Abstract: PO1241
Analysis of Calcium Signaling in Autosomal Dominant Polycystic Kidney Disease (ADPKD)
Session Information
- Cystic Kidney Disease - I
November 04, 2021 | Location: On-Demand, Virtual Only
Abstract Time: 10:00 AM - 12:00 PM
Category: Genetic Diseases of the Kidneys
- 1001 Genetic Diseases of the Kidneys: Cystic
Authors
- Van Giel, Dorien, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium
- Decuypere, Jean-Paul, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium
- Mekahli, Djalila, Katholieke Universiteit Leuven, Leuven, Belgium
- Vennekens, Rudi, Katholieke Universiteit Leuven, Leuven, Flanders, Belgium
Background
ADPKD is an inheritable kidney disease characterized by the development of fluid-filled renal cysts, mainly caused by mutations in the PKD1 and PKD2 genes, leading to loss of renal function. Molecular mechanisms underlying cystogenesis are poorly characterized but it is postulated that disturbed calcium homeostasis is a primary event in cystogenesis. The precise molecular players that cause this disturbance are still a poorly explored area, especially in relevant human cell types. We therefore aim to characterize the profile of calcium-coupled G-protein coupled receptors (GPCRs) in a human renal epithelial cell models, to identify which receptors are present, whether their function is affected in ADPKD and whether they can be used to modulate cyst formation and growth.
Methods
Urine-derived conditionally immortalized proximal tubule epithelial cells (ciPTECs) of ADPKD patients and healthy controls were screened for calcium-coupled GPCRs, using an agonist library on Fura-2 loaded cell populations seeded in 96-well format. Validation of specific hits was done using single-cell measurements with a fluorescence microscope and built-in perfusion system in the ciPTECs as well as in tissue-derived conditionally immortalized cystic cells (ciCCs). Matrigel-based 3D cell culture was used to grow ciCCs to assess their ability to form cystic structures. Structures were stained with nuclear and cytosolic stains and imaged via confocal microscopy.
Results
From a library of 418 GPCR agonists a selective amount of calcium-coupled GPCRs was found functionally active in ciPTECs. ciPTECs from both healthy controls and ADPKD patients were found to functionally express purinergic -, histamine -, serotonin and dopamine receptors. In single-cell experiments, we did not find any significant differences in functionality between healthy controls and ADPKD patients, but observed that response characteristics are mainly donor-specific, suggesting patient-specific disease mechanisms. ciCCs grown in in 3D cell culture were found to form hollow, cell-lined cyst-like structures.
Conclusion
We describe the first thorough characterization of calcium-coupled GPCRs in a human proximal tubule epithelial cell model. We established a 3D cyst growth assay using tissue-derived cystic cells to explore the possibility to use the identified GPCRs to modulate cyst formation and growth.
Funding
- Government Support – Non-U.S.