Project Summary/Abstract Kir4.1/Kir5.1 in distal convoluted tubule (DCT) plays a key role in mediating the effect of low K (LK) or high K (HK) and low sodium (LS) or high sodium (HS) intake on NCC through Cl--sensitive with-no-lysine kinase (WNK) which regulates ste20-proline-alanine rich kinase (SPAK), a key kinase for controlling NCC activity. Although many details of this signaling mechanism explaining the effects of chronic dietary K+ or Na+ intake have been elucidated, we have demonstrated that chronic perturbations remodel the distal nephron. Thus, the observed changes may not reflect the initial dynamic response of Kir4.1/Kir5.1 and NCC in the DCT. Importantly, dietary intake is typically episodic, leading to highly dynamic electrolyte excretory patterns. The DCT comprises an early part (DCT1) and a late part (DCT2). While basolateral Kir4.1/Kir5.1 and apical NCC are expressed along both DCT1 and DCT2, the epithelial sodium channel (ENaC) and ROMK are only detected in the DCT2. This raises the possibility that the response of Kir4.1/Kir5.1 and NCC to short-term dietary K+ or Na+ may differ between DCT1 and DCT2, contributing to rapid homeostatic effects. We now hypothesize that Overnight but not chronic dietary K+-induced regulation of Kir4.1/Kir5.1 and NCC occurs predominantly in the DCT1, whereas overnight but not chronic dietary Na+ or AngII perfusion- induced regulation of Kir4.1/Kir5.1 and NCC occurs predominantly in the DCT2 via AT1aR. This hypothesis is supported by several lines of preliminary data: 1) Overnight LK intake increases Kir4.1/Kir5.1 currents only in the DCT1 but not in DCT2; 2) Overnight LS intake increases Kir4.1/Kir5.1 currents predominantly in the DCT2 but to a less degree in DCT1; 3) Acute application of AngII stimulates Kir4.1/Kir5.1 only in the DCT2 and enhances NCC function; 4) Overnight AngII infusion robustly stimulates Kir4.1/Kir5.1 channel activity and increases whole-cell K+ currents in the DCT2 ; 5) Overnight AngII infusion stimulates NCC expression/activity in Kcnj10+/+ mice but not in inducible kidney-specific Kir4.1 knockout mice. The physiological significance of the proposal is to elucidate the mechanisms underlying dual regulation of the DCT, such that DCT1 behaves like a “K+-sensor” and DCT2 behaves like a “Na+-sensor”. This differential response is essential for normal physiological homeostasis during short-term alterations in K+ or Na+ intake. The application has three aims:1) Test hypothesis that the effect of overnight dietary K+ on NCC expression/activity is initiated by modulating Kir4.1/Kir5.1 predominately in the DCT1, whereas the effect of overnight dietary Na+ on NCC expression/activity is initiated through regulating Kir4.1/Kir5.1 predominantly in the DCT2; 2)Test the hypothesis that acute or overnight AngII treatment activates Kir4.1/Kir5.1 and NCC predominately in the DCT2 but not in the DCT1 and that the activation of Kir4.1/Kir5.1 is essential for acute but not chronic AngII-induced s...