Antibodies and reagents
Primary antibodies used in this study are listed in the Supplementary Table S1. The D1R and D5R antibodies have been thoroughly validated66,67,68,69, using the methods advocated by the ad hoc International Working Group for Antibody Validation70. The commercial NPFF-R1 and NPFF-R2 antibodies were characterized by immunoblotting the kidney cortices from mice infused with specific Npff-r1 and Npff-r2 siRNA underneath the kidney capsule (Supplementary Fig. S5). Normal mouse (Cat. No. sc-2025), rabbit IgG (Cat. No. 2729), and chicken IgY (Cat. No. AC146) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), Cell Signaling Technology (Danvers, MA) and Sigma-Aldrich (St. Louis, MO), respectively. Appropriate secondary Alexa Fluor antibodies were purchased from Thermo Fisher Scientific (Gaithersburg, MD). Fenoldopam (Cat. No. 1659), NPFF (Cat. No. 3137), and RF-9 (Cat. No. 3672) were purchased from Tocris (Minneapolis, MN). RFRP-2 (Cat. No. 048-44) and RFRP-3 (Cat. No. 048-46) were purchased from Phoenix Pharmaceuticals (Burlingame, CA). The scrambled peptide was synthesized by GenScript (Piscataway, NJ). AC-263093 (Cat. No. orb611290) was purchased from Biorbyt (St. Louis, MO). Forskolin (Cat. No. F3917) and other reagents were purchased from Sigma (St. Louis, MO).
Cell culture
hRPTCs67,68,69,71 were verified of their RPTC origin by the expression of γ-glutamyl transpeptidase and NHE3, as previously reported67,68. mRPTCs, kindly supplied by Dr. Ulrich Hopfer (Case Western Reserve University School of Medicine), were isolated from C57BL/6 mice and characterized, as previously described72. Immortalized RPTCs with low passages were cultured in a 1:1 mixture of DMEM and Ham’s F-12 medium, supplemented with 5% fetal bovine serum, selenium (5 ng/mL), insulin (5 μg/mL), transferrin (5 μg/mL), hydrocortisone (36 ng/mL), triiodothyronine (4 pg/mL), and epidermal growth factor (10 ng/mL).
RT-PCR
Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed, as previously described72. Briefly, RNA of hRPTCs was extracted with Trizol (Invitrogen, Carlsbad, CA) and further purified, using the RNeasy RNA Extraction Mini kit (Qiagen). RNA samples were converted into first-strand cDNA using an RT2 First Strand kit (SABiosciences-Qiagen). The transgenes were amplified (Taq DNA polymerase, Invitrogen) with specific NPFF, NPFF-R1, and NPFF-R2 primer pairs (Supplementary Table S2) at 95 °C for 3 min, followed by 35 cycles at 94 °C for 30 s, 53 °C for 30 s, 72 °C for 45 s, and 60 °C for 10 min. The PCR products of NPFF, NPFF-R1, and NPFF-R2 were resolved in 1.5% agarose gel in Tris/Borate/EDTA buffer, containing 0.5 µg/mL ethidium bromide and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE). The gels were photographed under ultraviolet light.
Western blot
Western blotting was performed as previously described69,71. Briefly, kidney cortices were lysed in 1 × RIPA lysis buffer (Millipore, Billerica, MA), containing protease and phosphatase inhibitor cocktail (Thermo Fisher Scientific, Rockford, IL), and the samples were adjusted to have the same protein concentration. The proteins were separated by SDS-PAGE, transferred onto nitrocellulose membranes, and then probed with primary antibodies and appropriately conjugated secondary antibodies. The images were visualized by a LiCor Odyssey Imaging system.
Immunofluorescence imaging
Immunofluorescence imaging was performed, as previously described69,71,74. HRPTCs or mRPTCs were grown on poly-d-lysine-coated coverslips and fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 20 min at room temperature. After washing with PBS, the cells, fixed on coverslips, were incubated with primary anti-NPFF, anti-NPFF-R1, anti-NPFF-R2, anti-D1R, or anti-D5R antibodies overnight at 4 °C. The coverslips were then incubated with the proper Alexa Fluor-488 and -555 secondary antibodies for 2 h at 4 °C. The coverslips were mounted in a proper antifade mounting medium and sealed onto glass slides.
Human kidney sections (Imgenex, San Diego, CA, USA) were prepared for antigen retrieval, using heat and pressure and immunostained for NPFF-R1, NPFF-R2, D1R, and D5R antibodies. Wheat germ agglutinin, conjugated with Alexa Fluor 647, was used to target the lectin-rich brush border and plasma membranes of RPTs. DAPI was used to visualize nuclei. For negative controls, the primary antibodies were replaced with normal rabbit serum at the appropriate dilutions.
