Development Of DOCA-salt Hypertension Research Articles

Role of the median preoptic nucleus in the development of chronic DOCA-salt hypertension

Lesions of the anteroventral third ventricle (AV3V region) are known to prevent many forms of experimental hypertension, including mineralocorticoid (DOCA-salt) hypertension in the rat. However, AV3V lesions include the organum vasculosum of the lamina terminalis (OVLT), portions of the median preoptic nucleus (MnPO), as well as efferent fibers from the subfornical organ (SFO), thereby limiting the ability to define the individual contribution of these structures to the prevention of experimental hypertension. We have previously reported that the SFO does not contribute to the chronic hypertensive response to DOCA-salt in rats, and yet the OVLT plays a significant role in the development of DOCA-salt hypertension. Since efferent fibers of the OVLT project to and through the MnPO, the present study was designed to test the hypothesis that the MnPO is necessary for DOCA-salt hypertension in the rat. Male Sprague-Dawley rats underwent unilateral nephrectomy followed by a week of recovery, and subsequent SHAM (n=5) or electrolytic lesion of the MnPO (MnPOx; n=7). Throughout another week of recovery and during the experimental protocol, rats consumed a 2% NaCl diet and 0.9% NaCl drinking solution. 24-hour mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg/rat; SQ). No differences in the 5-day average control MAP were observed between groups (MnPOx: 104 ± 1 mmHg; SHAM: 107±2 mmHg). The chronic pressor response to DOCA was attenuated in MnPOx rats by day 11 of treatment and continued such that MAP increased 25±3 mmHg in SHAM rats by day 21 of DOCA compared to 14±3 mmHg in MnPOx rats. These results support the hypothesis that the MnPO is an important brain site of action for the pathogenesis of DOCA-salt hypertension in the rat. University of Minnesota Grant In Aid #546839 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract MP46: Knockout Of Add3 Impairs Autoregulation Of Renal Blood Flow And Promotes Hypertension-induced Renal Injury By Increasing Glomerular Capillary Pressure And Podocyte Loss

Recently, we reported that a mutation in γ-Adducin (ADD3) alters the actin cytoskeleton and is associated with an impaired myogenic response of the afferent arteriole and enhanced hypertension-induced renal disease. However, it remains to be determined whether the loss of ADD3 function promotes renal injury by increasing glomerular capillary pressure (Pgc) and podocyte loss or other mechanisms. The present study compared the time course of changes in renal hemodynamics and the progression of renal injury during the development of DOCA-salt hypertension in FHH 1 BN rats (WT) with an intact myogenic response vs. FHH 1 BN Add3KO rats (Add3KO) in which the myogenic response is impaired. When transmural pressure rose from 40 to 100 mmHg, the inner diameter of the preglomerular artery constricted by 19% (47.7 ± 4.3 to 38.4 ± 3.4 μm, n = 5) in WT, but it dilated by 28% (53.0 ± 2.2 to 67.9 ± 4.3 μm, n = 7) in Add3KO. Pgc was similar (50.1 ± 0.4 vs. 51.2 ± 0.8 mmHg, n = 6) at 100 mmHg, but rose by 6 and 14 mmHg in WT vs. Add3KO when perfusion pressure rose to 150 mmHg. Mean arterial pressure increased similarly and reached 177.7 ± 3.5 vs. 182.6 ± 2.3 mmHg (n = 9) after 3 weeks of DOCA-salt hypertension in WT vs. Add3KO. After 1 week of DOCA-salt hypertension, glomerular filtration rate (GFR) increased by 38% (1.2 ± 0.1 to 1.6 ± 0.1 ml/min/kidney, n = 6) and glomerular nephrin expression decreased by 20% (165.4 ± 4.5 to 131.614 ± 5.2 RFU, n = 7) in Add3KO. Both were unaltered in WT. Proteinuria increased 2 folds in WT (56.9 ± 4.7 to 168.6 ± 26.7 mg/day, n = 12) in the first week of hypertension vs. a 6-fold increase in Add3KO (64.6 ± 4.1 to 446.0 ± 41.9 mg/day, n = 9). After 3 weeks of hypertension, the degree of glomerulosclerosis (3.4 ± 0.1 vs. 2.4 ± 0.1 glomerular injury score, n = 9~12), protein cast formation (9.0% ± 0.8% vs. 4.8% ± 0.4% of area, n = 6), epithelial-mesenchymal transition, interstitial fibrosis (17.9% ± 0.8% vs. 9.5% ± 0.3% of area, n = 6), and inflammation was significantly greater in Add3KO vs. WT. GFR and Pgc were 28% and 19% lower in Add3KO than WT. These results indicate that the impaired myogenic response increases the transmission of pressure to the glomerulus to induce the loss of podocytes, which accelerates the progression of renal injury during the development of hypertension in Add3KO.

