Renal NPFF and Receptors NPFF‐R1 & NPFF‐R2: Novel Negative Regulators of Sodium Transport and Blood Pressure
Authors: Bishop Michael D , Gomes Jerald , Mazhar Momina , Asico Laureano , Jose Pedro A , Villar Van Anthony M 
Published: November 2023

Hypertension is characterized by sustained elevated blood pressure (BP) and is a major risk factor for cardiovascular and cerebrovascular mortality. It is a multifactorial disorder that results from environmental, behavioral, and genetic factors. Although a myriad of genes have been implicated in its development, very few of them have been shown to be causative of hypertension. Neuropeptide FF (NPFF), a peptide implicated in hormonal modulation and nociception, was shown to inhibit cAMP production and increase BP when administered into the rat brain. In this study, we demonstrated for the first time the expression of NPFF and its receptors, NPFF‐R1 and NPFF‐R2, in renal proximal tubules and evaluated their role in sodium transport and BP regulation using several biological and biophysical approaches in human renal proximal tubule cells (hRPTCs) and C57Bl/6J mice. We now report that NPFF and its receptors are expressed (both mRNA and protein) in hRPTCs. We next determined the ability of NPFF receptors to interact with renal D1‐like dopamine receptors (D1R and D5R), which are the cognate receptors for dopamine, a hormone that increases cAMP production, promotes Na+ excretion by inhibiting Na+/K+‐ATPase, and thus prevents BP elevation. NPFF‐R1 and NPFF‐R2 colocalized with D1R and D5R in the proximal tubules of the human kidney and co‐ immunoprecipitated with the D1R and D5R in hRPTCs, indicating a possible counter‐regulation among these receptors. We next evaluated the physiological relevance of this interaction. The D1R/D5R agonist fenoldopam (1 mM/30 min) increased cAMP production (4.23±0.56 pmol/mg/min vs. 2.54±0.19, vehicle; P<0.05), which was abrogated by NPFF (2.36±0.29) (1 mM/30 min). Treatment with NPFF alone tended to decrease cAMP production (2.11±0.79). Moreover, fenoldopam treatment of the basolateral side of polarized hRPTCs grown in Transwells® increased the intracellular Na+ (117.54±3.77 % of vehicle‐treated control), indicating inhibition of sodium exit from the cell, which was prevented by co‐treatment with NPFF (98.56±7.03%). Treatment with NPFF alone decreased the intracellular Na+ (86.10±4.6%), indicating that renal NPFF may be a negative regulator of D1R/D5R action. siRNA‐mediated silencing of NPFF and its receptors increased the intracellular levels of reactive oxygen species (ROS) (2.5‐fold for NPFF, and 2‐fold for both receptors) compared to mock siRNA‐treated and vehicle‐treated controls. Furthermore, an acute renal subcapsular infusion of NPFF (10 mg in 10 ml/kidney) increased the BP (140±10 mm Hg vs. 100 mm Hg) in C57Bl/6J mice within 15 minutes, which normalized within one hour, demonstrating the ability of NPFF to increase BP. Further uncovering the functional relevance of renal NPFF and the role of ROS will allow a better understanding of the intricate and dynamic genetic regulation of sodium transport and BP.Support or Funding InformationThe work was funded by grants from the US National Institutes of Health, P01HL074940, P01HL068686, R01HL092196, R37HL023081, R01DK039308, and DK090918.