Different modes of Piezo1 Ca2+ signaling in principal and intercalated cells of the collecting duct

Abstract

It has been recognized that mechanical stress arising from elevated fluid flow and variations in osmolarity is an important regulator of the water‐electrolyte transport in the real tubule. The collecting duct is the terminal segment of the renal tubule, which is commonly exposed to the highest level of mechanical forces. It consists of electrically uncoupled principal and intercalated cells having different morphology and discrete functional roles. Using genetic animal models, we previously demonstrated that mechano‐activated TRPV4 and TRPC3 Ca2+‐permeable channels mediate responses to high flow and hypotonicity, respectively. However, both channels are not considered to be truly mechanosensitive, but rather being activated in response to a primary putative mechanosensor. Piezo1 has been recently identified as a Ca2+‐permeable channel directly activated by the plasma membrane stretch. Piezo1 expression has been found in the renal epithelia, but its role in mechanosensitive Ca2+ signaling is not known. Here, we used freshly isolated split‐opened cortical collecting ducts to show that selective Piezo1 activators, YODA‐1(20uM) and Jedi2 (500uM) 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. The subsequent immunofluorescent analysis revealed that the cells with rapid responses to Piezo1 stimulation express AQP2, a selective marker of principal cells. In contrast, AQP2‐negative intercalated cells matched the population of cells with the slow response. Genetic deletion and pharmacological inhibition of TRPV4 and TRPC3 (with GSK2193874 and Pyr10, respectively) did not affect Piezo1‐dependent elevations in [Ca2+]i arguing against a functional link between channel activation with flow‐ and osmo‐sensitive Ca2+ signaling. In summary, our results demonstrate that Piezo1 is functional in both principal and intercalated cells of the collecting duct with a greater expression in the former. Activation of the channel is not directly associated with mechanosensitive Ca2+ signaling via TRPV4 and TRPC3. Future studies are necessary to unravel the significance and physiological roles of different Piezo1 expression in principal and intercalated cells of the collecting duct.