Mouse brains were coronally sectioned at 10 μm thickness using a cryostat, and the sections, including the entire forebrain regions underwent standard immunohistochemistry, as previously reported73 to study brain distribution of NPFF-R1 and NPFF-R2. For NPFF-R1 and NPFF-R2 immunostaining, the primary antibodies were diluted 1:100 in blocking buffer (3% BSA and 0.3% Triton X-100 in PBS) and the slides were incubated for 2 days in a cold room. Alexa Fluor 488-conjugated secondary antibody was diluted 1:1000 in blocking buffer and the slides were incubated for 2 h at room temperature to visualize both immunoreactive signals. The slides were cover-slipped and subjected to microscopy, using a BX43F Olympus fluorescent microscope (Center Valley, PA) with a DP80 camera. Preliminary experiments to determine the appropriate working concentration and incubation time for each antibody were performed. For negative controls, the primary antibodies were removed, and the slides were only incubated with the blocking buffer followed by Alexa Fluor 488-conjugated secondary antibody for the appropriate time period.
Cyclic AMP assay
Cyclic AMP (cAMP) was assayed using a direct immunoassay kit (Arbor Assays, Ann Arbor, MI), as previously described69,71. Briefly, hRPTCs and mRPTCs were grown in 12-well plates. The RPTCs at ~ 75% confluence were pretreated with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX; 1 M; Sigma-Aldrich, St. Louis, MO, USA), before the addition of a D1-like receptor agonist, fenoldopam (1 µM/30 min), NPFF, RFRP-2, RFRP-3, and/or AC-263093, at the indicated concentrations and time, and re-challenged with forskolin (10 µM) or PBS for 30 min. The cell lysates were prepared to determine the protein concentration, using the BCA protein assay kit (Thermo Scientific, Rockford, IL, USA). After quantification, the same amount of cell lysates (Supplementary Fig. S3) or culture supernatants (Figs. 3 and 6) were used to determine cAMP concentrations with reading the optical density at 450 nm by an ELISA plate reader.
Co-immunoprecipitation assay
Co-immunoprecipitation was performed using a Dynabeads kit (Thermo Fisher Scientific), as previously described74. Briefly, ~ 90% confluent hRPTCs were harvested, and the cell pellets were lysed in a lysis buffer (20 mM Tris·HCl, pH 8.0/1 mM EDTA/1 mM NaN3 /2 mM DTT/0.25 M sucrose), with 0.2 mM phenylmethylsulfonyl fluoride, and protease and phosphatase inhibitor cocktail. Five µg of anti-D1R, anti-D5R, anti-NPFF-R1, or anti-NPFF-R2 antibodies were conjugated with Dynabeads in 0.5 mL of slurry. The cell lysates were then incubated with the conjugated antibodies at 4 °C for 4 h, followed by proper washing. The controls were normal rabbit IgG and chicken IgY. Proteins bound to the beads were eluted in 60 µL of loading buffer at 65 °C for 15 min, separated by 10% SDS-PAGE, and transferred onto a nitrocellulose membrane for incubation with the detecting antibody, followed by the appropriate secondary antibody, before visualization with a LiCor Odyssey Imaging system.
Targeted quantification of NPFF with Liquid chromatography- tandem mass spectrometry (LC–MS/MS)
Targeted LC–MS/MS method was used to measure NPFF in the mouse serum and kidney as previously described75,76. Briefly, C57BL/6 mouse serum and kidney samples were homogenized and sonicated in ice-cold acidified 90% methanol buffer to precipitate large proteins and extract NPFF in the supernatant. Supernatant samples were collected after centrifugation at 18,000×g for 30 min at 4 °C. Molecular weight cutoff (MWCO, 10 K) ultra-centrifugal filter (Sigma) was used to enrich molecules lower than 10 K MW, which were then dried down and desalted by Waters HLB solid phase extraction plate. 13C5, 15N folic acid was spiked into the sample as the internal standard (I.S.). LC–MS–MS analysis was conducted using a Dionex Ultimate 3000 RSLCnano system coupled with a Thermo Scientific Q-Exactive HFX mass spectrometer as described previously75. An Easy-spray PepMap C18 LC column (2 μM, 100 Å, 75 μM × 15 cm) was used to separate peptide samples with a 1 h LC gradient. Serial concentrations of NPFF standards (Cayman) were spiked into a highly diluted sample matrix (no detectable NPFF signal) with I.S. to generate calibration curves. A parallel reaction monitoring (PRM) method was established by using the a2, b2, y4, y6 fragment ions from NPFF peptide, normalized to the fragment ion from the I.S. Data analysis was conducted with Skyline software76 and R studio.
