Regulation by the cAMP Pathway of Primary Cilia Length in LLC-PK1 Renal Epithelial Cells

CANTERO MR; PÉREZ PL; CANTIELLO HF; SCARINCI MN

Rosario

Congreso; Reunión Anual SAFIS 2019; 2019

The primary cilium (PC) is a non-motile sensory organelle whose dysfunction leads to the onset of autosomal dominant polycystic kidney disease (ADPKD). Previous studies demonstrated that the PC of renal epithelial cells express a functional polycystin-2 (PC2, TRPP2) channel, and the type-2 vasopressin receptor (V2R), whose activation mediates the local elevation of cAMP. Although PC2 contributes to the regulation of Ca2+ transport in renal epithelial cells, little is known as to how PC2 contributes to the regulation of PC length in renal epithelial cells. In this study we explored how the Ca2+- and cAMP- signaling pathways crosstalk and regulate PC length in LLC-PK1 renal epithelial cells. The length of the PC was obtained by tracing the immunochemical signal obtained with a specific antibody against -acetylated tubulin with the ImageJ software. Under normal Ca2+ conditions (1.2 mM), arginine-vasopressin (AVP, 10 µM) increased PC length by 15.47% respect to its control condition (4.72 ± 0.05 µm, n = 510, vs. 5.45 ± 0.09 µm, n = 120, p < 0.001). Treatment of cells with the cAMP analog, 8-Br-cAMP (1 mM) in the presence of normal Ca2+ also increased PC length by 16.31 ± 1.46% (5.49 ± 0.08 µm, n = 157, p < 0.001). Despite the elongating effect of the cAMP signals (both AVP and 8Br-cAMP) on PC length in normal Ca2+, cells exposed to high external Ca2+ (6.2 mM) had shorter PC lengths both in the absence (13,56%, 4.08 ± 0.06 µm, n = 653, p < 0.001 ) or presence of AVP (12.72%, 4.12 ± 0.11 µm, n = 140, p < 0.001) compared to their respective controls in normal Ca2+. Cells exposed to 8-Br-cAMP, however, reverted the effect of high Ca2+, with a 13.77% increase (5.37 ± 0.12 µm, n = 136, p < 0.001) on PC length. The encompassed evidence indicates a crosstalk between Ca2+ and cAMP signals on the PC length of renal epithelial cells. The differential effects of 8Br-cAMP and AVP in high Ca2+, however, suggests the presence of a possible secondary pathway not associated with a V2R-mediated increment in cAMP but possibly a Ca2+-associated V1R activation as well. The functional interaction between signaling pathways could be relevant in the final outcome of the control of kidney function, and the onset of mechanisms that trigger cyst formation in ADPKD.