Abstract
Genome-wide association studies (GWAS) have prioritized a transcription factor, nuclear receptor 2 family 2 (NR2F2), as being associated with essential hypertension in humans. Here we provide evidence that validates this association and indicates that Nr2f2 is a genetic determinant of blood pressure (BP). Using the zinc-finger nuclease technology, the generation of a targeted Nr2f2-edited rat model is reported. The resulting gene-edited rats have a 15 bp deletion in exon 2 leading to a five-amino-acid deletion in the hinge region of the mutant Nr2f2 protein. Both systolic and diastolic blood pressures of the Nr2f2mutant rats are significantly lower than controls. Because the hinge region of Nr2f2 is required for interaction with Friend of Gata2 (Fog2), protein–protein interaction is examined. Interaction of Nr2f2mutant protein with Fog2 is greater than that with the wild-type Nr2f2, indicating that the extent of interaction between these two transcription factors critically influences BP.
Similar content being viewed by others
Introduction
Blood pressure (BP) is a complex polygenic trait influenced by multiple genetic elements, the number, magnitude of effect and identities of which are largely unknown. Genome-wide association studies have prioritized genes, on the basis of their association with hypertension, as potential candidates for BP regulation. The Wellcome Trust Case–Control Consortium (WTCCC) was one of the first large scale genome-wide association studies to report a suggestive association on a region on human chromosome 15 to hypertension1. Subsequently, by a haplotype-based re-analysis of this data, the gene nuclear receptor subfamily 2, group F, member 2 (NR2F2) also known as chicken ovalbumin upstream promoter transcription factor (COUP-TFII) was highly prioritized as a candidate gene associated with hypertension2. Four other linkage studies in humans have reported the genomic region containing NR2F2 as being linked to essential hypertension3,4,5,6. Similar lines of evidence were obtained with multiple linkage and substitution mapping studies using rat models of hypertension7,8,9,10,11. Nr2f2 is a nuclear receptor transcription factor vital for angiogenesis and heart development12, but its role in BP regulation remains undefined.
The current study was conducted to directly test the involvement of Nr2f2 in BP regulation. For this purpose, a targeted gene-edited rat model was generated, in which the Nr2f2 protein was mutated in the hinge region. Both systolic and diastolic BPs of the Nr2f2mutant rats are significantly lower than controls, indicating that Nr2f2 is indeed a genetic determinant of BP. Further, the structural mechanism by which Nr2f2 influences BP is delineated to the hinge region, through which it interacts with another transcription factor, Friend of Gata2 (Fog2). The extent of interaction of Nr2f2 with Fog2 is enhanced when the Nr2f2 protein is mutated in the hinge region. These data provide evidence for the extent of interaction between two transcription factors, Nr2f2 and Fog2, as being important for the regulation of blood pressure.
Results and Discussion
Targeted-editing of the Nr2f2 locus
Gene deletion mouse models serve as genetic tools to validate observations documented through genetic linkage and association studies. Nr2f2 knockout mice are, however, embryonic lethal13. To examine the role of Nr2f2 in BP regulation, we therefore focused on constructing a Nr2f2mutant rat model without disrupting the two important domains of the Nr2f2 protein, the DNA-binding domain and the ligand-binding domain. Custom zinc-finger nucleases (ZFNs) targeting exon 2 of Nr2f2 were designed. Exon 2 of Nr2f2 codes for amino acids in the hinge region of the Nr2f2 protein, which is between the DNA-binding and the ligand-binding domains. ZFNs targeting Nr2f2 were microinjected into single-cell embryos of the hypertensive rat strain—the Dahl salt-sensitive (S) rat. Embryonic lethality was bypassed by the hypomorphic founder Nr2f2mutant rats. These rats had a shorter (345 bp) PCR-amplified genomic DNA fragment encompassing exon 2 of Nr2f2 compared with the wild-type Nr2f2 (360 bp) genomic fragment (Fig. 1a). Sequencing of the PCR products confirmed the deletion to be 15 bp from 1,571 to 1,556 bp of the mRNA of Nr2f2 (Fig. 1b). Two different antibodies against Nr2f2 were used to clarify the protein status of Nr2f2. Immunoblotting with an amino (N)-terminal antibody detected the presence of Nr2f2 protein in both the Nr2f2mutant rats and the wild-type hypertensive rats, whereas antibodies to the hinge region of Nr2f2 cross-reacted exclusively with the wild-type Nr2f2 protein but not the mutant Nr2f2 protein (Fig. 2, Supplementary Fig. 1).
