Genetic Model Of Hypertension Research Articles

Abstract 145: Knockout of Gamma- Adducin Impairs Vascular Function in Renal Arterioles in Association With Htn-Induced Renal Injury

The FHH rat is a genetic model of hypertension that develops proteinuria, glomerulosclerosis, and chronic kidney disease. However, the genetic causes are unknown. We have previously identified an inactivating mutation of Add3 in FHH rats in association with reduced ADD3 expression and impaired myogenic reactivity of the renal arterioles. The vascular impairment and renal dysfunction were restored with knock-in of wild type (WT) Add3 in FHH rats. The present study examined whether knockout (KO) of Add3 in normal WT rats impairs the myogenic response of renal afferent arteriole (Af-art), interlobular arteriole (IA), arcuate arteriole (AA), renal blood flow (RBF) autoregulation, and if the reduction of expression of ADD3 promotes hypertension-induced renal dysfunction. Blood pressure was similar in 24-week old Add3 KO vs. WT rats, and after induction of 3 weeks of DOCA-salt-hypertension in these strains. Proteinuria was significantly higher in both normotensive and hypertensive Add3 KO compared with WT rats. KO of Add3 abolished the myogenic response of Af-art compared with WT rats. Similarly, the diameters of IA and AA increased 46 % (from 55.28 ± 2.95 um to 80.39 ± 0.41 um) and 25 % (from 92.57 ± 9.51 um to 115.49 ± 11.68 um) respectively in Add3 KO rats (n=4), while they remained unchanged in WT (n=5). Moreover, KO of Add3 abolished the RBF autoregulation compared with WT rats. These results indicate that KO of Add3 plays an important role to reduce the myogenic reactivity of renal arterioles and RBF autoregulation, and enhances renal damage after the onset of hypertension.

Endogenous AT1 receptor-protein kinase C activity in the hypothalamus augments glutamatergic input and sympathetic outflow in hypertension.

The angiotensin AT1 receptor expression and protein kinase C (PKC)-mediated NMDA receptor phosphorylation levels in the hypothalamus are increased in a rat genetic model of hypertension. Blocking AT1 receptors or PKC activity normalizes the increased pre- and postsynaptic NMDA receptor activity of hypothalamic presympathetic neurons in hypertensive animals. Inhibition of AT1 receptor-PKC activity in the hypothalamus reduces arterial blood pressure and sympathetic nerve discharges in hypertensive animals. AT1 receptors in the hypothalamus are endogenously activated to sustain NMDA receptor hyperactivity and elevated sympathetic outflow via PKC in hypertension. Increased synaptic N-methyl-d-aspartate receptor (NMDAR) activity in the hypothalamic paraventricular nucleus (PVN) plays a major role in elevated sympathetic output in hypertension. Although exogenous angiotensin II (AngII) can increase NMDAR activity in the PVN, whether endogenous AT1 receptor-protein kinase C (PKC) activity mediates the augmented NMDAR activity of PVN presympathetic neurons in hypertension is unclear. Here we show that blocking AT1 receptors with losartan or inhibiting PKC with chelerythrine significantly decreased the frequency of NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and the amplitude of puff NMDA currents of retrogradely labelled spinally projecting PVN neurons in spontaneously hypertensive rats (SHRs). Also, treatment with chelerythrine abrogated the potentiating effect of AngII on mEPSCs and puff NMDA currents of labelled PVN neurons in SHRs. In contrast, neither losartan nor chelerythrine had any effect on mEPSCs or puff NMDA currents in labelled PVN neurons in Wistar-Kyoto (WKY) rats. Furthermore, levels of AT1 receptor mRNA and PKC-mediated NMDAR phosphorylation in the PVN were significantly higher in SHRs than in WKY rats. In addition, microinjection of losartan or chelerythrine into the PVN substantially reduced blood pressure and renal sympathetic nerve discharges in SHRs but not in WKY rats. Chelerythrine blocked sympathoexcitatory responses to AngII microinjected into the PVN. Our findings suggest that endogenous AT1 receptor-PKC activity is essential for presynaptic and postsynaptic NMDAR hyperactivity of PVN presympathetic neurons and for the augmented sympathetic outflow in hypertension. This information advances our mechanistic understanding of the interplay between angiotensinergic and glutamatergic excitatory inputs in hypertension.

Antisense Oligonucleotides Immobilized on Silicon-Organic Nanoparticles as a Tool for Prolonged Correction of Hypertensive States.

