Salt‐evoked molecular responses of NPFFR2 via a “Sodium Response Element” and antagonism of the D1 dopamine receptor
Authors: Rozyyev Selim , Asico Laureano D. , Hunt Jessica , Crusan Annabelle P. , Jose Pedro A. , Villar Van Anthony M. 
Published: April 2020

The causal role of dietary sodium chloride in hypertension and sodium sensitivity has long been recognized. But the exact molecular mechanisms by which relevant genes respond to changes in body sodium and/or extracellular sodium concentrations are not completely understood. We now show two distinct mechanisms by which the neuropeptide FF receptor R2 (NPFFR2) responds to alterations in sodium levels. NPFFR2 is one of two receptors for NPFF and is endowed with pro‐hypertensive and anti‐natriuretic activities in the kidney. First, we searched for regulatory elements in the NPFFR2 gene promoter. Human renal proximal tubule cells (hRPTCs) acutely exposed to low (90 mmol NaCl) salt had increased mRNA (2.3±0.4‐fold, P<0.05) and protein (155±5% vs. 100±4% for normal salt, P<0.05) levels of NPFFR2, while exposure to high (170 mmol) salt decreased the mRNA (−0.70±0.05‐fold, P<0.05) and protein (~50±4%, P<0.05) levels. Promoter analysis of the mouse and human NPFFR2 genes for regulatory elements identified a single 8‐bp region, aptly called “NaRE” for Na+ Response Element, at ~2.3 kb upstream of +1 position, and was identical to the “Dehydration Responsive Element” in the Rdc2a gene which is involved in the rapid response to dehydration, cold, and salinity in Arabidopsis thaliana. To confirm the involvement of NaRE in the NPFFR2 response to sodium, we measured the promoter activity of NPFFR2 gene. With wild‐type NaRE, the NPFFR2 promoter responded well to low (4.1±0.05‐fold increase, P<0.05) and high (0.52±0.5‐fold decrease, P<0.05) salt levels, but not in the absence of NaRE in a mutant NPFFR2 promoter construct. In vivo correlates using an innovative antigene RNA to block the NaRE selectively in the kidneys of C57Bl/6 mice resulted in the inability of NPFFR2 to increase in response to low sodium intake (<0.04 G sodium/day) and reduced further the decreased systolic blood pressure caused by the low sodium diet (65±0.6 mm Hg vs. 83.3±1.3, P<0.05, n=3–4/group). Another mechanism involved the ability of NPFFR2 to dynamically interact with the D1 dopamine receptor. NPFFR2 and D1R co‐immunoprecipitated (co‐IP) and colocalized in hRPTCs and kidney. NPFF and the D1R/D5R agonist fenoldopam had antagonistic effects on cAMP production (2.54±0.1 pmol/mg/min for fenoldopam vs. 1.23±0.2 for vehicle vs. 1.11± 0.2 for both, P<0.05, n=4/group) and sodium transport (1.78±0.1‐fold with fenoldopam, 1±0.11 for vehicle, and 1.1±0.2 for both, P<0.05, n=4/group) in hRPTCs. C57Bl/6 mice fed a 4% NaCl markedly increased (>2.5‐fold) the co‐IP between renal NPFFR2 and D1R, thus enabling the D1R to antagonize more the NPFFR2 effects. Our data are the first to identify (i.e., NaRE) and demonstrate novel transcriptional and post‐translational mechanisms by which mammalian genes respond to sodium.Support or Funding InformationThe work was funded by grants from the US National Institutes of Health, P01HL074940, P01HL068686, R01HL092196, R37HL023081, R01DK039308, and DK090918. It was also supported partly by minigrants (VA Villar & LD Asico) from the National Kidney Foundation of Maryland (NKF‐MD).