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Abstract: PO2155

NaRE: A Novel "Salt Response Element" in the NPFFR2 Gene Promoter and Antagonist of the D1 Dopamine Receptor

Session Information

Category: Hypertension and CVD

  • 1403 Hypertension and CVD: Mechanisms

Authors

  • Rozyyev, Selim, Children's National Hospital, Washington, District of Columbia, United States
  • Asico, Laureano D., The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States
  • Hunt, Jessica, The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States
  • Jose, Pedro A., The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States
  • Villar, Van Anthony M., The George Washington University School of Medicine and Health Sciences, Washington, District of Columbia, United States
Background

The neuropeptide FF receptor R2 (NPFFR2) is one of two receptors for NPFF and is endowed with pro-hypertensive and anti-natriuretic activities. We looked for regulatory elements in the NPFFR2 gene promoter and how it interacts with an anti-hypertensive and natriuretic protein, the dopamine type receptor (D1R).

Methods

Human renal proximal tubule cells (hRPTCs) treated with low NaCl (90 mM) increased mRNA (2.3±0.4-fold) and protein (155±5% vs. 100±4%) levels of the salt-retaining NPFFR2, whereas treating the cells with high NaCl concentration (170 mM) decreased the mRNA (-0.70±0.05-fold) and protein (~50±4%, P<0.05) levels.

Results

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. This is 75% identical to the “Dehydration Responsive Element” (“TACCGACAT”) in the Rdc2a gene of Arabidopsis thaliana which is a cis-acting element which responds to dehydration, low temperature, and salinity. In the presence of the wild-type NaRE, the NPFFR2 promoter responded well to both low (4.1±0.05-fold increase) and high (0.52±0.5-fold decrease) NaCl concentrations, but not in the absence of NaRE in a mutant NPFFR2 promoter construct. Antigene RNA to block NaRE expression in the kidneys of C57Bl/6 mice resulted in the inability of the mice to respond to a low sodium diet (<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). We then studied a mechanism by which NPFFR2 dynamically interacts with the D1R. NPFFR2 and D1R co-immunoprecipitated and colocalized in hRPTCs and mouse 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 fenoldopam and NPFF) and sodium transport (1.78±0.1-fold with fenoldopam) in hRPTCs. C57Bl/6 mice fed a 4% NaCl markedly increased (>2.5-fold) the co-immunoprecipitation between renal NPFFR2 and D1R, thus increasing the ability of NPFFR2 to antagonize the D1R effects.

Conclusion

Our data are the first to identify NaRE and demonstrate novel transcriptional and post-translational mechanisms by which mammalian genes respond to sodium.

Funding

  • NIDDK Support