Impaired renal hemodynamics and glomerular hyperfiltration contribute to hypertension-induced renal injury

Recently, we reported a mutation in γ-adducin (ADD3) was associated with an impaired myogenic response of the afferent arteriole and hypertension-induced chronic kidney disease (CKD) in fawn hooded hypertensive (FHH) rats. However, the mechanisms by which altered renal blood flow (RBF) autoregulation promotes hypertension-induced renal injury remain to be determined. The present study compared the time course of changes in renal hemodynamics and the progression of CKD during the development of DOCA-salt hypertension in FHH 1BN congenic rats [wild-type (WT)] with an intact myogenic response versus FHH 1BN Add3KO (Add3KO) rats, which have impaired myogenic response. RBF was well autoregulated in WT rats but not in Add3KO rats. Glomerular capillary pressure rose by 6 versus 14 mmHg in WT versus Add3KO rats when blood pressure increased from 100 to 150 mmHg. After 1 wk of hypertension, glomerular filtration rate increased by 38% and glomerular nephrin expression decreased by 20% in Add3KO rats. Neither were altered in WT rats. Proteinuria doubled in WT rats versus a sixfold increase in Add3KO rats. The degree of renal injury was greater in Add3KO than WT rats after 3 wk of hypertension. RBF, glomerular filtration rate, and glomerular capillary pressure were lower by 20%, 28%, and 19% in Add3KO rats than in WT rats, which was associated with glomerular matrix expansion and loss of capillary filtration area. The results indicated that impaired RBF autoregulation and eutrophic remodeling of preglomerular arterioles increase the transmission of pressure to glomeruli, which induces podocyte loss and accelerates the progression of CKD in hypertensive Add3KO rats.

Ileal cross transplantion between OVLT lesioned and sham rats alters OVLT‐sympathetic‐gut microbiome axis function in DOCA‐salt hypertension.

Recently, there has been growing interest in understanding the gut microbe‐host interaction in the maintenance of health or disease states, but relatively few studies have shown an association between the gut microbiome and specific types of hypertension. The deoxycorticosterone acetate (DOCA)‐salt hypertensive model in rats has a well described neurogenic component linked to increased sympathetic nervous system acitivity. As previously demonstrated by our laboratory, the hypertensive response in DOCA‐treated rats involves the organum vasculosum of the lamina terminalis (OVLT), a circumventricular organ that connects directly with the paraventricular nucleus (PVN) to increase sympathetic activity. In addition, it has been shown that celiac ganglionectomy supports a role of splanchnic innervation to the gut in the hypertensive response to DOCA. Indeed, we have previously shown that the OVLT plays a pivotal role in the development of DOCA‐salt hypertension. Moreover, we have also observed that this nucleus is able to mediate changes in the intestinal microbiome with the concomitant increase in blood pressure during DOCA salt hypertension. However, these changes were mitigated and offset by lesion of the OVLT and bacterial populations in OVLT lesioned rats with attenuated hypertension more closely resembled those in normal control rats. Therefore, we currently hypothesize that ileal transplantation from OVLT lesioned rats to SHAM operated rats will reverse or attenuate DOCA‐salt hypertension. Adult male Sprague‐Dawley rats were randomly selected for lesion of OVLT (OVLTx; n=3) or SHAM operation (OVLT SHAM; n=3), subsequently uninephrectomized and instrumented with telemetric blood pressure transducers as well as chronic indwelling ileal catheters. Rats were then subjected to DOCA‐salt for 16 days. 10 days after DOCA treatment, rats underwent daily ileal transplantation: approximately 2 ml of the ileal content was withdrawn from the ileal catheter in all rats and cross transplanted (between OVLTx and respective SHAM rats) on days 10–16 of DOCA treatment. Mean arterial pressure (MAP) was measured during 5 control days and 16 days of DOCA treatment. On days 3–6 and 9–10 of DOCA treatment, the chronic pressor response to DOCA was attenuated in OVLTx rats such that by Day 10 MAP increased to 125±1.8 mmhg in SHAM rats when compared to only 112±2.5 mmHg in OVLTx rats. After ileal cross transplantation, a progressive decrease in MAP in SHAM rats was observed. In fact, on the sixth day after transplantation, MAP was decreased to 114±1.2 mmHg in these animals, whereas in OVLTx rats MAP increased to 120±1.8 mmHg. Therefore, after ileal cross transplantation, a powerful reversal of the chronic hypertensive response was observed. Additionally, an opposite effect was observed in OVLTx rats in that blood pressure increased. These results support a prominent role of the OVLT‐sympathetic‐gut microbiome axis in the development of hypertension. As such, targeting these alterations in gut microbiota may have promising therapeutic potential to improve health and a role in the prevention and treatment of hypertension.Support or Funding InformationAmerican Heart Association Award: 19AIREA34380525

Lesion of the OVLT markedly attenuates chronic DOCA-salt hypertension in rats.

