Interaction Between NCC and ENaC α, γ Subunits are Differentially Regulated‐ Role of SGK1

Abstract

Thiazide‐sensitive NCC and ENaC are paramount in the regulation of sodium and water homeostasis. Previous data from this laboratory has extensively demonstrated a novel anatomical and biochemical interaction between these two transporters in the late distal convoluted tubule (DCT2). In this study, we sought to further our understanding of the physiological mechanisms regulating this interaction. Knowing the important role of aldosterone in their regulation, we hypothesized two downstream mediators‐ SGK1 and WNK4‐ would alter their association. We used a mammalian two‐hybrid (M2H) assay, using vectors (pBind/pAct with NCC and/or ENaC α or γ, controls and SGK1, WNK4 or NEDD4‐2) which allow for activation of the luciferase reporter vector (firefly) when interaction occurs. COS‐7 cells were transfected at 50‐70% confluency, and allowed to grow for 48 hours. Cells were lysed; M2H determined by analysis of firefly and renilla luciferase; renilla used as a transfection control. Data were normalized (firefly/renilla) and expressed as fold change (of negative, ±SEM, ANOVA, n=8‐12). Our data demonstrate a significant interaction between both ENaC α (7.7±0.8, p<0.05) or γ (4.3±0.7, p<0.05); however, with SGK1 a greater increase in NCC and ENaC α interaction was observed (14.7±1.5, p<0.05). No significant changes were observed with NCC/ENaC γ interactions (SGK1, WNK4 or NEDD4‐2). Here we confirm the direct interaction between NCC and ENaC α/γ. Furthermore, these data suggest that increases in SGK1, possibly via aldosterone, may strengthen or increase NCC and ENaC α interaction.