Attenuation of hypertension in the Nr2f2mutant rats
Compared with the wild-type hypertensive rats, Nr2f2mutant rats maintained on a high-salt (2% NaCl) diet had lower systolic BP (179±3 versus 197±5 mm Hg; t-test, P<0.05) as measured by the tail-cuff method. To further confirm these observations, rats were surgically implanted with radio transmitters and BP was continuously recorded by telemetry. Both the Nr2f2mutant rats and the wild-type hypertensive rats maintained normal diurnal rhythms of systolic and diastolic BP. However, throughout the observation period, both systolic and diastolic BP of the Nr2f2mutant rats were consistently lower than that of the wild-type hypertensive rats (Fig. 3a,b). Nr2f2mutant rats maintained on a low-salt (0.3% NaCl) diet did not, however, demonstrate a similar BP-lowering effect (Supplementary Fig. 2). Figure 3c,d demonstrate the blunted increase in salt-induced blood pressure in the mutant rats compared with the hypertensive rats. These data constitute strong evidence to indicate that the structural alterations within the hinge region of Nr2f2 contribute importantly to salt-sensitive BP regulation.
Improved cardiac and renal function of the Nr2f2mutant rats
Nr2f2mutant rats exhibited superior cardiac function compared with the wild-type hypertensive rats. As measured by echocardiography, Nr2f2mutant exhibited superior left ventricular function as demonstrated by increased fractional shortening (FS) (56%±3 versus 41%±2; t-test, P<0.05, Fig. 4a), increased velocity of circumferential shortening (Vcf) (8.6±0.3 versus 6.1±0.4; t-test, P<0.05, Fig. 4b) and increased FS/MPI (fractional shortening/myocardial performance index) (1.48±0.22 versus 0.78±0.08; t-test, P<0.05, Fig. 4c). Nr2f2mutant rats also exhibited superior renal function compared with the wild-type hypertensive rats. Urinary protein excretion (UPE) of Nr2f2mutant rats was lower compared with the wild-type hypertensive rats (87.97±7.07 versus 122.63±7.07; t-test, P<0.05; Fig. 5). These additional physiological parameters that provide evidence to the overall improvement in cardiovascular and renal function that are associated with the observed lowering of BP in the Nr2f2mutant rats.
Interaction between Nr2f2 and Fog2 is linked to BP regulation
Next, we sought to explore the molecular mechanism by which Nr2f2 mediates the observed alterations in blood pressure and we focused on the sole difference between the two strains, which is in the hinge region of Nr2f2. Amino acids 117–414 of Nr2f2 are required for protein–protein interaction with another transcriptional regulator, Fog2 (ref. 14). Fog2 is a multi-zinc-finger transcriptional co-repressor protein required for cardiac development. Fog2 regulates cardiac morphogenesis by interaction with multiple cardiac specific transcriptional factors. Nr2f2 and Fog2 null mice have similar defects related to mesenchymal–epithelial and endothelial interaction, which are important for heart and blood vessel formation14. Of further interest, Ciullo et al., identified a new locus strongly linked to hypertension in human chromosome 8q22–23 region, within which, Fog2 is prioritized as one of the candidate genes for essential hypertension15. We therefore hypothesized that the protein–protein interaction between the hinge region of Nr2f2 and Fog2 is important for BP regulation. To test this hypothesis, wild-type and mutant forms of Nr2f2 cDNA, with Histidine tags, were cloned into pCDNA 3.1(B) myc/His (−) expression vector and overexpressed in NRK52E or COS-7 cells (Fig. 6a, Supplementary Fig. 3). The overexpressed Nr2f2 proteins along with their binding partners were immunoprecipitated with the Nr2f2 N-terminal antibody or with histidine tags and probed with the antibody against Fog2. Compared with the wild-type Nr2f2 protein, the binding of the mutant form of Nr2f2 to Fog 2 was more pronounced (Fig. 6b). To further assess the downstream effect of this interaction, we conducted a promoter chromatin immunoprecipitation experiment. As shown in Fig. 7, an enrichment of Nr2f2 binding to the promoter of atrial natriuretic factor, Anf, was noted in the Nr2f2mutant rats. Anf is a direct target gene known to be influenced by the Nr2f2–Fog2 interaction14. Similar enrichments were not observed with promoters for renin and ApoB, which are other targets of Nr2f2 (refs 16, 17) that are not described in the literature to require Fog2. Because Anf is a vasorelaxant18,19, rats were further tested for vasoreactivity. Vasorelaxation responses of the mesenteric arteries from Nr2f2mutant rats were significantly higher than that of the wild-type rats (Fig. 8). These data indicate that the extent of interaction between Nr2f2 and Fog2 through the hinge region of Nr2f2 is linked to the extent of blood pressure and that modulation of transcription of Anf, is perhaps, one of the potential underlying mechanisms.
