Intercalated cells (ICs) secrete H+/HCO3- into the aldosterone-sensitive distal nephron (ASDN). 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 the flow- induced [Ca2+]i response in cortical collecting ducts (CCDs), whereas an activator increases [Ca2+]i in CCDs perfused at slow flow rates. These observations suggest that Piezo1 mediates flow-induced early basolateral Ca2+ entry. Mucin 1 (or Muc1) is an apical surface glycoprotein expressed in distal aspects of the nephron, and is most robust in type A (H+-secreting) ICs and in type B (HCO3--secreting) ICs. The physiologic role of Muc1 in ICs is uncertain. In preliminary studies, we found that the cytoplasmic (V1) and transmembrane (V0) domains of V-ATPase dissociate in ICs of Muc1 knockout (KO) mice. Muc1 KO animals have impaired urinary acidification following an acid load. These data suggest that Muc1 regulates V-ATPase V1/V0 domain assembly in ICs. Based on these observations, we hypothesize that (i) Piezo channels function as mechanosensors in the ASDN and enable FIKS by facilitating basolateral Ca2+ entry in ICs, and (ii) Muc1 modulates specific signaling pathways that regulate the assembly of V1/V0 domains and functional V-ATPase in specific H+-secreting epithelia, including ICs. Experiments proposed in Aim 1 will define the role of mechano-activated Piezo1 channels in the cellular response to an increase in tubular flow and to dietary K+ adaptation. The functional impact of pharmacologic (activators and inhibitors) and genetic (KO) manipulations of Piezo1 will be assessed at the level of single tubules ([Ca2+]i, JK and JNa) and animals (clearance studies) subjected to variable dietary K+. In Aim 2, we will define the role of Muc1 in the regulated assembly and functional expression of the V-ATPase in H+- secreting epithelia. We will determine whether Muc1 is required for the assembly of V1/V0 domains of the V- ATPase in H+-secreting ICs and determine the functional consequences of a lack of assembly of V-ATPase V1/V0 domains in H+-secreting ICs. Muc1-dependent signaling pathways that regulate V1/V0 domain assembly and V-ATPase functional activity in H+-secreting ICs will be defined. We expect that the results of our proposed studies will uncover novel and unexpected pathways involved in urinary K+ and H+ excretion and identify potential targets for novel therapies to treat K+ and acid-base imbal...