A high content 3D in vitro screening assay that uses primary patient material to model Autosomal Dominant Polycystic Kidney Disease

Background

Autosomal Dominant Polycystic Kidney Disease (ADPKD) Is a genetic disorder that has a prevalence of 1:2500. The disease is characterized by the formation of fluid-filled cysts in the kidney that over time will severely compromise renal function and will lead to renal failure. Although it is known for some time which mutation are causing ADPKD, is not yet understood why these lead to cyst formation. Currently there is only one drug on the market for ADPKD, a vasopressin receptor 2 inhibitor Tolvaptan. Tolvaptan does not prevent cysts from forming but slows down the cyst swelling. Also, Tolvaptan has side effects such as liver damage and polyuria.

Here, we report the development of an ex vivo 3D screening assay that uses primary cells from resected kidneys from ADPKD patients to facilitate in vitro studies for ADPKD and identify new druggable targets and test potentially therapeutic molecules

Results

Tissue was harvested directly from resected kidneys and cultured in an optimized protein hydrogel and optimized culture medium for cryopreservation. Cultures propagated from these cryopreserved vials developed a cystic phenotype and could be stably maintained in 3D culture. Swelling of the cystic structures could be induced using forskolin, which activates adenylyl cyclase and using desmopressin, a vasopressin analog which activates the vasopressin 2 receptor. This sensitivity was maintained throughout the culture period. 3D cultures were seeded in 384 well plates to test a panel of reference compounds, amongst which tolvaptan and rapamycin in combination with desmopressin or forskolin. Compound effects were visualized with high content imaging of the nuclei and F-actin cytoskeleton.  Using in house developed Ominer® analysis software, the entire z-stack of each well was recapitulated for 3D quantification and morphometric analysis. Measurement of phenotypic characteristics such as cyst size, shape and thickness of the cyst wall aided to distinguish between reduction of cyst swelling or an adverse response leading to cell death. To date, a collection of 5 patients is available for large compound screens.

Conclusion

To address the need for more representative in vitro assays to study ADPKD, we have developed an imaging based 3D screening assay that uses low-passage patient material as cell model. With this assay, we have successfully tested a panel of reference compounds. Using our image-based approach we are able to discriminate between efficacy or toxicity inducing compounds. This assay offers a powerful tool for future drug and target discovery as well as mechanistic studies for ADPKD.