In summary, our study provides an important step forward in our current understanding of Nr2f2 as a genetic factor associated with essential hypertension in humans and in rodent models of hypertension. Specifically, combined with the available linkage studies in rat models, by utilizing a novel mutant model of Nr2f2, we have not only demonstrated that Nr2f2 is a genetic determinant of BP, but also documented the significance of the relatively less-explored hinge region of Nr2f2 as an important molecular feature, which through its interaction with Fog2, is linked to BP regulation.
Methods
Animals
All animal procedures and protocols used in this report were approved by the University of Toledo Health Science Campus Institutional Animal Care and Use Committee.
Generation of a ZFN-mediated Nr2f2mutant rat
ZFN construct specific for the rat Nr2f2 gene ( NM_080778.2) were designed, assembled and validated by Sigma-Aldrich, to target the first exon 2 of Nr2f2 (Target sequence: 5′-CAGGATGCCGCCCACCCAgcctacTCACGGGCAGTTTGCACTG-3′; ZFN binds to each underlined sequence on opposite strands). mRNA encoding the Nr2f2 ZFN pairs at a concentration of 10 ng μl−1 were injected into one cell SS/McwiHsd (SS) rat embryos20. Out of 147 embryos injected, 66 healthy embryos were transferred to pseudo-pregnant female SD:Crl rats (Charles River), of which five pups were born. At 14 days of age, the pups were tagged, tail clipped and DNA was extracted and amplified using primers flanking the above target sequence Nr2f2-F (5′-GGGGAAGTGATTTGTGAGGA-3′) and Nr2f2-R (5′-GCCCATGATGTTGTTAGGCT-3′). PCR products were analysed for ZFN-induced mutations using a Cel-I assay as described previously20. Among the five pups born, two positive founders (male/female) were identified. PCR products were TOPO cloned and sequenced to reveal the same 15-bp deletion 5′-CCTACTCACGGGCAG-3′ in exon 2 of the Nr2f2 gene in both mutant animals. The founder male/female rats were backcrossed to the parental SS/JrHsdMcwi strain. Multiple separate pairs of mutation-carrying progeny were then intercrossed to generate an F2 population that was used for phenotyping and breeding to homozygosity. The DNA sequencing of the homozygous Nr2f2mutant rats confirmed the 15-bp deletion in F2 offspring, which results in a predicted loss of five amino acids PTHGQ.