Lesions of the anteroventral third ventricle (AV3V region) are known to prevent many forms of experimental hypertension, including mineralocorticoid [deoxycorticosterone acetate (DOCA)-salt] hypertension in the rat. However, AV3V lesions include the organum vasculosum of the lamina terminalis (OVLT), portions of the median preoptic nucleus, and efferent fibers from the subfornical organ (SFO), thereby limiting the ability to define the individual contribution of these structures to the prevention of experimental hypertension. Having previously reported that the SFO does not play a significant role in the development of DOCA-salt hypertension, the present study was designed to test the hypothesis that the OVLT is necessary for DOCA-salt hypertension in the rat. In uninephrectomized OVLT-lesioned (OVLTx; n = 6) and sham-operated ( n = 4) Sprague-Dawley rats consuming a 0.1% NaCl diet and 0.9% NaCl drinking solution, 24-h mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg sc per rat). No differences in control MAP were observed between groups. The chronic pressor response to DOCA was attenuated in OVLTx rats such that MAP increased to 133 ± 3 mmHg in sham-operated rats by day 21 of DOCA compared with 120 ± 4 mmHg (means ± SE) in OVLTx rats. These results support the hypothesis that the OVLT is an important brain site of action for the pathogenesis of DOCA-salt hypertension in the rat.

Role of the OVLT‐gut microbiome axis on the development of DOCA‐salt hypertension

Limited studies suggest an association of gut microbiome changes with certain forms of hypertension, but a cause/effect relationship has yet to be fully defined. The deoxycorticosterone acetate (DOCA)‐salt model of hypertension in rats is known to have a neurogenic component linked to increased sympathetic nervous system activity. Indeed, we have recently shown the hypertensive response in DOCA‐treated rats requires the organum vasculosum of the lamina terminalis (OVLT), a circumventricular organ that has direct connections to the paraventricular nucleus (PVN) to increase sympathetic activity. Furthermore, celiac ganglionectomy also supports a role of splanchnic sympathetic innervation to the gut in the hypertensive response to DOCA. Taken together, we hypothesize an OVLT‐sympathetic‐gut (OSG) microbiome axis is involved in the development of hypertension. Having previously reported that the OVLT plays a significant role in the development of DOCA‐salt hypertension, the present study was designed to test the hypothesis that the OVLT mediates changes in the gut microbiome associated with concomitant hypertension. In uninephrectomized OVLT lesioned (OVLTx; n = 5) and SHAM (n = 2) Sprague‐Dawley rats consuming a 2% NaCl diet and 0.9% NaCl drinking solution, 24‐hour mean arterial pressure (MAP) was recorded telemetrically 5 days before and 21 days after DOCA implantation (100 mg/rat; SQ). Microbiome analyses were performed on regional gut samples at the end of the study and compared to normal rats (n=2). The chronic pressor response to DOCA was attenuated in OVLTx rats such that MAP increased to 153±4 mmHg in SHAM rats by Day 21 of DOCA compared to 127±5 mmHg in OVLTx rats. Moreover, DOCA hypertension in SHAM rats was associated with decreased levels of Lactobacillus species in the jejunum and ileum, and increased Escherichia levels in the cecum compared with normal rats. However, these changes in bacterial genera were mitigated and offset by lesion of the OVLT and these bacterial populations in OVLT lesioned rats with attenuated hypertension more closely resembled those in normal control rats. We conclude that DOCA‐salt hypertension is associated with specific microbiome changes in the gut, and the attenuated hypertensive effects of DOCA‐salt in OVLT lesioned rats is mediated in part through counteracting changes in these bacterial populations.Support or Funding InformationUniversity of Minnesota Grant‐In‐Aid # 107426This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract P113: Angiotensin Converting Enzyme 2 Modulates Bradykinin B1 Receptor Function During DOCA-Salt Hypertension

DOCA-salt hypertension is associated with reduced angiotensin converting enzyme 2 (ACE2) and increased bradykinin B1 receptor (B1R) expression in the brain. ACE2 hydrolyzes des-Arg(9)-bradykinin, the endogenous B1R agonist, into inactive metabolites. Therefore, we hypothesized that ACE2 overexpression or deletion modulates B1R function in the brain during neurogenic hypertension. To test this hypothesis, we used mice overexpressing ACE2 in neurons (SA), ACE2 knockout and B1R knockout mice. Blood pressure (BP) was monitored using telemetry probes in conscious animals. While baseline BP was not different between strains, DOCA-salt treatment (1 mg/g body weight DOCA, 1% NaCl for 3 weeks) resulted in a significantly lower BP in B1R knockout mice (121 ±2 mmHg, n=5) compared to wildtype (WT) mice (138 ±3 mmHg, n=8). DOCA-salt hypertension resulted in 27% decrease (74 ±6 vs. 54 ±2 Fluorescence Units (FU)/min/μg of protein, p<0.05) in ACE2 activity in the hypothalamus, but not in B1R knockout mice with DOCA (69 ±6 vs. 65 ±3 FU/min/μg of protein). In DOCA-treated WT mice, B1R mRNA (Real time PCR) and protein expression (Western blot) in the hypothalamus were increased by 3 and 2 fold (n=6, p<0.01), respectively. This increased B1R expression was blunted in SA mice with DOCA (3.2 ±0.4 vs. 0.9 ±0.1 fold, p<0.001) but not in ACE2 knockout mice with DOCA (3.2 ±0.4 vs. 3.1 ±0.9 fold). Moreover, DOCA-salt treatment resulted in an increased expression of pro-inflammatory ADAM17 in the brain by 2.2 fold (n=6, p<0.01 vs. WT). ACE2 overexpression or B1R knockdown blunted this ADAM17 expression (1.4 ±0.1 and 1.1 ±0.6 fold, respectively, n=3, p<0.01 vs. WT+DOCA), while it was remain increased in ACE2 knockout mice (2.6 ±0.7 fold, n=3, p<0.01) compared to control mice. Together, our data provide novel evidence to support a role for ACE2 in the modulation of central B1R function in the development of DOCA-salt hypertension.

