Regulation of Na+/H+ exchanger‐3 (NHE3) by adrenomedullin (AM) in human proximal tubule cells: Role for receptor activity‐modifying proteins (RAMPs) and Na+/H+ exchanger‐regulatory factor‐1 (NHERF1)

AM functions as both a potent vasodilator and natriuretic/diuretic peptide in the proximal tubules of the kidney. A functional AM receptor (AM‐R) is comprised of a heterodimer between the calcitonin receptor‐like receptor (CL‐R) and RAMP3. RAMPs are single transmembrane proteins critical to AM‐R for plasma membrane expression, phenotype, and trafficking. Given AM's natriuretic/diuretic role in the proximal tubules and documented receptor interaction with NHERF1, we hypothesized that AM is regulating NHE3, via cAMP activation, to control natriuresis in human proximal tubule cells (hPTE). To test this hypothesis, we employed confocal microscopy, overlay interaction assays, and ion transport assays. We report apical membrane distribution of RAMP3 in polarized hPTE cells that is dependent on NHERF1 expression. RAMP3 was demonstrated to interact with endogenous NHERF1 and NHE3 via PDZ interactions, with the RAMP3‐NHE3 interaction being dependent on NHERF1 expression. AM was demonstrated to dose‐dependently inhibit NHE3‐regulated ion transport. This inhibition of ion transport was alleviated when cells were pretreated with an AM‐R antagonist or the multi‐protein complex was disrupted, by RNAi knockdown of NHERF1. This study proposes a molecular mechanism for AM‐mediated inhibition of sodium reabsorption in the proximal tubules, through a multi‐protein complex of RAMP3‐NHERF1‐NHE3. In addition, this finding provides a therapeutic target for the well‐documented protective effect of AM in hypertension.