Blood pressure measurements by the tail-cuff and radiotelemetry methods
Each set of wild-type hypertensive SS/JrHsdMcwi rats, hitherto referred as hypertensive rats (n=20 males) and Nr2f2mutant rats (n=17 males), hypertensive rats (n=6 females) and Nr2f2mutant rats (n=6 females) were bred, housed and studied concomitantly to minimize environmental effects. All rats were fed with 0.3% NaCl low-salt diet (Harlan Teklad), weaned at 30 days of age. To assess salt sensitivity, at the age of 6 weeks, rats were divided into two groups. The low-salt group was continued to be fed with the 0.3% NaCl low-salt diet, whereas the high-salt group was switched to a diet containing 2% NaCl. After 24 days on these diets, systolic blood pressure (BP) of the rats was measured by using the tail-cuff method21. Conscious restrained rats were warmed to 28 °C. The BP of each rat was measured for two consecutive days by two masked operators. BP values for each day were the mean of three or four consistent readings. The final BP value used was the mean of the two daily BP values. BP was also collected using a telemetry system (Data Sciences International, St Paul, MN)22. Four days after the BP measurements by the tail-cuff method, Nr2f2mutant (n=8 males) and wild-type hypertensive rats (n=7 males) rats were surgically implanted with C40 radiotelemetry transmitters such that the body of the transmitter is placed into the left flanks and the probe is inserted into their femoral arteries all the way into the lower abdominal aortae. Rats were allowed to recover from surgery for 4 days before the transmitters were turned on for recording BP.
Urinary protein excretion
Within 5 days following BP measurements, Nr2f2mutant (n=17) and wild-type hypertensive (n=18) rats were housed individually in metabolic cages and urine samples were collected over a 24 h period. The pyrogallol-based QuanTtest Red Total Protein Assay from Quantimetrix (Redondo Beach, CA) was used to determine protein concentrations of the urine samples. A VERSAmax microplate reader from Molecular Devices (Sunnyvale, CA) was used to determine absorbance at 600 nm. Protein concentrations were determined by reading against the absorbance of the QuanTtest human protein standards (25–200 mg dl−1). UPE data are presented as milligrams of protein per kg of body weight over a 24 h period (mg of protein per kg bwt per 24 h).
Echocardiography
Nr2f2mutant (n=12) and wild-type hypertensive rats (n=12) were evaluated by transthoracic echocardiography for the determination of fractional shortening (FS), velocity of circumferential shortening (Vcf), and myocardial performance index(MPI) using a Sequoia C512 System (Siemens Medical) with a 15-MHz linear array transducer23,24. The rats were anaesthetized with 1.5–2.0% isoflurane by mask, the chest was shaved, the animal was situated in the supine position on a warming pad and ECG limb electrodes were placed. Two-dimensional guided M-mode images and Doppler studies of aortic and trans-mitral flows were obtained from the parasternal long and foreshortened apical windows, respectively. Study duration was typically 15–20 min per animal.
All data were analysed offline with software resident on the ultrasound system in an investigator-masked manner. LV end-diastolic (LVDd) and end-systolic (LVSd) dimensions were determined from M-mode tracings recorded at the mid-papillary level, and LV FS was calculated as [(LVDd−LVSd)/LVDd] × 100%. Ejection time and the isovolumic contraction and relaxation times were measured from colour-flow directed Doppler pulsed-wave traces of mitral and aortic flow obtained at the level of the LV outflow tract from the apical four-chamber view. Aortic outflow and mitral inflow waveforms were recorded when the mitral and aortic waveforms were distinct and aortic and mitral valve clicks were clearly visible. Ejection, contraction and relaxation times were determined from three consecutive beats and averaged to calculate MPI. MPI was calculated as the sum of the isovolumic contraction and relaxation times divided by the ejection time. The Vcf was calculated as FS/ejection time. Values are means±s.e.. Groups were compared with Student's t-test.
