School of Medicine and Health Sciences Poster Presentations

Title

Cooperative Interaction between the Renal Neuropeptide FF Receptors and the Angiotensin II Type 1 Receptor

Document Type

Poster

Keywords

hypertension; dopamine; renin-angiotensin-aldosterone system; neuropeptide FF

Publication Date

Spring 2017

Abstract

Despite breakthroughs in medical genetics and molecular biology, the genetic basis for essential hypertension has proven to be difficult to decipher. Understanding the mechanisms that regulate blood pressure is a challenge that requires the characterization of novel regulators of renal sodium transport and blood pressure. The neuropeptide FF (NPFF) is a mammalian neuropeptide encoded by the NPFF gene and believed to be expressed in neurons. This study aims to demonstrate the presence of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the renal parenchyma and determine their interactions with dopamine and angiotensin II receptors. We report that NPFF and its receptors are expressed in human renal proximal tubule cells (hRPTCs) and that they interact with the D1-like dopamine receptors (D1R and D5R) and the angiotensin II type 1a receptor (AT1Ra). The NPFF receptors co-immunoprecipitated and colocalized with the D1R and D5R in hRPTCs and human kidney. NPFF treatment inhibited cAMP production, while the D1R/D5R agonist fenoldopam stimulated cAMP production in hRPTCs. Co-treatment of NPFF abrogated the effect of fenoldopam, indicating antagonism between these receptors. Fenoldopam treatment at the basolateral side of polarized hRPTCs grown in Transwells® increased the intracellular Na+ concentration, indicating inhibition of sodium exit from the cell. This was prevented by co-treatment with NPFF, which did not change the intracellular Na+ by itself. We further evaluated the interaction of NPFF and its receptors with the renin-angiotensin-aldosterone (RAS) system, which counteracts the physiological effects of dopamine. The NPFF receptors co-immunoprecipitated and colocalized with the AT1R in hRPTCs and human kidney. Additionally, C57Bl/6 mice under pentobarbital anesthesia were treated with NPFF (10 µg/100 µl per kidney, 1 hour) alone, angiotensin II (1 μg/kg/min, I.V., 1 hour) alone, or NPFF and angiotensin II together (1 hour), and blood pressure and heart rate were monitored over time. Treatment with NPFF alone increased the blood pressure (106 vs. 125 mm Hg, n=1) and heart rate (339 vs. 391 beats/min) which normalized within 1 hour, while co-treatment with angiotensin II sustained the increase in blood pressure (125 mm Hg) and heart rate (462 beats/min). Angiotensin II infusion resulted in an increase in blood pressure (124 mm Hg) and heart rate (385 beats/min). Our preliminary findings indicate that the renal NPFF may be a negative regulator of D1R/D5R and also a positive modulator of AT1R. Further uncovering the functional relevance of renal NPFF will allow a better understanding of the dynamic regulation of Na+ transport and blood pressure homeostasis.

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Creative Commons License
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Poster to be presented at GW Annual Research Days 2017.

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Cooperative Interaction between the Renal Neuropeptide FF Receptors and the Angiotensin II Type 1 Receptor

Despite breakthroughs in medical genetics and molecular biology, the genetic basis for essential hypertension has proven to be difficult to decipher. Understanding the mechanisms that regulate blood pressure is a challenge that requires the characterization of novel regulators of renal sodium transport and blood pressure. The neuropeptide FF (NPFF) is a mammalian neuropeptide encoded by the NPFF gene and believed to be expressed in neurons. This study aims to demonstrate the presence of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the renal parenchyma and determine their interactions with dopamine and angiotensin II receptors. We report that NPFF and its receptors are expressed in human renal proximal tubule cells (hRPTCs) and that they interact with the D1-like dopamine receptors (D1R and D5R) and the angiotensin II type 1a receptor (AT1Ra). The NPFF receptors co-immunoprecipitated and colocalized with the D1R and D5R in hRPTCs and human kidney. NPFF treatment inhibited cAMP production, while the D1R/D5R agonist fenoldopam stimulated cAMP production in hRPTCs. Co-treatment of NPFF abrogated the effect of fenoldopam, indicating antagonism between these receptors. Fenoldopam treatment at the basolateral side of polarized hRPTCs grown in Transwells® increased the intracellular Na+ concentration, indicating inhibition of sodium exit from the cell. This was prevented by co-treatment with NPFF, which did not change the intracellular Na+ by itself. We further evaluated the interaction of NPFF and its receptors with the renin-angiotensin-aldosterone (RAS) system, which counteracts the physiological effects of dopamine. The NPFF receptors co-immunoprecipitated and colocalized with the AT1R in hRPTCs and human kidney. Additionally, C57Bl/6 mice under pentobarbital anesthesia were treated with NPFF (10 µg/100 µl per kidney, 1 hour) alone, angiotensin II (1 μg/kg/min, I.V., 1 hour) alone, or NPFF and angiotensin II together (1 hour), and blood pressure and heart rate were monitored over time. Treatment with NPFF alone increased the blood pressure (106 vs. 125 mm Hg, n=1) and heart rate (339 vs. 391 beats/min) which normalized within 1 hour, while co-treatment with angiotensin II sustained the increase in blood pressure (125 mm Hg) and heart rate (462 beats/min). Angiotensin II infusion resulted in an increase in blood pressure (124 mm Hg) and heart rate (385 beats/min). Our preliminary findings indicate that the renal NPFF may be a negative regulator of D1R/D5R and also a positive modulator of AT1R. Further uncovering the functional relevance of renal NPFF will allow a better understanding of the dynamic regulation of Na+ transport and blood pressure homeostasis.