We propose an original method for controlling BP by administration of Si~ODN nanocomposites containing antisense oligonucleotides fixed on silicon-organic nanoparticles. ODN in nanocomposites are targeted to mRNA of the genes encoding angiotensin-converting enzyme (ACE1) and type 1 angiotensin-II receptor (AT1A). The experiments were performed on hypertensive ISIAH rats, a genetic model of hypertension. Single inhalation or intraperitoneal administration of the nanocomposites targeted to ACE1 mRNA or ATA1 mRNA, respectively, led to a pronounced decrease (by ~30 mm Hg) in systolic BP in ISIAH rats over a week. The use of scrambled ODN in the nanocomposites had no effect. A decrease in the expression of ACE1 and AT1A genes under the effect of the corresponding antisense ODN was demonstrated, which attested to directed effect of the test preparations.

Treatment of Hypertension with Stable Analogue of 14, 15‐Epoxyeicosatrienoic Acid (EET‐A) in Spontaneously Hypertensive Rats

IntroductionHypertension and concomitant complications, such as blood circulation disorders, stroke, heart attack or kidney failure, became one of the leading causes of death in the world. Recent reports indicate on epoxyeicosatrienoic acids (EETs) as significant factors in blood pressure regulation. These cytochrome P‐450 dependent metabolites of arachidonic acid, exhibit vasodilator and natriuretic activity. It has been shown that EETs facilitate the production of vasodilatory NO, protect the endothelium, exhibit antioxidant properties and might inhibit inflammation. All of the above‐mentioned features should contribute to the reduction of blood pressure and adverse effect of hypertension, hence we postulate the usefulness of EETs as novel drugs for the prevention and/or treatment of hypertension. In the present study the potential hypotensive efficiency of a stable analogue of 14,15‐EET: disodium (S)‐2‐(13‐(3‐entyl)ureido)‐tridec‐8(Z)‐enamido) succinate, called EET‐A, in genetic model of hypertension (spontaneously hypertensive rat, SHR) was evaluated.MethodsMale SHR (n = 6) in the established stage of hypertension (16 weeks) were treated for four weeks with EET‐A dissolved in drinking water (10 mg/kg/day); aged‐matched control group received water only (n = 6). Systolic blood pressure (SBP) was measured by telemetry technique every three days. Once a week observations in metabolic cages (food and water intake, feces, diuresis) were performed; urine and blood samples were collected for further analysis. Nitric oxide metabolites and vascular endothelial growth factor (VEGF) content in urine was measured with commercial ELISA kit.ResultsPreliminary results showed that EET‐A did not impact the blood pressure of adult SHR. We did not observe any changes in diuresis, water and food intake. Also sodium excretion did not change after EET‐A treatment, however, surprisingly, it significantly increased in the control group (EET‐A day 0: 0.5 ± 0.1 vs day 28: 0.5 ± 0.1, NS; control group day 0: 0.4 ± 0.2 vs day 28: 1.0 ± 0.1#* mmol/24h, *p<0.05 within the control group; # values on day 28th between groups, p<0.05). The excretion rates of NO metabolites (nitrates/nitrites; NOx) and VEGF‐A increased significantly in the EET‐A treated group (NOx day 0: 0.99 ± 0.18 vs day 28: 1.56 ± 0.13 μmol/24h, p<0.05; VEGF‐A day 0: 3.2 ± 0.7 vs day 28: 9.5 ± 2.0 ng/24h, p<0.05).SummaryAnalysis of the obtained data suggests that EET‐A in the applied dose does not lower blood pressure. Further studies are necessary to investigate if, despite the lack of blood pressure decrease, EET‐A improved kidney function.Support or Funding InformationNational Science Centre 2017/26/M/NZ5/00367This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of Early Postnatal Gut Microbiota on Immune System in SHR Hypertension