Abstract 079: (pro)renin Receptor Knockdown In The Paraventricular Nucleus Of Hypothalamus Attenuates The Development Of Doca-salt Hypertension

We previously reported that neuron-specific PRR deletion prevents the development of deoxycorticosterone acetate (DOCA)-salt induced hypertension. However, it is not clear which brain region is responsible for the effect of PRR knockout on the development of hypertension. In present study, we bilaterally micro-injected adeno-associated virus (AAV2)-mediated Cre recombinase with an eGFP tag (AAV-Cre, 50 nl/side) into the paraventricular nucleus of hypothalamus (PVN) of PRR-Floxed mice to knockdown the PRR expression in this region. Deoxycorticosterone acetate-salt (DOCA, 50mg, 0.9%NaCl drinking solution, 21 days) treatment was started 3 days after AAV-Cre or control virus (AAV-eGFP) injection. Blood pressure (BP) was monitored using radio telemetric probes in conscious animals. Autonomic functions were also assessed using pharmacological methods. PVN was micro-punched for PRR real time PCR to verify PRR knockdown efficiency. Three days after virus injection, AAV-Cre significantly decreased PRR expression (fold change: 0.69 ± 0.1 vs. 1.0 ± 0.05) in the PVN compared to AAV-eGFP before DOCA-salt treatment. Three weeks after DOCA-salt treatment, PRR mRNA levels were increased in the AAV-eGFP group (fold change: 3.0 ± 0.1), and this increase was abolished by AAV-Cre (fold change: 1.2 ± 0.04, P<0.05). AAV-Cre expression in the PVN had no effect on PRR expression in the cerebral cortex or brain stem. Blood pressure (mmHg) and heart rate (HR, bpm) were recorded at baseline and following DOCA-salt treatment. AAV-Cre or control virus had no effect on baseline BP or HR. Following DOCA-salt treatment, BP was significantly increased in mice treated with AAV-eGFP (138.5 ± 7.4); while BP was significantly lower in AAV-Cre treated mice (118.9 ± 3.4). The cardiac sympathetic tone (ΔHR to propranolol: -123 ± 46 vs. -181 ± 16) and vasomotor sympathetic tone (ΔBP to chlorisondamine: -47 ± 9.2 vs. -69 ± 4.7) were significantly lower in AAV-Cre treated mice after DOCA-salt treatment. The cardiac parasympathetic tone (ΔHR to methylatropine: 82 ± 18 vs. 40 ± 7.3) was greater in AAV-Cre treated mice. In summary, PRR knockout in the PVN attenuates the development of DOCA-salt hypertension and is associated with the improvement of autonomic function.

Inhibition of the bradykinin B1 receptor attenuates blood pressure and cardiac hypertrophy in DOCA‐salt hypertensive mice (856.12)

DOCA‐salt hypertension is associated with increased activity of the kallikrein‐kinin system. We sought to explore whether bradykinin B1 receptor (B1R)‐mediated inflammation contribute to DOCA‐salt hypertension. While baseline blood pressure (BP; telemetry) was not different, DOCA‐salt treatment (1 mg/g body weight DOCA, 1% NaCl for 28 d) resulted in significantly lower BP in B1R knockout mice (121 ±2 mmHg, n=5) compared to non‐transgenic (NT) mice (138 ±3 mmHg, n=8). In DOCA‐treated NT mice, B1R expression (Western blot) in the brain was increased by 2 fold (n=4, p<0.01). To dismiss potential developmental alterations in knockout mice, we complemented our study using a pharmacological approach with inhibition of B1R in the brain of NT mice. ICV infusion of R715, a specific B1R antagonist, attenuated the DOCA‐salt‐induced increase in BP in NT mice (125 ±3 mmHg, n=3, p<0.01). Cardiac hypertrophy was also attenuated in NT+R715 mice (HW/BW: 4.1 ±0.1 vs. 5.6 ±0.2; HW/tibia length: 7.9 ±0.3 vs. 9.2 ±0.7; p<0.01 vs. NT+DOCA). NT+DOCA mice had increased inflammation (qRT‐PCR), as shown by elevated gene expression of TNF, IL‐1β and IL‐6 (3‐, 8‐, and 5‐fold, respectively, n=9, p<0.01 vs. NT) in the hypothalamic paraventricular nucleus, which was attenuated in NT+R715 mice. Taken together, these data provide evidence that B1R contributes to neuroinflammation, thereby the development of DOCA‐salt hypertension.Grant Funding Source: Supported by AHA 12EIA8030004 and 13POST16500025