Vascular reactivity
After recording blood pressure by radiotelemetry, the wild-type hypertensive (n=9) and Nr2f2mutant rats (n=4) were euthanized with CO2 inhalation method. The mesentery was immediately excised and placed in ice-cold physiological salt solution (PSS) consisting of mmoles l−1 of 118.5 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4, 25.0 NaHCO3 and 5.5 D-glucose aerated with 95% O2–5% CO2. Using a dissecting microscope, the mesenteric arteries were gently cleaned of adipose and connective tissue and cut into segments of approximately 2 mm in length. The segments were then mounted in a Mulvany–Halpern style small vessel wire myograph chamber (Model 620M, Danish Myotechnology, Aarhus, Denmark) filled with 6 ml PSS, maintained at 37 °C, and continuously aerated with 95% O2, 5% CO2 to measure vascular reactivity25,26,27. After 30 min of equilibration period, a standardized computer-assisted normalization procedure was performed to set the pretension of the arteries using LabChart8 software (AD Instruments, Colorado Springs, CO). This involves setting a circumference of the arteries equivalent to 90% of that which they would have at a transmural pressure of 100 mm Hg. Segments were washed with PSS and left to equilibrate for another 30 min. Vessel contractility was then assessed with the high-K+ (120 mmol l−1) solution. After washing the segment three to four times with PSS, cumulative concentration-response curve to phenylephrine (0.1–100 μM) was obtained. After another 30-min wash, the segments were precontracted with submaximal dose of phenylephrine until the plateau was achieved. Dose-dependent relaxation responses to ACh (0.001–1 μM) and sodium nitroprusside (1 nM–100 μM) were assessed by constructing cumulative concentration-response curve. Relaxation in response to ACh and sodium nitroprusside were expressed as a percentage of the level of pre-constriction induced by submaximal dose of phenylephrine.
Western blotting
Antibodies were from the following sources: Anti- Nr2f2 mouse monoclonal (Nr2f2-Ab1, Abcam, 1:2,000 dilution), anti-CoupTF II (Nr2f2-Ab2, Cell Signaling, 1:500 dilution), anti-Fog2 (GeneTex, 1:1,000 dilution), donkey anti-mouse IgG-HRP conjugate (SC-2020, 1:5,000 dilution), anti-rabbit IgG-HRP conjugate (Cell Signaling, 1:5,000 dilution). Samples were homogenized in ice-cold RIPA lysis buffer with protease inhibitor cocktail (Pierce). Immunoblotting for Nr2f2 was carried out using tissue lysates from Nr2f2mutant and wild-type hypertensive rat homogenates. In brief, 40 μg of proteins were boiled with Laemmli sample buffer (Bio-Rad) for 5 min at 95 °C. Protein samples were resolved using 4–15% Criterion Tris-HCl Gel (Bio-Rad) at room temperature, and transferred on to polyvinylidene difluoride membrane (Millipore), blocked with 5% fat-free milk powder in phosphate-buffered saline with Tween 20 and incubated with anti-Nr2f2 and secondary antibodies conjugated with horseradish peroxidase. Immunoreactivity was detected by autoradiography using SuperSignal West Pico Chemiluminescent Substrate (Pierce). Full-length blots are shown in Supplementary Fig. 1.
Chromatin Immunoprecipitation (ChIP)
ChIP16,28 was performed using heart tissue from wild-type male hypertensive (n=3) and Nr2f2mutant rats (n=3). Tissues were minced and fixed with 1% formaldehyde. After crosslinking, nuclei were isolated and the cross-linked chromatin was subjected to sonication to obtain DNA fragments. Chromatin (50 μg) was immunoprecipitated with 8 μg of Nr2f2 antibody (Nr2f2-Ab1). As a negative control, chromatin was precipitated with 8 μg of mouse IgG (Cell Signaling). The immune complexes were precipitated with protein G agarose beads. Precipitated chromatin was eluted from the beads and crosslinks were reversed overnight at 65 °C. Chromatin was treated with RNase A, proteinase K and the DNA was column purified (PCR Purification kit, Qiagen). Purified DNA was PCR amplified using primers (Supplementary Table 1) targeting the Anf, Renin and Apob promoter regions, and Hbb used as a negative control.
Cell culture, transfection and immunoprecipitation
NRK52E cells were obtained from ATCC (Catalogue number CRL-1571) and cultured in DMEM supplemented with 5% fetal bovine serum, 100 units ml−1 penicillin and 100 μg ml−1 streptomycin in a humidified atmosphere at 37 °C with 5% CO2. Both wild-type and mutant forms of Nr2f2 cDNA were cloned into the pCDNA 3.1(B) myc/His (−) expression plasmid. NRK52E cells were transfected with both wild-type and mutant forms of Nr2f2 plasmids using X-tremeGENE HP DNA transfection reagent (Roche Applied Science). Forty-eight hours after transient transfection, cells were treated with the cell lysis buffer and used for subsequent experiments. For immunoprecipitation, lysates (500 μg of protein) were pre-cleared with protein G agarose beads (Pierce) at 4 °C for 2 h. After removal of the beads by centrifugation, lysates were incubated overnight with anti-Nr2f2 antibody (5 μg per IP experiment, Nr2f2-Ab1, Abcam) at 4 °C. The immunoprecipitated complex was captured by incubating with 50 μl of protein G agarose beads. Western blotting was performed with Anti-Fog2 antibody (1:1,000 dilution) as detailed above.