Hypertension exhibits a prominent immunological component. We have recently shown that spontaneously hypertensive rats (SHR) have a genetic abnormality whereby they are born with a large population of proinflammatory CD161+ immune cells. Although early postnatal maternal environment is important for development of hypertension, its impact on the immune system is not known. Cross‐fostering SHR pups by normotensive Wistar‐Kyoto (WKY) dams lowers the blood pressure in adulthood only if the cross‐fostering is initiated at a very young age. In this study, we used SHR as a genetic model of hypertension to test whether early postnatal gut microbiota affect the immune system and blood pressure of SHR. Since cross‐fostering permanently changes the gut microbiota of the fostered offspring, we first examined the microbial populations in the fecal samples of SHR and normotensive control WKY using 16s rDNA sequencing. We found that the newborn SHR (1‐week old) have abnormal gut microbiota that is qualitatively and quantitatively different from the newborns of normotensive WKY. The representation of predominant bacterial phylum Proteobacteria was less in 1‐week old SHR pups (94% Proteobacteria in WKY vs. 65% in SHR neonates). Even within the phylum Proteobacteria, the colonizing genera in WKY and SHR differed dramatically. In contrast, the representation of phylum Fermicutes in the neonatal SHR gut was greater than WKY (5% Fermicutes in WKY vs. 31% in SHR). We also observed a dramatic shift in 1‐week old SHR gut microbiota when we cross‐fostered WKY and SHR pups beginning at 1‐day of age. Two major bacterial genera of phylum Proteobacteria present in self‐fostered SHR pups, but absent in WKY, were depleted after cross‐fostering, and were replaced by other bacteria predominant in WKY gut. Investigation of immune cells in the spleen of cross‐fostered SHR using cell surface markers and flow cytometry showed that there was a clear reduction in CD161+ cells (30.7% in self‐fostered SHR vs. 12.6% in cross‐fostered SHR at 30 weeks of age), a marker associated with inflammation. Thus, cross‐fostering of SHR pups by WKY dams shifted the composition of their gut microbiota toward WKY within one week. Additionally, preliminary results suggest lower systolic blood pressure (tail cuff pressure) in cross‐fostered SHR. In summary, SHR are born with a defect in their immune system that results in greater proinflammatory response associated with hypertension. Moreover, the gut microbiota of the SHR is remarkably different from that of normotensive WKY, and cross‐fostering of SHR by WKY dams favorably changes the gut microbiome and immune regulation.Support or Funding InformationNational Institutes of Health Program Project Grant to FMA (HL 14388) VA Merit Review Award to MWC (1 I01 BX001414), American Heart Association Innovative Research Grant to MVS (16IRG27260323).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Metabolic Changes in Spontaneously Hypertensive Rat Hearts Precede Cardiac Dysfunction and Left Ventricular Hypertrophy.

BackgroundSustained pressure overload leads to changes in cardiac metabolism, function, and structure. Both time course and causal relationships between these changes are not fully understood. Therefore, we studied spontaneously hypertensive rats (SHR) during early hypertension development and compared them to control Wistar Kyoto rats.Methods and ResultsWe serially evaluated myocardial glucose uptake rates (Ki) with dynamic 2‐[18F] fluoro‐2‐deoxy‐D‐glucose positron emission tomography, and ejection fraction and left ventricular mass to body weight ratios with cardiac magnetic resonance imaging in vivo, determined glucose uptake and oxidation rates in isolated perfused hearts, and analyzed metabolites, mammalian target of rapamycin activity and endoplasmic reticulum stress in dissected hearts. When compared with Wistar Kyoto rats, SHR demonstrated increased glucose uptake rates (Ki) in vivo, and reduced ejection fraction as early as 2 months of age when hypertension was established. Isolated perfused SHR hearts showed increased glucose uptake and oxidation rates starting at 1 month. Cardiac metabolite analysis at 2 months of age revealed elevated pyruvate, fatty acyl‐ and branched chain amino acid‐derived carnitines, oxidative stress, and inflammation. Mammalian target of rapamycin activity increased in SHR beginning at 2 months. Left ventricular mass to body weight ratios and endoplasmic reticulum stress were elevated in 5 month‐old SHR.ConclusionsThus, in a genetic hypertension model, chronic cardiac pressure overload promptly leads to increased myocardial glucose uptake and oxidation, and to metabolite abnormalities. These coincide with, or precede, cardiac dysfunction while left ventricular hypertrophy develops only later. Myocardial metabolic changes may thus serve as early diagnostic markers for hypertension‐induced left ventricular hypertrophy.

Newly manufactured Marukome MK-34-1 miso with angiotensin-converting enzyme inhibitory activity and its antihypertensive effects in genetic hypertensive rat models

We newly manufactured miso rich in angiotensin-converting enzyme (ACE) inhibitory activity (Marukome MK-34-1, shinki miso) and investigated its antihypertensive properties in rat models of genetic hypertension. ACE inhibitory activity was tenfold higher in shinki miso than in commercially available Marukome Nenrin miso (nenrin miso). The inhibitory activity of shinki miso was confined to <3 kDa fractions and was detected in several fractions with high polarity by C18 high-performance liquid chromatography. Systolic blood pressure (SBP) increased age-dependently in stroke-prone spontaneously hypertensive rats (SHRSP/Izm) given a 0.6% (w/v) NaCl solution (salt solution group) that matched the salt content of the miso solutions. This SBP increase was attenuated in both the 5% nenrin and 5% shinki miso solution groups compared to the salt solution group. The reduction in SBP was greater in rats fed shinki than in rats fed nenrin miso. Similarly, in a salt-induced hypertension model with Dahl rats, the 5% nenrin miso solution attenuated the rising SBP observed in the salt solution group. Moreover, combining 5% nenrin miso with 5% shinki miso (2:1, v/v) (awase miso group) significantly decreased the SBP per gram salt intake by 8% compared with the nenrin miso treatment. However, there were no differences in urinary Na excretion between the nenrin and awase miso groups. In conclusion, we produced a new miso with potent ACE inhibitory activity that reduced spontaneous and salt-induced hypertension. These results suggest that salt sensitivity is decreased by the addition of shinki miso to nenrin miso.