In situ RNA hybridization by RNAScope
In situ RNA hybridization was performed using RNAscope technology (Advanced Cell Diagnostics, Newark, CA), as previously described72. Briefly, thin sections (5 µm) of formalin-fixed, paraffin-embedded mouse kidneys were deparaffinized in xylene and rehydrated with step-down concentrations of ethanol. The tissues were then treated serially with the following: 10-min immersion in pretreatment 1 solution (endogenous H2O2 blocker); 100 °C, 15-min immersion in pretreatment 2 solution; and protease digestion, 40 °C for 10 min. The tissues were rinsed with water after each pretreatment step and then hybridized with specific Npff, Npff-r1, and Npff-r2 RNAscope probes at 40 °C for 2 h (Advanced Cell Diagnostics). The specific probes were targeted for mice: Npff mRNA, Mm-Npff1 (NM_018787.1., Cat. No. 479901) with region designed against 117–313 nt; Npff-r1 mRNA, Mm-Npff1 (NM_001177511.1., Cat. No. 410161) with region designed against 14–1298 nt; Npff-r2 mRNA, and Mm-Npff2 (NM_133192.3., Cat. No. 410171) with region designed against 233–1342 nt. Mm‐PPIB, Mus musculus peptidylprolyl isomerase B (Ppib, Cat. No. 313911) was the positive control. Bacillus subtilis dihydrodipicolinate reductase (dapB, Cat. No. 310043) was the negative control. After the wash and buffer steps, the signal was amplified, using a multistep process (Each RNAscope 2.5 HD Reagent Kit—BROWN, Cat. No. 322300; HybEZ Hybridization System, Cat. No. 310010). Horseradish peroxidase (HRP)-labeled probes were visualized by the application of 3, 3′-diaminobenzidine (DAB). The sections were then counterstained with hematoxylin.
Urinary sodium excretion and blood pressure measurement
Adult C57BL/6 mice (male, 8-week-old), purchased from Jackson Laboratory (Bar Harbor, ME), were housed in a temperature-controlled facility with a 12:12-h light–dark cycle and fed with regular mouse chow and water ad libitum for at least 2 weeks before any studies were performed. Renal Npff-r1 and Npff-r2 were silenced by the chronic renal subcapsular infusion of specific Npff-r1 and Npff-r2 siRNA (Cat. No. SI01037379 and SI04925039 respectively, Qiagen, Germantown, MD), via an osmotic minipump, as previously described69,71,72. Briefly, the mice were uninephrectomized 1 week prior to the implantation of the minipump. For the minipump implantation, the mice were anesthetized with pentobarbital (50 mg/kg body weight, intraperitoneally). The osmotic minipumps (100 µL; flow rate: 0.5 µl/h) were filled with validated Npff-r1-specific siRNA, Npff-r2-specific siRNA (Cat. No. SI01037379, Cat. No. SI04925039, Qiagen), or non-silencing mock siRNA (Cat. No. 03650318, Qiagen), as control. The siRNAs were dissolved in an in vivo transfection reagent (TransIT In Vivo Gene Delivery System, Mirus), under sterile conditions. The minipumps were fitted with a polyethylene delivery tubing (Alzet #0,007,701) and the tip of the tubing was inserted within the subcapsular space of the remaining kidney. The efficiency of siRNA infusion was analyzed by real-time PCR, performed on an Applied Biosystems® ViiA™ 7 Real-Time PCR System (Foster City, CA). The primers (SABiosciences-Qiagen) used for qRT-PCR are in Supplementary Table S3. The data were analyzed using the ΔCt method68,72, the gene and protein expressions of Npff-r1 or Npff-r2 after the 7-days siRNA infusion are shown in Supplementary Figure S6 and Supplementary Figure S5, respectively.
Twenty-four urine samples were collected from mice individually housed in metabolic cages. The mice were acclimatized in the metabolic cages for 24 h before the collection of urine. Urine sodium concentration was measured using Easylyte Analyzer (Medica Corporation, Bedford, MA). Urinary sodium excretion (UNaV) was calculated as urine volume × sodium (mEq/liter).
Blood pressures of mice with acute renal subcapsular infusion of NPFF were measured (Cardiomax II; Columbus Instruments, Columbus OH) from the aorta via the carotid artery under pentobarbital sodium anesthesia (50 mg/kg)71,72 and by tail cuff69 with a CODA system (Kent scientific corporation, Torrington, CT, USA) in conscious mice as described previously.
Blood pressure of chronic NPFF (9.25 μmol, 0.5 μL/h, 0.05 nmol/day) and saline (0.9%NaCl) infusion groups was recorded by telemetry in conscious mice, as previously described35,72.
The studies were conducted by following the guidelines set by the US National Institutes of Health for the ethical treatment and handling of animals in research and approved by the Institutional Animal Care and Use Committee (IACUC) of The George Washington University. All experiments were performed in accordance with relevant guidelines and regulations and the recommendations in the ARRIVE guidelines.
Statistical analysis
Data are presented as mean ± standard deviation (SD). All the cell experiments were performed using a minimum of triplicate wells and repeated at least twice, which is our laboratory routine for cAMP assays. However, the inter- and intra-assay variability was not assessed in the LC–MS/MS NPFF quantification.
Differences between two groups were assessed by Student’s t-test and differences among three or more groups were assessed by one-way ANOVA with the Newman-Keuls or Holm-Sidak test. P values < 0.05 were considered statistically significant (SigmaPlot, San Jose, CA).
Ethics approval and consent to participate
The mouse experiments were performed according to a protocol (A353) approved on December 8, 2017, and protocol A2022-014 that is good until March 23, 2025) by the George Washington University Institutional Animal Care and Use Committee. The use of hRPTCs followed a protocol, HSR#13310, approved by the University of Virginia Institutional Review Board, which is renewed annually.
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