Renal autocrine neuropeptide FF (NPFF) signaling regulates blood pressure

Abstract

The kidney and brain play critical roles in the regulation of blood pressure. Neuropeptide FF (NPFF), originally isolated from the bovine brain, has been suggested to contribute to the pathogenesis of hypertension. However, the roles of NPFF and its receptors, NPFF-R1 and NPFF-R2, in the regulation of blood pressure, via the kidney, are not known. In this study, we found that the transcripts and proteins of NPFF and its receptors, NPFF-R1 and NPFF-R2, were expressed in mouse and human renal proximal tubules (RPTs). In mouse RPT cells (RPTCs), NPFF, but not RF-amide-related peptide-2 (RFRP-2), decreased the forskolin-stimulated cAMP production in a concentration- and time-dependent manner. Furthermore, dopamine D1-like receptors colocalized and co-immunoprecipitated with NPFF-R1 and NPFF-R2 in human RPTCs. The increase in cAMP production in human RPTCs caused by fenoldopam, a D1-like receptor agonist, was attenuated by NPFF, indicating an antagonistic interaction between NPFF and D1-like receptors. The renal subcapsular infusion of NPFF in C57BL/6 mice decreased renal sodium excretion and increased blood pressure. The NPFF-mediated increase in blood pressure was prevented by RF-9, an antagonist of NPFF receptors. Taken together, our findings suggest that autocrine NPFF and its receptors in the kidney regulate blood pressure, but the mechanisms remain to be determined.

Keywords: Blood pressure; Brain; Dopamine D1-like receptor; Kidney; Neuropeptide FF.

Figure 1

Gene expression of NPFF(Npff) and its receptors in human and mouse kidneys. (a) Agarose gel electrophoresis of RT-PCR products of NPFF, NPFF-R1, and NPFF-R2 in human renal proximal tubule cells (hRPTCs). (b) Gene expressions of Npff, Npff-r1, and Npff-r2 in the RPTs of C57BL/6 mice analyzed by RNAScope. The mouse kidney sections were hybridized using specific Npff, Npff-r1, and Npff-r2 probes (Advanced Cell Diagnostics); Npff, Npff-r1, and Npff-r2 RNA appear as deep brown dots, some of them indicated by arrows (right panel). (c) Negative control images: no visible signals can be seen in kidney sections hybridized using a non-specific probe, dapB, a bacterial gene (Advanced Cell Diagnostics). Bar scale, 20 μm. (d) mRPTCs were prepared and stained as described in the Materials and Methods section. The mRPTCs were incubated with anti-FMRF (for NPFF), anti-NPFF-R1, and anti-NPFF-R2 antibodies, as indicated (left panel), and counterstained for DNA with DAPI (4′,6-diamidino-2-phenylindole, middle panel). The merged images (right panel) show possible nuclear staining of NPFF and its receptors. Bar scale, 10 μm.

Figure 2

Analysis of NPFF peptide by targeted LC–MS/MS. (a) Extracted LC–MS ion chromatograms (m/z 541.3010) of NPFF peptide standard and NPFF extracted from the mouse kidney and serum. (b) MS spectrum of NPFF peptide. (c) MS/MS peptide fragmentation spectrum of NPFF peptide detected in mouse serum (red) versus standard (blue). (d) NPFF calibration curves using a2, b2, y4, and y6 fragment ions based on the targeted parallel reaction monitoring (PRM) assay.

Figure 3

Inhibition of cAMP production by NPFF in mRPTCs. (a) mRPTCs were exposed to the indicated concentrations of NPFF for 15 min, followed by 10 µM forskolin for 30 min. (b) mRPTCs were exposed to 10⁻⁷ M NPFF at the indicated time points, followed by 10 µM forskolin for 30 min. n = 4. * p < 0.05 versus 0 M or 0 min, one-way ANOVA, Newman-Keuls test. (c) mRPTCs were treated with RFRP-2, an RF-amide with no known function, at the indicated concentrations for 15 min, n = 4. (d) mRPTCs were treated with RFRP-2 (10⁻⁷ M) at the indicated time points, followed by 10 µM forskolin for 30 min (right). n = 4. * p < 0.05 versus 0 M or 0 min, one-way ANOVA, Newman-Keuls test. (e) mRPTCs were exposed to RFRP-3, an NPFF-R1 agonist, in the absence or presence of RF-9, a dual NPFF-R1 and NPFF-R2 antagonist, as indicated, for 15 min, followed by 10 µM forskolin (Forsk) for 30 min. (f) mRPTCs were exposed to AC-263093, an NPFF-R2 agonist, in the absence or presence of RF9, as indicated, for 15 min, followed by 10 µM forskolin (Forsk) for 30 min. n = 6, * p < 0.05 versus Forsk alone, # p < 0.05 versus Forsk plus NPFF, one-way ANOVA, Newman-Keuls test. NPFF, an agonist for both NPFF-R1 and NPFF-R2, served as positive control to inhibit Forsk-induced increase in cAMP production in (a) and (b).