Prostatic blood flow as prominent targets on benign prostatic hyperplasia

Benign prostatic hyperplasia/benign prostatic enlargement (BPH/BPE) is a common proliferative disease, and giving rise to associate with lower urinary tract symptoms (LUTS). However, the pathogenesis is not well clarified, and thought to be multifactorial. There are some lines of evidence that impairment in the blood supply of the lower urinary tract causes development of BPH/BPE. Clinical data showed an association between the development of BPH/BPE and atherosclerotic disease such as hypertension, diabetes and hyperlipidemia. The spontaneously hypertensive rat (SHR) has been used as model of genetic hypertension. SHR also shows decreased blood flow and hyperplastic morphological abnormalities in the ventral prostate. Our previous studies demonstrated that chronic treatment with vasodilative drugs nicorandil (ATP sensitive potassium channel opener) and silodosin (alpha1 adrenoceptor antagonist) increased blood flow and suppressed the growth factor and morphological abnormalities in the SHR ventral prostate. These data suggested that prostatic blood flow could be therapeutic targets for BPH/LUTS.

Disparate effects of antibiotics on hypertension.

Gut microbiota are associated with a variety of complex polygenic diseases. The usage of broad-spectrum antibiotics by patients affected by such diseases is an important environmental factor to consider, because antibiotics, which are widely prescribed to curb pathological bacterial infections, also indiscriminately eliminate gut commensal microbiota. However, the extent to which antibiotics reshape gut microbiota and per se contribute to these complex diseases is understudied. Because genetics play an important role in predisposing individuals to these modern diseases, we hypothesize that the extent to which antibiotics influence complex diseases depends on the host genome and metagenome. The current study tests this hypothesis in the context of hypertension, which is a serious risk factor for cardiovascular diseases. A 3 × 2 factorial design was used to test the blood pressure (BP) and microbiotal effects of three different antibiotics, neomycin, minocycline, and vancomycin, on two well-known, preclinical, genetic models of hypertension, the Dahl salt-sensitive (S) rat and the spontaneously hypertensive rat (SHR), both of which develop hypertension, but for different genetic reasons. Regardless of the class, oral administration of antibiotics increased systolic blood pressure of the S rat, while minocycline and vancomycin, but not neomycin, lowered systolic blood pressure in the SHR. These disparate BP effects were accompanied by significant alterations in gut microbiota. Our study highlights the need to consider an individualized approach for the usage of antibiotics among hypertensives, as their BP could be affected differentially based on their individual genetic and microbiotal communities.

Therapeutic Attenuation of Cardiac Remodeling After Acute Myocardial Infarction: A Conversation With Marc A. Pfeffer, MD, PhD.

Marc Pfeffer, MD, PhD, is the Dzau Professor of Medicine at Harvard Medical School; and Senior Physician, Cardiovascular Division at Brigham and Women’s Hospital. He was born in Brooklyn, NY, and obtained his MD and PhD at the University of Oklahoma. He married his late wife and research collaborator, Janice Pfeffer, PhD, in 1970. He has won numerous awards, including the American Heart Association James B. Herrick Award for Outstanding Achievements in Clinical Cardiology (2013) and the American Heart Association Distinguished Scientist Award (2015), the American College of Cardiology Distinguished Scientist Award (Translational Domain; 2017), and an Honorary Doctoral Degree from Sahlgrenska Academy, University of Gothenburg, Sweden (2014). ### Dr Rutherford asks: In the early 1970s, you, and your late wife Janice, studied adrenergic mechanisms and cardiac hypertrophy and ventricular function, as well, in spontaneously hypertensive rats. What led to your initial animal studies on acute myocardial infarction and your interest in the renin-angiotensin-aldosterone system? Dr Pfeffer replies: As graduate students in the Department of Physiology and Biophysics at the University of Oklahoma, my late wife, Janice, and I had the privilege to work under the tutelage of Edward D. Frohlich, MD, who is a pioneer in the hemodynamics of hypertension with a major emphasis on the response of the heart to the augmented workload imposed by hypertension. He was keenly interested in the functional consequences of developing left ventricular hypertrophy and obtained breeding stock of the newly developed Japanese strain of the SHR. We developed methods to assess hemodynamics and left ventricular function to compare normotensive with hypertensive rats during aging. We found a marked age-associated deterioration in contractile performance in the hypertensive rats that could be prevented by a variety of antihypertensive agents. Our studies of the longitudinal changes in ventricular structure and function in an animal model of genetic hypertension caught the attention of Eugene Braunwald, MD, who was organizing a Program Project Grant on the Pathophysiology of Left Ventricular Hypertrophy at the then Peter Bent Brigham Hospital. We moved to the Brigham in 1976 as his postdoctoral fellows (me as a medical intern) …

