MO007ANALYSIS OF CALCIUM SIGNALING IN AUTOSOMAL DOMINANT POLYCYSTIC KIDNEY DISEASE (ADPKD)

Abstract

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inheritable kidney disease characterized by the development of fluid-filled cysts in all nephron segments, leading to loss of renal function. Mutations in PKD1 or PKD2, which encode polycystin-1 and polycystin-2, are the most common cause of ADPKD. The 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 receptors and channels in a human renal epithelial cell model, to identify which receptors and channels are present and whether their function is affected in ADPKD. Method Human urine-derived conditionally immortalized proximal tubule epithelial cells (ciPTECs) of ADPKD patients and healthy controls were screened for calcium-coupled GPCRs, using a GPCR agonist library on Fura-2 loaded cell populations seeded in 96-well format using the Flexstation3 (Molecular Devices). Validation of specific hits was done using single-cell measurements with a fluorescence microscope and built-in perfusion system. The expression of TRP channels and STIM/Orai proteins was determined via qPCR. 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. Through qPCR we found expression of various TRP channels, including TRPML1, TRPC1/3, TRPM3/4/7, TRPV4 and TRPA1, as well as high expression of STIM1/2 and Orai1/2/3. Conclusion We describe the first thorough characterization of molecular players involved in calcium signalling mechanisms in human renal epithelial cells, including the profile of calcium-coupled GPCRs and the expression of TRP channels and STIM/Orai proteins, of further interest to investigate disturbed calcium dynamics in ADPKD.