Figure 4

Colocalization of NPFF receptors with D1-like receptors (D₁R and D₅R) in human kidney sections. (a) Strong co-localization of NPFF-R1 and NPFF-R2 with D₁R in human kidney sections. (b) Strong co-localization of NPFF-R1 with D₅R but minimal colocalization of NPFF-R2 with D₅R in human kidney sections. NPFF-R1 or NPFF-R2, green; D₁R or D₅R, red; wheat germ agglutinin (WGA, plasma membrane marker), magenta; DAPI (4′,6-diamidino-2-phenylindole), blue. Merge 1, NPFF-R1 (or NPFF-R2) with D₁R (or D₅R), the colocalization of NPFF receptors with D₁R or D₅R is denoted in yellow; Merge 2, WGA with DAPI. Bar scale, 20 µm.

Figure 5

Co-immunoprecipitation of NPFF receptors with D1-like receptors (D₁R and D₅R) in hRPTCs. (a) Co-immunoprecipitation of NPFF-R1 and NPFF-R2 with D₁R in hRPTCs. (b) Co-immunoprecipitation of NPFF-R1 but not NPFF-R2 with D₅R in hRPTCs. hRPTC lysates were immunoprecipitated (IP) with anti-NPFF-R1 or anti-NPFF-R2 antibodies coupled to Dynabeads for 4 h at 4 °C. The protein complexes bound to the beads were eluted and separated by SDS-PAGE, transferred onto nitrocellulose membranes, and immunoblotted (IB) with anti-D₁R (a) or anti-D₅R (b) antibodies, as indicated. The expected bands for D₁R and D₅R are at 70 kDa and 55 kDa, respectively. Normal IgG was used for negative control and immunoblotting of D₁R or D₅R in cell lysates for positive control.

Figure 6

Antagonism between NPFF receptors and D1-like receptors on cAMP production. hRPTCs were treated with fenoldopam (FEN, 10⁻⁷ M), a D1-like receptor agonist, without or with NPFF (10⁻¹¹ M) (a), RFRP-3 (10⁻¹⁰ M) (b), or AC-263093 (10⁻⁷ M) (c) for 30 min. VEH, vehicle. n = 6/group, * p < 0.05 versus VEH, # p < 0.05 versus FEN, One-way ANOVA, Newman-Keuls test.

Figure 7

Renal protein expression of Na⁺/H⁺ exchanger type 3 (a) and Na⁺/K⁺-ATPase (b) after the renal subcapsular infusion of mock, Npff-r1 siRNA, or Npff-r2 siRNA in C57BL/6 mice fed a normal salt diet. Left panels, representative immunoblots of the protein expressions of NHE3 (upper panel in a), phospho-NHE3 (middle panel in a), and Na⁺/K⁺-ATPase (upper panel in b) as indicated. Immunoblots of GAPDH served as loading control. Densitometric analysis of immunoblots. n = 3/group, * p < 0.05 versus Mock, # p < 0.05 versus Npff-r1 siRNA, one-way ANOVA, Newman-Keuls test.

Figure 8

Effect of the renal subcapsular infusion of NPFF on urinary sodium excretion and systolic blood pressure in C57BL/6 mice. (a) NPFF (9.25 μmol, 0.5 μL/h), chronically infused underneath the renal capsule, decreased renal sodium excretion in conscious C57BL/6 mice fed a normal salt diet. UNaV, urinary sodium excretion. n = 5–6, * p < 0.05, Student’s t test. (b) Acute effect of NPFF on systolic blood pressure, measured from the aorta, via the carotid artery, caused by a single-dose injection (10 µg in 100 µL) of NPFF underneath the renal capsule. n = 4, * p < 0.05 versus basal, one-way ANOVA, Holm-Sidak post-hoc test. (c) Chronic effect of NPFF on systolic blood pressure, measured by carotid artery in conscious mice with chronic renal subcapsular infusion of NPFF (9.25 μmol, 0.5 μL/h) for seven days. n = 4, * p < 0.05 versus vehicle (0 day), one-way ANOVA, Holm-Sidak post-hoc test. (d) Acute effect of NPFF on systolic blood pressure (measured by tail cuff) caused by a single renal subcapsular injection of vehicle (100 µL saline), NPFF (10 µg/100 µL), RF9 (10 µg/100 µL), RF9 + NPFF (10 µg each/100 µL), or scrambled peptide (Scrm-pep, 10 µg/100 µL) in pentobarbital-anesthetized mice. n = 4–7, * p < 0.05 versus vehicle (saline, 0.9% NaCl), RF9, NPFF-R1 and NPFF-R2 antagonist; Scrm-pep, NPFF scrambled peptide; one-way ANOVA, Student–Newman–Keuls test.