Abstract P124: Role of Add3 Dysfunction in Renal Epithelial Cells in the Development of Renal Injury in Fhh Rats

The Fawn Hooded Hypertensive Rat (FHH) is a genetic model of hypertension, in which Add3 has been identified as a candidate gene for renal injury but the mechanism is unknown. The present study examined the effects of knockdown of Add3 on rat renal epithelial (NRK) cells which are the model system for podocytes and proximal tubular cells. Knockdown of the expression of Add3 using a 27-mer Dicer-substrate RNAi (DsiRNA) decreased cell viability using an MTS assay by 50% in comparison to cells treated with vehicle or scrambled DSiRNA (Add3DsiRNA, OD 0.5±0.1; untreated cells 0.9±0.1; NC-1 0.9±0.1; n=8, P&lt;0.001). F-actin immunostaining revealed that Add3 DSiRNA markedly disrupted the cytoskeleton, and the F-actin staining intensity/cell ratio was significantly reduced (untreated 543±47, n=552 cells; NC-1 592±39, n=566 cells; Add3DsiRNA 430±58, n=385 cells, P&lt;0.05). The maximum mitochondrial respiratory rate was reduced in Add3 DsiRNA treated cells (untreated 444±42; NC-1 414±32; Add3 DsiRNA 275±16 pmole/min/μg, P&lt;0.01). Proton leak was higher (untreated 42±3; NC-1 40±4; Add3 DsiRNA 63±5 pmole/min/μg, P&lt;0.002) and oxygen consumption coupled to ATP production was decreased in Add3 DsiRNA treated cells (untreated 229±12, n=9; NC-1 200±12, n=8; Add3 DsiRNA 146±11 pmole/min/μg, n=9, P&lt;0.001). Spare respiratory capacity, an index of cell fitness, was reduced by knockdown of Add3 (untreated 154±42, n=9: NC-1 143±32, n=8; Add3DsiRNA 33±16 pmole/min/μg, n=9, P&lt;0.001). Non-mitochondrial oxygen consumption rate (OCR/μg protein) increased in cells treated with Add3 DsiRNA (untreated 57±7; NC-1 80±11; Add3DsiRNA 116±15 pmole/min/μg, P&lt;0.01) suggesting a possible metabolic switch to glycolytic oxygen consumption. In conclusion, knockdown of Add3 in NRK cells disrupts the actin cytoskeleton, promotes mitochondrial dysfunction and decreases cell viability. Loss of Add3 function may contribute to proteinuria in FHH rats by altering the cytoskeleton and promoting effacement and loss of podocytes and the reuptake of filtered protein in the proximal tubule.

A novel genetic model to explore the Brenner hypothesis: Linking nephron endowment and number with hypertension

Nephron endowment, the total number of nephrons an individual is born with, is determined by both genetic and environmental factors during embryonic development. In 1988, Brenner hypothesized that there was an inverse relationship between nephron number and hypertension. Over the course of one’s lifetime it is predicted that even healthy individuals will lose a significant percentage of nephrons as part of normal aging. Thus, a low nephron endowment at birth or in combination with age- or disease-related nephron loss could pre-dispose individuals to the development of hypertension. Currently, it is not clear what minimal number (ie, threshold) of nephrons is associated with susceptibility to glomeruli injury or hypertension, due in part to the lack of relevant animal models. The BPH2 mouse is a unique genetic model of hypertension that has a normotensive line (BPN3 mice) as well as a hypotensive line (BPL1 mice) derived from the original breeding of eight common inbred strains of mice. Thus, we hypothesize that the differences in blood pressure observed in BPH2, BPN3, and BPL1 mice will correlate inversely with nephron number as predicted by the Brenner hypothesis. If our hypothesis is true, then the BPH2 mouse model will provide a unique experimental model to study the impact of nephron endowment and nephron number on susceptibility to renal injury and hypertension.

Adverse remodeling of the obtuse marginal artery in compensatory hypertrophied myocardium from spontaneously hypertensive rats.