Abstract 103: ACE2 Overexpression Prevents DOCA-Salt Hypertension by Modulating Oxidative Stress and Nitric Oxide in the Central Nervous System

Elevated reactive oxygen species (ROS) in the central nervous system (CNS) through NADPH oxidase and diminished Nitric oxide (NO) levels are involved in the pathogenesis of hypertension. We previously reported that central Angiotensin Converting Enzyme 2 (ACE2) overexpression prevents the development of hypertension induced by DOCA-salt in a transgenic mouse model (syn-hACE2; SA) with human ACE2 targeted selectively to neurons in the CNS. While baseline blood pressure (BP; telemetry) was not different among genotypes, DOCA-salt treatment (1mg/g body wt DOCA, 1% saline in drinking water for 3 weeks) resulted in significantly lower BP level in SA mice (122 ±3 mmHg, n=12) compared to non-transgenic (NT) littermates (138 ±3 mmHg, n=8). To elucidate the mechanisms involved in this response, we investigated the paraventricular nucleus (PVN) expression of Nox-2 (catalytic subunit of NADPH oxidase), 3-nitrotyrosine, and endothelial nitric oxide synthase (eNOS) and anti-oxidant enzymes superoxide dismutase (SOD) and catalase in the hypothalamus. DOCA-salt treatment resulted in decreased catalase (95.2 ±5.6 vs. 113.8 ±17.6 mmol/min/ml, p<0.05) and SOD (4.1 ±0.4 vs. 5.9 ±0.2 U/ml, p<0.01) activities in hypothalamic homogenates of NT mice, which was prevented by ACE2 overexpression (141.8 ±9.9 vs. 142.1 ±9.2 mmol/min/ml and 5.9 ±0.3 vs. 7.9 ±0.2 U/ml, respectively). NT mice treated with DOCA-salt showed increased oxidative stress as indicated by increased expression of Nox-2 (61 ±5 % increase, n=9, p<0.001 vs. NT) and 3-nitrotyrosine (89 ±32 % increase, n=9, p<0.01 vs. NT) in the PVN which was attenuated in SA mice. Furthermore, DOCA-salt hypertension resulted in decreased phosphorylation of eNOS-ser1177 in the PVN (33 ±5 % decrease, n=9, p<0.05 vs NT) and this decrease was prevented by ACE2 overexpression. Taken together, these data provide evidence that brain ACE2 regulates the balance between NO and ROS levels, thereby preventing the development of DOCA-salt hypertension.

Abstract 25: Prorenin Elevates Blood Pressure via the (Pro)renin Receptor: Who Needs Renin When You Have Prorenin in the Brain

Growing evidence supports that the brain renin-angiotensin (Ang) system (RAS) plays an important role in blood pressure (BP) regulation and hypertension. This concept has been perplexed by extremely low renin activity in the brain. We recently reported the presence of prorenin in the mouse brain at a level of 10-fold higher than that of renin. In addition, we and others have identified the localization of the (pro)renin receptor (PRR) in various brain nuclei involved in BP regulation. The binding of prorenin to PRR leads to Ang II formation in vitro. Our hypothesis: Brain prorenin binding to PRR mediates Ang II formation, leading to an increase in BP. To test this, neuron-specific PRR knockout mice (Nefh-PRR) and wildtype (WT) littermates (N=5/group) were each implanted with a telemetric probe for BP recording and an intracerebroventricular (ICV) cannula for infusion of mouse prorenin (100ng/ul), mouse renin (100ng/ul), or Ang II type 1 receptor (AT1R) blocker (losartan, 10ug/ul) at 0.3ul/minute for 10 minutes. Mouse prorenin infusion increased the BP (mmHg) in WT mice (ΔMAP: 41 ± 5); however, the prorenin-induced pressor response was abolished in Nefh-PRR mice (ΔMAP: 5 ± 1). Infusion of mouse renin similarly increased BP in Nefh-PRR (ΔMAP: 27 ± 2) and WT (ΔMAP: 31 ± 5) mice. The pressor response induced by prorenin or renin was completely blocked by the infusion of losartan. The data suggests that ICV prorenin via PRR mediates Ang II-dependent pressor response in WT mice. To determine whether PRR contributes to the development of brain RAS-dependent hypertension, Nefh-PRR and WT littermates (N=8/group) were treated with 50mg of deoxycorticosterone acetate (DOCA) subcutaneously, plus 0.9% NaCl drinking water for 21 days. The baseline BP was similar between Nefh-PRR (101 ± 2) and WT (101 ± 3) mice. BP was increased in WT mice (132 ± 6) by DOCA-salt treatment, while Nefh-PRR mice remained normotensive (108 ± 3). In summary, prorenin via PRR mediates AngII/AT1R-dependent pressor response in the brain. Neuron-specific PRR deletion attenuates the development of DOCA-salt hypertension likely due to the lack of Ang II/AT1R activation. We conclude that prorenin/PRR may be the key factors to initiate the brain RAS and play an essential role in neurogenic hypertension.

