to induce iPSCs into mature post-mitotic kidney tubules that for the first time allows us inactivating VHL in a cell culture system in its native environment. This permits to effectively investigate the underlying mechanisms how loss of VHL causes cellular changes in the proximal tubules and how it triggers additional genetic insults that ultimately lead to clear cell renal cell carcinoma, the most abundant cancer in the kidney.