Spontaneously hypertensive rats (SHR) serve as a model of genetic hypertension. Adverse remodeling of a coronary artery has been reported in SHR. This model is used to study new therapies in regression vascular remodeling. However, no data are available that show remodeling of the intramyocardial branch of the obtuse marginal artery in 10-month-old SHR. This study was designed to assess remodeling (changes in vascular structure and fibrosis) of this coronary artery. The study was performed on 10-month-old male SHR (n=7) and normotensive control Wistar Kyoto rats (WKY) (n=7). Using histology, we show that the external diameter, lumen diameter, wall width, and cross-sectional area of the intramyocardial artery were significantly greater in SHR than in WKY. The wall-to-lumen ratio was similar in SHR and WKY. The collagen volume density of the intramyocardial artery in SHR was significantly greater than in WKY. Our results show hypertrophic outward remodeling in the intramyocardial branch of the obtuse marginal artery of the left ventricle in SHR. This artery can serve as a new vascular bed from adult SHR to study novel therapies in regression coronary artery remodeling.

Abstract 11603: Structural Insights Into C-terminus and N-terminus of a Designer cGMP Activating ANP-Based Antihypertensive Peptide: ZD100

Introduction: As importantly underscored by the SPRINT Trial, innovative blood pressure (BP) lowering and multiorgan protective drugs are needed for treating hypertension (HTN). We engineered a Mayo Clinic designer atrial natriuretic peptide (ANP)-based drug, ZD100, which possesses a unique 12 amino acid (AA) carboxy-terminus (CT) extension while maintaining the N-terminus (NT) of ANP. ZD100 is more potent than ANP in reducing BP, inducing natriuresis and inhibiting aldosterone via pGC-A and its second messenger cGMP. To date, the in vivo actions of ZD100 in a model of essential HTN are undefined. Further, in attempt to enhance and/or minimize pGC-A activation, the importance of specific AAs in the CT and NT of ZD100 is unknown and is critical to define. Hypothesis: We hypothesize that ZD100 is a potent anti-HTN drug activating pGC-A/cGMP system and that specific AA variations in the CT and/or NT of ZD100 will modify the ability of activating pGC-A. Methods: Spontaneously hypertensive rats (SHRs) received intravenous (IV) ZD100 or vehicle and BP, plasma and urinary cGMP and the natriuretic effects were determined. We then engineered 15 strategic ZD100 variants (Vs) consisting of mutated peptides in the CT or NT or both. We stimulated HEK293 cells overexpressing pGC-A with ZD100 and the 15 Vs (10-8 M) and measured intracellular cGMP generation. Results: In SHRs, acute IV ZD100 reduced BP, increased plasma and urinary cGMP generation and induced natriuresis demonstrating efficacy in HTN. In vitro, we found two more potent pGC-A activators (+100% cGMP activation) only among the CT-only mutated ZD100 Vs. Importantly, key mutations of either the CT or NT resulted in 6 loss-of-function Vs identifying importance of peptide-receptor dependency. Conclusions: We demonstrate for the first time in a genetic model of HTN that ZD100 reduces BP, activates systemic and renal pGC-A/cGMP and induces natriuresis. Enhanced or reduced pGC-A activation with targeted AA mutations in the CT and NT of ZD100 provides key structure function insights into ZD100 which may lead to next generation ZD100 therapeutic peptides. These findings support ZD100 as a novel peptide therapeutic strategy for HTN and will help guide the selection of best-in-class ZD100 analogues.

Phenylethanolamine N-methyltransferase gene expression in adrenergic neurons of spontaneously hypertensive rats

Epinephrine is synthesised by the catecholamine biosynthetic enzyme, phenylethanolamine N-methyltransferase (PNMT), primarily in chromaffin cells of the adrenal medulla and secondarily in brainstem adrenergic neurons of the medulla oblongata. Epinephrine is an important neurotransmitter/neurohormone involved in cardiovascular regulation; however, overproduction is detrimental with negative outcomes such as cellular damage, cardiovascular dysfunction, and hypertension. Genetic mapping studies have linked elevated expression of PNMT to hypertension. Adrenergic neurons are responsible for blood pressure regulation and are the only PNMT containing neurons in the brainstem. The purpose of the current study was to determine whether elevated blood pressure found in adult spontaneously hypertensive rats (SHR) is associated with altered regulation of the PNMT gene in catecholaminergic neurons. C1, C2, and C3 adrenergic regions of 16 week old Wistar Kyoto (WKY) and SHR rats were excised using micropunch microdissection for mRNA expression analyses. Results from the current study confirm high PNMT mRNA expression in all three brainstem adrenergic regions (C1: 2.96-fold; C2: 2.17-fold; C3 1.20-fold) of the SHR compared to normotensive WKY rats. Furthermore, the immediate early gene transcription factor (Egr-1) mRNA was elevated in the C1 (1.84-fold), C2 (8.57-fold) and C3 (2.41-fold) regions in the brainstem of the SHR. Low mRNA expression for transcription factors Sp1 and GR was observed, while no change was observed for AP-2. The findings presented propose that alterations in the PNMT gene regulation in the brainstem contribute to enhanced PNMT production and epinephrine synthesis in the SHR, a genetic model of hypertension.