Abstract 213: Neuron-specific (Pro)renin Receptor Deletion Attenuates Neurogenic Hypertension and is Associated With Improvement of Autonomic Function

Although (pro)renin receptor (PRR) is highly expressed in the brain, the physiological importance of brain PRR remains to be determined. We previously showed that PRR mRNA levels were up-regulated in the brain regions involved in blood pressure (BP) regulation including the subfornical organs (SFO), paraventricular nuclei (PVN), and rostral ventrolateral medulla (RVLM) in the DOCA-salt-induced hypertensive mice. To elucidate whether the increase of PRR expression contributes to the development of hypertension, we developed a neuron-specific PRR knockout mouse (Nefh-PRR) model by crossing PRR-floxed mice with mice that express Cre-recombinase with neurofilament-H (Nefh-Cre) promoter. Immunostaining showed that PRR expression was significantly reduced in cardiac regulatory brain regions including SFO (0.35± 0.15), PVN (0.34 ± 0.09), and RVLM (0.41 ± 0.1), where the Cre-recombinase was expressed, in Nefh-PRR compared to Nefh-cre mice (1 ± 0.1, 1 ± 0.19 and 1 ± 0.06). Nefh-PRR mice and Nefh-cre mice with wild type PRR gene (WT, n=8/group) were implanted with telemetric probes. BP (mmHg) and heart rate (HR, bpm) were recorded for baseline and following DOCA-salt treatment (50mg DOCA, 0.9%NaCl drinking solution, 21 days). Nefh-PRR mice exhibited normal baseline BP and HR. Following DOCA-salt treatment, BP was significantly lower in Nefh-PRR mice (108 ± 3) compared to WT mice (132 ± 6, P<0.05). The cardiac sympathetic tone (HR response to propranolol, ΔHR: -128±46vs. -180±45) and the vasomotor sympathetic tone (BP response to chlorisondamine, ΔBP: -46 ±17vs.-68± 12) were lower in Nefh-PRR than WT mice after DOCA-salt treatment. Moreover, HR response to methylatropine (ΔHR: 98±38 vs. 43±19) was greater in Nefh-PRR. In addition, DOCA-salt treatment increased plasma vasopressin (AVP) levels (33.6 ± 5.3 vs.3.0 ± 0.3pg/ml) in WT mice. PRR deletion significantly attenuated the increase in plasma AVP levels (19.0 ± 2.0pg/ml) induced by DOCA-salt. The data suggest PRR deletion prevents the development of DOCA-salt hypertension and is associated with reduction of plasma AVP levels and improvement of autonomic function. We conclude that brain PRR may be a novel therapeutic target for the treatment of hypertension.

Impaired flow-induced arterial remodeling in DOCA-salt hypertensive rats

Arteries from young healthy animals respond to chronic changes in blood flow and blood pressure by structural remodeling. We tested whether the ability to respond to decreased (-90%) or increased (+100%) blood flow is impaired during the development of deoxycorticosterone acetate (DOCA)-salt hypertension in rats, a model for an upregulated endothelin-1 system. Mesenteric small arteries (MrA) were exposed to low blood flow (LF) or high blood flow (HF) for 4 or 7 weeks. The bioavailability of vasoactive peptides was modified by chronic treatment of the rats with the dual neutral endopeptidase (NEP)/endothelin-converting enzyme (ECE) inhibitor SOL1. After 3 or 6 weeks of hypertension, the MrA showed hypertrophic arterial remodeling (3 weeks: media cross-sectional area (mCSA): 10±1 × 10(3) to 17±2 × 10(3) μm(2); 6 weeks: 13±2 × 10(3) to 24±3 × 10(3) μm(2)). After 3, but not 6, weeks of hypertension, the arterial diameter was increased (Ø: 385±13 to 463±14 μm). SOL1 reduced hypertrophy after 3 weeks of hypertension (mCSA: 6 × 10(3)±1 × 10(3) μm(2)). The diameter of the HF arteries of normotensive rats increased (Ø: 463±22 μm) but no expansion occurred in the HF arteries of hypertensive rats (Ø: 471±16 μm). MrA from SOL1-treated hypertensive rats did show a significant diameter increase (Ø: 419±13 to 475±16 μm). Arteries exposed to LF showed inward remodeling in normotensive and hypertensive rats (mean Ø between 235 and 290 μm), and infiltration of monocyte/macrophages. SOL1 treatment did not affect the arterial diameter of LF arteries but reduced the infiltration of monocyte/macrophages. We show for the first time that flow-induced remodeling is impaired during the development of DOCA-salt hypertension and that this can be prevented by chronic NEP/ECE inhibition.

