Regulation of ciliary length in LLC-PK1 renal epithelial cells

CANTERO MR; PEREZ PL; CANTIELLO HF; SCARINCI N

San Francisco, California

Congreso; 62th Annual Meeting of the Biophysical Society; 2018

Polycystin-2 (PC2, TRPP2) is a Ca2+ permeable nonselective cation channel whose dysfunction generates autosomal dominant polycystic kidney disease (ADPKD). PC2 is present in different cellular locations, including the primary cilium of LLC-PK1 renal epithelial cells, where it may contribute to ciliary Ca2+ transport. Little is known, however, as to how renal epithelial cells control the length of the primary cilium, and in particular how PC2 function may contribute to this regulation. Here, we explored the effect of maneuvers that either control Ca2+ transport through PC2 or modulate its function, on the ciliary length of LLC-PK1 cells. Cells (2-3 wk old) were labeled with a specific antibody against alpha-acetylated tubulin to identify primary cilia, and DAPI to locate cellular nuclei. Images were collected after overnight exposure of cells to the various experimental conditions. The length of the primary cilium was obtained with the ImageJ software. Primary cilia length measurements did not follow a normal distribution, in any of the conditions tested. Comparisons between groups were conducted either by group (Mann-Whitney) or in between groups (Kruskall Wallis) rendering significant differences for all conditions respect to Control (normal Ca2+, 1.2 mM, n = 199). Cells exposed either to low Ca2+ (n = 212) or high Ca2+ (n = 331) had a decreased primary cilium length by 23.8% and 14.4%, respectively (p < 0.001). Amiloride (200 microM, n = 287) and LiCl (10 mM, n = 92), two chemicals that reduce PC2 function, both induced an increase in primary cilium length of 12.1% and 20.0% (p < 0.001), respectively. The data indicate that PC2 function controls ciliary length in renal epithelial cells, suggesting a Ca2+-mediated regulatory mechanism. Dysregulation of this mechanism may be essential in the onset of ADPKD.