Contribution of Orexin to the Neurogenic Hypertension in BPH/2J Mice.

BPH/2J mice are a genetic model of hypertension associated with an overactive sympathetic nervous system. Orexin is a neuropeptide which influences sympathetic activity and blood pressure. Orexin precursor mRNA expression is greater in hypothalamic tissue of BPH/2J compared with normotensive BPN/3J mice. To determine whether enhanced orexinergic signaling contributes to the hypertension, BPH/2J and BPN/3J mice were preimplanted with radiotelemetry probes to compare blood pressure 1 hour before and 5 hours after administration of almorexant, an orexin receptor antagonist. Mid frequency mean arterial pressure power and the depressor response to ganglion blockade were also used as indicators of sympathetic nervous system activity. Administration of almorexant at 100 (IP) and 300 mg/kg (oral) in BPH/2J mice during the dark-active period (2 hours after lights off) markedly reduced blood pressure (-16.1 ± 1.6 and -11.0 ± 1.1 mm Hg, respectively;P<0.001 compared with vehicle). However, when almorexant (100 mg/kg, IP) was administered during the light-inactive period (5 hours before lights off) no reduction from baseline was observed (P=0.64). The same dose of almorexant in BPN/3J mice had no effect on blood pressure during the dark (P=0.79) or light periods (P=0.24). Almorexant attenuated the depressor response to ganglion blockade (P=0.018) and reduced the mid frequency mean arterial pressure power in BPH/2J mice (P<0.001), but not BPN/3J mice (P=0.70). Immunohistochemical labeling revealed that BPH/2J mice have 29% more orexin neurons than BPN/3J mice which are preferentially located in the lateral hypothalamus. The results suggest that enhanced orexinergic signaling contributes to sympathetic overactivity and hypertension during the dark period in BPH/2J mice.

Sex differences in the blood antioxidant defense system in juvenile rats with various genetic predispositions to hypertension.

This study investigated the contribution of blood oxidative stress (OS) to the development of hypertension, as well as sex differences in the antioxidant defense system (ADS) in genetic models of hypertension. Nine-week-old normotensive Wistar-Kyoto (WKY) rats, borderline hypertensive rats (BHR) and spontaneously hypertensive rats (SHR) of both sexes were used. Systolic blood pressure (SBP) was determined by tail-cuff plethysmography, the trolox equivalent antioxidant capacity (TEAC) and the concentration of lipid peroxides (LP) were determined in plasma. The activity of the antioxidant enzymes Cu/Zn-superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT) was determined in erythrocytes. SBP was significantly elevated in BHR and SHR in both sexes. BHR and SHR males had a higher SBP than the respective females. Sex-dependent differences in the ADS were found only in SHR, in which TEAC, SOD and CAT were significantly higher in males than in females. No differences in TEAC, SOD, CAT and GPx were observed between BHR (males and females) and WKY controls. LP levels were similar in all the groups investigated. Significant positive correlations were observed between SBP and both SOD and CAT. TEAC correlated positively with SOD and LP. As no signs of oxidative damage to lipids were found in young BHR and SHR of either sex, OS in the blood does not seem to be causatively related to the development of hypertension in these rats. However, despite activated antioxidant defenses, the positive correlation between plasma TEAC and LP suggests that oxidative damage is progressing slowly and therefore it seems to be a consequence rather than the cause of hypertension.

Cardiac phenylethanolamine N-methyltransferase: localization and regulation of gene expression in the spontaneously hypertensive rat.

Phenylethanolamine N-methyltransferase (PNMT) is the terminal enzyme in the catecholamine biosynthetic pathway responsible for adrenaline biosynthesis. Adrenaline is involved in the sympathetic control of blood pressure; it augments cardiac function by increasing stroke volume and cardiac output. Genetic mapping studies have linked the PNMT gene to hypertension. This study examined the expression of cardiac PNMT and changes in its transcriptional regulators in the spontaneously hypertensive (SHR) and wild type Wistar-Kyoto (WKY) rats. SHR exhibit elevated levels of corticosterone, and lower levels of the cytokine IL-1β, revealing systemic differences between SHR and WKY. PNMT mRNA was significantly increased in all chambers of the heart in the SHR, with the greatest increase in the right atrium. Transcriptional regulators of the PNMT promoter show elevated expression of Egr-1, Sp1, AP-2, and GR mRNA in all chambers of the SHR heart, while protein levels of Sp1, Egr-1, and GR were elevated only in the right atrium. Interestingly, only AP-2 protein-DNA binding was increased, suggesting it may be a key regulator of cardiac PNMT in SHR. This study provides the first insights into the molecular mechanisms involved in the dysregulation of cardiac PNMT in a genetic model of hypertension.

