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Kidney Week

Abstract: FR-PO744

Primary Patient Material as Model for a High Content 3D In Vitro Screening Assay to Study ADPKD

Session Information

Category: Genetic Diseases of the Kidneys

  • 1001 Genetic Diseases of the Kidneys: Cystic

Authors

  • Bange, Hester, OcellO, Leiden, Netherlands
  • Van de water, Bob, Leiden Academic Centre for Drug Research/LACDR, Leiden, Netherlands
  • Peters, Dorien J.M., Leiden University Medical Center, Leiden, Netherlands
  • Price, Leo Sebastian, OcellO, Leiden, Netherlands
Background

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is characterized by the formation of fluid-filled cysts in the kidney. Currently only one drug is on the market for ADPKD, a vasopressin receptor 2 inhibitor Tolvaptan. However, Tolvaptan does not prevent cysts from forming but slows down cyst swelling. Identifying the mechanisms involved in cystogenesis and identification of treatments is hampered by the lack of pathophysiologically relevant in vitro models. Here, we report the development of an ex vivo 3D cyst screening assay that uses primary cells from resected kidneys from ADPKD patients to facilitate in vitro studies for ADPKD.

Methods

Patient tissue was harvested directly from resected kidneys and cultured in 3D in an optimized hydrogel and culture medium until cryopreservation. Short expansion culture was performed in similar culture conditions. Compound testing was done in 384 well plates. After 24h pre-culture, compounds were added for 48h. Plates were fixed and stained for nuclei and F-actin. Plates were imaged and using Ominer ™ high throughput image analysis software the entire z-stack of each well was recapitulated for 3D quantification and morphometric analysis.

Results

Cultures propagated from cryopreserved tissue developed a cystic phenotype that was stably maintained in 3D culture. Swelling of these cysts could be induced using forskolin, which activates adenylyl cyclase and using desmopressin, which activates the vasopressin 2 receptor. Effect of Metformin, Rapamycin, Roscovtine and Tolvaptan was examined in combination with desmopressin or forskolin. Compound effects were visualized with high content imaging. Using Ominer ™ images were analyzed. Measurement of phenotypic characteristics such as nucleus morphology and thickness of the cyst wall enabled discrimination of compounds that reduced cyst swelling and compounds that were cytotoxic. All reference compounds showed the expected decrease in cyst swelling.

Conclusion

We have developed a 3D screening assay that uses low-passage patient material as model of renal cysts. 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.

Funding

  • Commercial Support