DOCA-salt hypertension does not require the ouabain-sensitive binding site of the α2 Na,K-ATPase

We have shown that the ouabain-sensitive α2 Na,K-ATPase is required for adrenocorticotropic hormone (ACTH)-induced hypertension and gestational blood pressure regulation. It is therefore of interest to explore whether this binding site participates in the development of other forms of hypertension, such as deoxycorticosterone acetate (DOCA)-salt using mutant mice with altered sensitivity to ouabain. Wild-type (α1 ouabain-resistant, α2 ouabain-sensitive: α(R/R)α2(S/S)), α1-resistant, α2-resistant (α1(R/R)α2(R/R)) and α1-sensitive, α2-resistant (α1(S/S)α2(R/R)) mice were uninephrectomized and implanted with DOCA pellets. The animals were given either tap water or 1% NaCl, and blood pressure was measured before and after DOCA. DOCA-salt-treated α1(R/R)α2(R/R) mice developed hypertension to the same extent as α1(R/R)α2(S/S) mice (wild type), and the α1(S/S)α2(R/R) mice given DOCA-salt also showed no difference from the other two genotypes. The expression of the α1 isoform was not changed by DOCA-salt treatment in either α1(R/R)α2(S/S) or α1(R/R)α2(R/R) mice. However, the α2 subunit was expressed at substantially higher levels in the hearts of α1(R/R)α2(R/R) than α1(R/R)α2(S/S) mice, regardless of treatment. Plasma levels of ouabain did not change consistently, but those of marinobufagenin were modestly higher in DOCA-salt treated mice relatively to those without salt. The ouabain-binding site of either the α1 or α2 Na,K-ATPase subunit does not play an essential role in the development of DOCA-salt hypertension in this mouse model. These findings indicate that the underlying mechanisms of hypertension induced by DOCA-salt treatment are different from those of ACTH-induced hypertension.

Effects of K+-Canrenoate on the Development of DOCA-salt Hypertension

The effects of K+-canrenoate, a digoxin antagonist, on the role of digoxin-like factor in the development of DOCA-salt hypertension has been examined. DOCA-salt rats treated with 66 mg kg-1 day-1 of K+-canrenoate (s.c.) presented a lower increase in blood pressure (P less than 0.01), less cardiac hypertrophy (P less than 0.05) and hypokalaemia (P less than 0.05) than non-treated DOCA-salt rats. K+-canrenoate treatment did not lead to significant changes in urinary volume, Na+ and K+ urinary excretion or suppression of plasma renin activity in DOCA-salt rats. None of the parameters were significantly different between uninephrectomized-salt rats treated or non-treated with K+-canrenoate. These data suggest a role for digoxin-like factor in DOCA-salt hypertension. However, the non-normalization of blood pressure observed in K+-canrenoate DOCA-salt treated rats indicates that other factors contribute to the initiating mechanisms in this type of hypertension. Moreover, these data suggest that digoxin-like factor plays no role in the suppression of plasma renin activity induced by DOCA and salt treatment.

Development of DOCA-salt Hypertension in Guinea-pigs: Effects of Indapamide

The effects of indapamide on the development of cardiac complications related to deoxycorticosterone acetate (DOCA)-salt-induced hypertension in guinea-pigs have been studied. Indapamide treatment (5 mg kg−1 daily) was initiated either before DOCA administration or five weeks after DOCA administration, for a period of five weeks. Increase in blood pressure was prevented or attenuated by indapamide (−19%, P<0·05). In parallel, development of cardiac hypertrophy assessed by an increase in left ventricular weight, thickness and volume was also prevented (−13, −25 and −25%, respectively, all P<0·05) or attenuated (-7·5, −15%, P<0·05 and −9·8%, respectively). DOCA-salt treatment over a longer period (10 weeks) led rapidly to the appearance of signs of heart failure with pleural or pericardial effusion. Indapamide-treated guinea-pigs presented no such signs of heart failure. These results demonstrate that the DOCA-salt guinea-pig is a suitable model for studying hypertensive disease. Indapamide prevents and produces a regression in hypertension and cardiac hypertrophy, and prevents the development of heart failure.

Sympathetic nerve activity during development of DOCA‐salt hypertension

Prior studies have provided indirect evidence suggesting sympathetic nerve activity (SNA) contributes to the development of hypertension. We hypothesized that evoked and resting SNA would be elevated 2‐3 weeks after inducing DOCA‐NaCl hypertension. We instrumented male Sprague‐Dawley rats to measure arterial pressure, ECG and renal SNA before inducing hypertension. We recorded resting SNA before and after implanting DOCA and administering 1% NaCl drinking water. Responses to acute stress were also recorded prior to inducing and during development of hypertension (2‐3 weeks). Background activity was determined with ganglionic blockade (trimethaphan). We observed that DOCA‐salt treatment (n=8) elicited an increase in arterial pressure that started within 2‐3 days. During the early phase, renal SNA did not seem to be elevated as the decrease in SNA in response to trimethaphan was unchanged (‐41± 6% control vs. ‐38 ± 6% on day 12). We examined the acute response to cold water stress (1 cm deep for 1 min) and observed that initial evoked responses during cold stress were progressively smaller in DOCA‐salt hypertension over 12 days. Our data suggest that SNA does not play a role in the development of DOCA‐salt hypertension, although it will be necessary to verify this in other sympathetic nerves. Supported by USPHS NIH R01 DA0017371, HL 091440 and 5T32 GM008306.

