Release of ATP by TRPV4 activation is dependent upon the expression of AQP2 in renal cells

BAZZI, ZAHER; CAPURRO, CLAUDIA; DI GIUSTO, GISELA; SCHWARZBAUM, PABLO J; PIZZONI, ALEJANDRO; RIVAROLA, VALERIA; FORD, PAULA

Rosario

Congreso; Reunión Anual de la Sociedad Argentina de Fisiología 2019; 2019

Sociedad Argentina de Fisiología

The involvement of purinergic signalling in kidney physiology and pathophysiology is rapidly gaining recognition. Purinergic signalling influences water and electrolyte transport in all segments of the renal tubule. In several tissues, there is increasing evidence that ATP release is dependent upon activation of the transient receptor potential cation channel (TRPV4). Because we have recently found that TRPV4 physical and functional interacts with the water channel AQP2 in cortical collecting ducts cells (CCD) (1), the aim of this work was to examine the possibility that TRPV4/AQP2 interaction influences ATP release in these cells. We used two rat CCD cell lines expressing AQP2 (AQP2-RCCD1) or not (WT-RCCD1). Extracellular ATP (ATPe) measurements were carried out with cells laid on coverslips that were mounted in the assay chamber of a custom-built luminometer. Cells were stimulated with the specific TRPV4 activator GSK1016790A (GSK, 10 nM) and ATPe was measured using firefly luciferase. We found that GSK stimulate ATP release only in AQP2- expressing cells (MaxAQP2 = 222.9 ± 32 nM (n=10)). ATP release stimulated by GSK in AQP2-RCCD1 cells was inhibited by the TRPV4 specific antagonist HC-067047 (1 μM) and by extracellular calcium removal. In order to identify if ATP release occurs via a conductive or an exocytic route, before stimulating cells with GSK, we incubated the cells with carbenoxolone (100 μM, to block pannexin 1 and connexin hemi-channels) or brefeldin A (5 μg/ml, an intracellular vesicular transport inhibitor). We found a similar reduction of ATP release with both inhibitors. Interestingly, when we tested both inhibitors together, an additive reduction was observed, suggesting that both mechanisms function independently. In addition, blocking purinoceptors with PPADS (20 μM) strongly reduced ATP release. In conclusion, these findings suggest that in CCD cells AQP2 is critical for the release of ATP induced by TRPV4 activation. Moreover, ATPe, in turn, acts in an autocrine and/or paracrine manner to stimulate PPADS-sensitive purinergic receptors leading to ATPe-induced ATP release.