Abstract MP14: A K572Q Mutation in Gamma-adducin Is Responsible for the Impaired Myogenic Response and Autoregulation of Renal and Cerebral Blood Flow in FHH Rats

The FHH rat is a genetic model of hypertension induced CKD, but the genes and pathways involved are unknown. We recently reported that the myogenic response of the renal and cerebral arteries is impaired in FHH rats and it was restored in a FHH.1BN congenic strain in which a small region of Chr. 1 containing 15 genes, including gamma-Adducin (Add3), from BN rats was transferred into the FHH background. We further identified a K572Q mutation in Add3 in FHH versus FHH.1BN rat. The present study examined the contribution of Add3 to the impaired myogenic reactivity using Add3 transgenic and KO rats. The diameter of the middle cerebral artery (MCA) decreased by 20-30% in SD, FHH.1BN and FHH.Add3 transgenic rats when perfusion pressure was increased from 40 to 140 mmHg. In contrast, the MCA of FHH and SD.Add3 knockout rats did not constrict. The myogenic response of the MCA is also impaired in MNS rats that share the same mutation in Add3 as in FHH rat and this phenotype was complemented in a F1 cross of FHH and MNS strain. Autoregulation of CBF was impaired in FHH rats and rose by 99 ± 7% when MAP was increased from 100 to 190 mmHg and was restored in FHH.Add3 and FHH.1BN rats. Similarly, the diameter of Af-art of FHH, MNS and a F1 cross of FHH and MNS rat increased in response to increase in perfusion pressure but decreased in FHH.1BN rats that have wild type Add3. FHH rats exhibited impaired autoregulation of RBF in comparison with FHH.1BN rats. Pgc estimated from the stopflow pressure increased by 20 mmHg in FHH rats when RPP was increased from 100 to 140 mmHg versus only 4 mmHg in the congenic strain. FHH rats developed severe renal injury and proteinuria rose from 37 ± 2 to 260 ± 32 mg/day as they aged from 12 to 21 weeks, but rose by a significant lesser extent in FHH.1BN and FHH.Add3 rats. BK current in VSMC isolated from the MCA was 5-fold higher in FHH vs. FHH.1BN rats. Administration of IBTX normalized the elevated BK channel current and restored the myogenic response in FHH rats but it had little effect in FHH.1BN. These results indicate that the K572Q mutation in Add3 plays a causal role in the impaired myogenic response and autoregulation of renal and cerebral circulation in FHH rats and may contribute to the development of renal and cerebral end organ damage with aging and after the onset of hypertension.

Abstracts From the 36th Annual Scientific Meeting of the HBPRCA

### CONTRIBUTION OF THE AREA POSTREMA TO THE INCREASED CARDIAC SYMPATHETIC NERVE ACTIVITY IN OVINE HEART FAILURE Abukar Y, Ramchandra R, May CN The Florey Institute of Neuroscience and Mental Health, Melbourne, Victoria, Australia Background : Heart failure (HF) is associated with an increase in cardiac sympathetic nerve activity (CSNA), which is directly linked to mortality in HF patients. The mechanisms responsible for the elevated CSNA remain unclear. Previous studies indicate that the area postrema (AP), a circumventricular organ in the brainstem, plays a role in the control of sympathetic nerve activity. We hypothesized that the elevated CSNA in HF is mediated by the AP and lesioning this region would reduce the increased CSNA in sheep with HF. Aims : To determine the effect of sham lesion or lesion of the AP on CSNA and hemodynamics in conscious sheep with HF. Methods : Studies were conducted in 2 groups of sheep with pacing-induced HF: sham (n=6) and AP lesion (n=6) sheep. Mean arterial blood pressure (MAP), heart rate (HR) and CSNA were recorded simultaneously in conscious sheep at least 4 days after surgery. Results : Heart failure was associated with a significant decrease in ejection fraction (from 74±2 % to 38±1 %; P<0.001), which was similar in both groups. There was a significant reduction in CSNA burst incidence in the AP lesion group compared with the sham group (45±10 and 89±3 bursts/100 heartbeats, respectively; P<0.01). Conclusions : In sheep with HF, the group with lesion of the AP had a significantly lower CSNA compared with the sham group. These data suggest that the AP plays a role in setting the detrimental high levels of CSNA in HF. ### G PROTEIN-COUPLED ESTROGEN RECEPTOR SIGNALING IMPROVES STROKE OUTCOME IN FEMALE MICE Broughton BRS, Jansen GL, Sobey CG Department of Pharmacology, Monash University, Clayton, Victoria, Australia Background: Estrogen has been assumed to provide neuroprotection following stroke entirely via classical estrogen receptors. Interestingly, there is recent evidence that activation of a novel G …