Role of piezo channels in intercalated cells

Intercalated cells (ICs) in the aldosterone-sensitive distal nephron (ASDN) secrete H+ and HCO3-. Emerging evidence has identified nontraditional roles for ICs, including absorption of filtered Na+ and Cl-, flow-induced K+ secretion (FIKS) and participation in innate immunity. Apical BK channels in ICs are activated by a flow- stimulated increase in intracellular Ca2+ concentration ([Ca2+]i). The rapid initial mechanoinduced increase in [Ca2+]i reflects basolateral Ca2+ entry and release of internal Ca2+ stores. Piezo1, a member of a family of mechanosensitive non-selective cation channels, is expressed along the basolateral membrane of ICs and principal cells (PCs) in the ASDN. In preliminary studies, we found that a Piezo1 inhibitor dampens both the flow- induced [Ca2+]i response in cortical collecting ducts (CCDs) as well as FIKS, whereas an activator increases [Ca2+]i in CCDs perfused at slow flow rates. The flow-induced IC [Ca2+]i response was largely absent in CCDs from mice with an IC-specific genetic deletion of Piezo1. These observations suggest that Piezo1 mediates flow- induced early basolateral Ca2+ entry into ASDN epithelial cells, a key factor in the activation of BK channels. Based on these observations, we hypothesize that Piezo1 channels function as mechanosensors in the ASDN and enable FIKS by facilitating basolateral Ca2+ entry and secondarily activating luminal Ca2+ entry pathways in ICs. Experiments proposed in Aim 1 will define the localization and developmental expression of Piezo1 in the ASDN, and cell type-specific changes in Piezo1 expression in CCDs in response to a low K+ (LK) or high K+ (HK) diet, or aldosterone. Studies in Aim 2 will determine the effects of Piezo1 activators and inhibitors on basal and flow-stimulated [Ca2+]i, effects on net transepithelial Na+ and K+ transport (JNa and JK) in CCDs isolated from control K+ (CK) and HK fed mice, and effects on IC BK channel activity as assessed by patch clamp. Studies proposed in Aim 3 will determine whether targeted deletion of Piezo1 in ICs alters K+ handling in mice, affects flow-induced increases in [Ca2+]i and JNa and JK in microperfused CCDs, and affects IC BK channel activity as assessed by patch clamp. We expect that the results of our proposed studies will uncover novel and unexpected pathways involved in urinary K+ excretion and identify potential targets for novel therapies to treat K+ imbalances.