Statistical analysis
All BP statistical analyses were done using the SPSS software (SPSS, Chicago, IL). Data were analysed by independent sample t-test. Data are presented as the mean±standard error. A P value of ≤0.05 was used as a threshold for statistical significance. For BP and UPE experiments measurements, the number of rats in each group ranged from 6 to 20.
Additional information
How to cite this article: Kumarasamy, S. et al. Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension. Nat. Commun. 6:6252 doi: 10.1038/ncomms7252 (2015).
References
Wellcome Trust Case Control Consortium. Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447, 661–678 (2007).
Browning, B. L. & Browning, S. R. Haplotypic analysis of Wellcome Trust Case Control Consortium data. Hum. Genet. 123, 273–280 (2008).
Krushkal, J. et al. Genome-wide linkage analyses of systolic blood pressure using highly discordant siblings. Circulation 99, 1407–1410 (1999).
Weder, A. B. et al. Erythrocyte sodium-lithium countertransport and blood pressure: a genome-wide linkage study. Hypertension 41, 842–846 (2003).
Xu, X. et al. An extreme-sib-pair genome scan for genes regulating blood pressure. Am. J. Hum. Genet. 64, 1694–1701 (1999).
Xu, X. et al. Mapping of a blood pressure quantitative trait locus to chromosome 15q in a Chinese population. Hum. Mol. Genet. 8, 2551–2555 (1999).
Joe, B. et al. Transcriptional profiling with a blood pressure QTL interval-specific oligonucleotide array. Physiol. Genomics. 23, 318–326 (2005).
Yagil, C., Hubner, N., Kreutz, R., Ganten, D. & Yagil, Y. Congenic strains confirm the presence of salt-sensitivity QTLs on chromosome 1 in the Sabra rat model of hypertension. Physiol. Genomics 12, 85–95 (2003).
Hubner, N. et al. Integrated transcriptional profiling and linkage analysis for identification of genes underlying disease. Nat. Genet. 37, 243–253 (2005).
Kato, N. et al. Isolation of a chromosome 1 region affecting blood pressure and vascular disease traits in the stroke-prone rat model. Hypertension 42, 1191–1197 (2003).
Cui, Z. H. et al. Exaggerated response to restraint stress in rats congenic for the chromosome 1 blood pressure quantitative trait locus. Clin. Exp. Pharmacol. Physiol. 30, 464–469 (2003).
Pereira, F. A., Qiu, Y., Zhou, G., Tsai, M. J. & Tsai, S. Y. The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development. Genes Dev. 13, 1037–1049 (1999).
Lee, C. T. et al. The nuclear orphan receptor COUP-TFII is required for limb and skeletal muscle development. Mol. Cell. Biol. 24, 10835–10843 (2004).
Huggins, G. S., Bacani, C. J., Boltax, J., Aikawa, R. & Leiden, J. M. Friend of GATA 2 physically interacts with chicken ovalbumin upstream promoter-TF2 (COUP-TF2) and COUP-TF3 and represses COUP-TF2-dependent activation of the atrial natriuretic factor promoter. J. Biol. Chem. 276, 28029–28036 (2001).
Ciullo, M. et al. New susceptibility locus for hypertension on chromosome 8q by efficient pedigree-breaking in an Italian isolate. Hum. Mol. Genet. 15, 1735–1743 (2006).
Weatherford, E. T., Liu, X. & Sigmund, C. D. Regulation of renin expression by the orphan nuclear receptors Nr2f2 and Nr2f6. Am. J. Physiol. Renal Physiol. 302, F1025–F1033 (2012).
