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 dietary K+-intake induced regulation of Kir4.1/Kir5.1, WNK and NCC is initiated predominantly in the DCT1, whereas dietary Na+-induced or AngII-application-induced regulation of Kir4.1/Kir5.1, WNK, NCC is started mainly in the DCT2 via type 1a angiotensin II receptor (AT1aR). This hypothesis is supported by several lines of preliminary data:1) Overnight LK intake stimulates Kir4.1/Kir5 activity in the DCT1 but not in the DCT2; 2) Overnight LS intake increases Kir4.1/Kir5.1 currents predominantly in the DCT2 but to a lesser degree in the DCT1; 3) Deletion of AT1aR in the kidney abolishes the overnight- LS-induced stimulation of Kir4.1/Kir5.1 of the DCT and NCC; 4) Acute application of AngII stimulates Kir4.1/Kir5.1 only in DCT2 and enhances NCC function; 5) Overnight AngII infusion robustly stimulates Kir4.1/Kir5.1 activity in DCT2 but to a less degree in the DCT1; 6) Overnight AngII infusion stimulates WNK4 and NCC expression/activity in Kcnj10-/- mice but not in Kir4.1 knockout mice. The three specific aims of the proposal are: 1) Test whether dietary K+ and Na+ induced regulation of Kir4.1/Kir5.1, WNK and NCC is different between DCT1 and DCT2; 2) Test whether 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 stimulation of WNK4 and NCC 3) Test whether deletion of AT1aR impairs the stimulatory effect of overnight LS but not overnight LK on Kir4.1/Kir5.1, WNK4 , pNCC and K+ homeostasis. 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...