LAUSR

Mechanical stress arising from elevated fluid flow is an important regulator of the water-electrolyte transport in the collecting duct (CD). It is well established that mechano-sensitive TRPV4 Ca2+-permeable channel mediates intracellular Ca2+ ([Ca2+]i) responses to high flow in these cells. Piezo1, a Ca2+-permeable channel directly activated by the plasma membrane stretch, is a recent arrival at this scene. Piezo1 expression has been reported in the CD. However, its functional significance is yet to be determined. Here, we applied a combination of Ca2+ imaging and confocal microscopy in freshly isolated split-opened CDs to investigate a potential interplay between TRPV4 and Piezo1 in mediating [Ca2+]i signaling in the CD. Immunofluorescent analysis of split-opened CDs showed presence of Piezo1 in both AQP2-positive (principal cells, PC) and -negative (intercalated cells, IC) with higher intensity in the former. Importantly, Piezo1 was apparent on both apical and basolateral membrane of CD cells. Piezo1 agonists, Yoda-1 (20μM) and Jedi2 (500μM) acutely increase [Ca2+]i in both principal and intercalated cells. The responses were abolished when extracellular Ca2+ was buffered with EGTA suggesting a direct Ca2+ influx via Piezo1. Interestingly, we detected two disparate calcium responses to Piezo1 activation with the majority (70%) cells showing a fast transient response with a sustained plateau, whereas the remaining (30%) population had much slower gradual calcium increase to the same plateau level. Subsequent immunofluorescent analysis revealed that fast Ca2+ responses were attributed to PC, whereas responses with slow kinetics corresponded to IC. Consistently, PCs have ~3 fold higher Piezo-1 signal when compared to neighbor ICs. We next tested whether Piezo1 activity depends on TRPV4 status. Genetic deletion and pharmacological inhibition of TRPV4 with GSK2193874 did not affect Piezo1-dependent elevations in [Ca2+]i. However, high K+ diet, which is known to elevate the flow in the CD and augment both TRPV4 activity and expression, led to diminished Piezo1 responses to Yoda-1. In summary, our results are consistent with the view that Piezo1 and TRPV4 are functional in both principal and intercalated cells of the collecting duct and operate independently to modulate mechanosensitive [Ca2+]i signaling. We hypothesize that augmented TRPV4-dependent [Ca2+]i is compensated by the diminished activity of Piezo1 to account for elevated flow rate upon increased K+ intake. This research was supported by NIH-NIDDK DK117865, DK119170, AHA EIA35260097 (to O. Pochynyuk) and AHA-19CDA34660148 (to V. N. Tomilin). This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.