Ladias, J. A. & Karathanasis, S. K. Regulation of the apolipoprotein AI gene by ARP-1, a novel member of the steroid receptor superfamily. Science 251, 561–565 (1991).
Ku, D. D., Guo, L., Dai, J., Acuff, C. G. & Steinhelper, M. E. Coronary vascular and endothelial reactivity changes in transgenic mice overexpressing atrial natriuretic factor. Am. J. Physiol. 271, H2368–H2376 (1996).
Kim, M. et al. GLP-1 receptor activation and Epac2 link atrial natriuretic peptide secretion to control of blood pressure. Nat. Med. 19, 567–575 (2013).
Geurts, A. M. et al. Generation of gene-specific mutated rats using zinc-finger nucleases. Methods. Mol. Biol. 597, 211–225 (2010).
Joe, B. et al. Positional identification of variants of Adamts16 linked to inherited hypertension. Hum. Mol. Genet. 18, 2825–2838 (2009).
Kumarasamy, S. et al. Refined mapping of blood pressure quantitative trait loci using congenic strains developed from two genetically hypertensive rat models. Hypertens. Res. 34, 1263–1270 (2011).
Morgan, E. E. et al. Validation of echocardiographic methods for assessing left ventricular dysfunction in rats with myocardial infarction. Am. J. Physiol. Heart Circ. Physiol. 287, H2049–H2053 (2004).
Gopalakrishnan, K. et al. Augmented rififylin is a risk factor linked to aberrant cardiomyocyte function, short-QT interval and hypertension. Hypertension 57, 764–771 (2011).
Pacurari, M. et al. Endothelial cell transfusion ameliorates endothelial dysfunction in 5/6 nephrectomized rats. Am. J. Physiol. Heart Circ. Physiol. 305, H1256–H1264 (2013).
Hilgers, R. H., Todd, J. Jr. & Webb, R. C. Regional heterogeneity in acetylcholine-induced relaxation in rat vascular bed: role of calcium-activated K+ channels. Am. J. Physiol. Heart Circ. Physiol. 291, H216–H222 (2006).
Lu, X. & Kassab, G. S. Assessment of endothelial function of large, medium, and small vessels: a unified myograph. Am. J. Physiol. Heart Circ. Physiol. 300, H94–H100 (2011).
Mehrotra, A., Joe, B. & de la Serna, I. L. SWI/SNF chromatin remodeling enzymes are associated with cardiac hypertrophy in a genetic rat model of hypertension. J. Cell. Physiol. 228, 2337–2342 (2013).
Acknowledgements
This work was funded by National Institutes of Health Grants HL076709, HL112641 and HL020176 (to B.J.). We thank Drs Howard Jacob and Aron Geurts at the Medical College of Wisconsin for assistance in developing the Nr2f2 mutant rat model; these models were developed, in part, through an American Reinvestment and Recovery Act (ARRA). Grand Opportunity award HL101681 to Dr Jacob. We also thank Dr Nitin Puri at the University of Toledo College of Medicine and Life Sciences for help with the vascular reactivity studies.
Author information
Authors and Affiliations
Contributions
B.J. conceived and designed the project. S.K., H.W., K.G., E.M. and B.M. performed the experiments. S.K., E.M. and H.W., analysed the data. B.J., S.K., K.G., E.M. and H.W. analysed the data. B.J., S.K., E.M. and H.W. prepared the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
Supplementary Figures 1-3 and Supplementary Table 1 (PDF 328 kb)
Rights and permissions
About this article
Cite this article
Kumarasamy, S., Waghulde, H., Gopalakrishnan, K. et al. Mutation within the hinge region of the transcription factor Nr2f2 attenuates salt-sensitive hypertension. Nat Commun 6, 6252 (2015). https://doi.org/10.1038/ncomms7252
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/ncomms7252
This article is cited by
-
A genome-wide by PM10 exposure interaction study for blood pressure in Korean adults
Scientific Reports (2023)
-
Rat models of human diseases and related phenotypes: a systematic inventory of the causative genes
Journal of Biomedical Science (2020)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.