DOCA‐salt hypertension persists in mice with ouabain‐resistant α1 and α2 Na, K‐ATPase (NKA)

It has been proposed that endogenous cardiac glycosides may play an important role in the development of various forms of human and experimental hypertension, including DOCA‐salt hypertension and ACTH‐induced hypertension. We have previously shown that ACTH‐induced hypertension is blunted in mutant mice that express a ouabain‐resistant isoform of the α2NKA. In the present study, therefore, we sought to explore whether the development of DOCA salt hypertension is altered in mutant mice variably expressing ouabain‐resistant and ‐sensitive α1 and α2 NKA isoforms. Mice were uninephrectomized and instrumented with telemetry implants for the continuous measurement of blood pressure. After recovery, DOCA pellets were implanted and animals were given saline to drink. Mean arterial pressure increased comparably in α1‐resistant/α2‐sensitive mice (wild type, 110±1 to 149±5 mmHg) vs α1‐resistant/α2‐resistant mice (108±4 to 146±3 mmHg). In separate groups of DOCA‐salt‐treated mice instrumented with indwelling arterial catheters, treatment with digoxin antibody (digibind) had no effect on blood pressure in either α1‐resistant/α2‐sensitive or α1‐resistant/α2‐resistant mice. We also evaluated DOCA‐salt hypertension by telemetry in α1‐sensitive/α2‐resistant mice, and found that blood pressure increased similarly to the other two genotypes (106±3 to 153±9). These data suggest that the elevated pressure in DOCA‐salt treated mice is not dependent on the interaction of endogenous cardiotonic steroids with α1 or α2 NKA.

The hemodynamic profile of the onset of hypertension in the DOCA‐salt rat model

The pathogenesis of various forms of experimental hypertension may be mediated by different hemodynamic mechanisms. In the present study we characterized the hemodynamic profile of the onset of hypertension in the DOCA-salt model. Rats were chronically instrumented for measurement of mean arterial pressure (MAP) by telemetry and cardiac output (CO) using a Transonics flow probe on the ascending aorta (N=4). Rats had free access to 0.9% saline and 0.1% NaCl food throughout the protocol. Control data was collected continuously for 7 days before and 10 days following subcutaneous administration of 50 mg of DOCA in a silicone implant. During the 7 day control period, basal values for MAP, CO, heart rate (HR) and total peripheral resistance(TPR) averaged 102 + 1.5 mm Hg, 21 + 1.6 ml/min/100g, 418 + 8.1 beats/min and 5.02 + 0.41 (arbitrary units) respectively. During the first 4 days of DOCA, MAP increased ~8% from baseline in parallel with a ~12% in CO. In contrast heart rate and TPR decreased by ~0.6% and ~8% respectively by Day 4 of DOCA. By Day 10 of DOCA, MAP increased to ~20% above control and CO returned to control levels whereas TPR increased to ~17% above baseline. Heart rate continued to decrease to ~7% below control on Day 10. We conclude that the development of DOCA-salt hypertension is mediated initially by an increase in CO followed by an increase in TPR as the hypertension progresses. Supported by NIH Grant HL 64178.

Role of renal nerves in development of hypertension in DOCA-salt model in rats: a telemetric approach

Centrally mediated hyperactivity of the autonomic nervous system contributes to DOCA hypertension; however, the targeted peripheral vascular bed(s) remain unclear. We propose that if renal sympathetic activity is a factor in the development of DOCA-salt hypertension, then renal denervation (RDNX) should attenuate the hypertensive response. In protocol 1, uninephrectomized RDNX (n = 9) and sham-denervated (n = 6) Sprague-Dawley rats were allowed free access to 0.9% NaCl solution and 0.1% NaCl diet. Mean arterial pressure (MAP) and heart rate were telemetrically recorded for 4 days before and 36 days after DOCA (100 mg/rat) implantation; sodium and water balances were recorded daily. Protocol 2 was similar except that saline intake in sham rats (n = 7) was matched to that observed in RDNX rats of protocol 1 for 30 days; for the last 10 days, the rats were allowed free access to saline. Before DOCA in protocol 1, MAP was lower (P < 0.05) in RDNX rats (99 +/- 1 mmHg) compared with sham rats (111 +/- 3 mmHg); however, heart rate and sodium and water balances were similar between groups. RDNX attenuated the MAP response to DOCA by approximately 50% (DeltaMAP = 22 +/- 3 mmHg, where Delta is change in MAP) when compared with sham rats (DeltaMAP = 38 +/- 6). RDNX rats consumed significantly less saline than sham rats, and cumulative sodium and water balances were reduced by 33% and 23%, respectively. In protocol 2, a similar pattern in MAP elevation was observed in RDNX and saline-restricted, sham-denervated rats even when saline restriction was removed. These results indicate that the renal sympathetic nerves are important in hypertension development but that other factors are also involved.