Baseline Renal Sympathetic Nerve Activity Research Articles

A NOVEL METHOD FOR THE ANALYSIS OF RENAL SYMPATHETIC NERVE ACTIVITY FROM MULTI-FIBER NERVE RECORDINGS

Objective: Measurement of renal sympathetic nerve activity (RSNA) via bipolar electrodes from multi-fiber preparations in various species has been used for decades in the context of autonomic regulation research. Traditionally, raw neurograms are usually integrated over short time intervals and this approach works perfectly for the analysis of dynamic RSNA changes due to acute intervention. However, comparability of baseline activities between groups is limited. We present a novel analysis method, based on the raw neurogram sampled at rate of 25 kHz. Design and method: For RSNA-burst analysis a software based programmable algorithm was used. Continuous activities longer than three single spikes (i.e., >8ms) were defined as bursts, followed by silent periods with some single spikes. Approximately 10.000 bursts/rat were analyzed and burst amplitude, burst duration, burst area (i.e., duration integral), as well as the burst frequencies were analyzed. Results: In rats with myocardial infarction (n=11) we found no difference in integrated baseline RSNA as compared to controls ((n=10). However, the new method revealed higher higher burst rate per cardiac cycle [CC] (2.65 ± 0.39 vs. 1.61 ± 0.38 bursts/CC; P<0.001) and burst frequency (15.09 ± 2.42 vs. 8.52 ±1.54 Hz; P<0.001) compared to healthy controls, indicating increased RSNA. Conclusions: Our new method of RSNA baseline analysis was able to detect subtle differences between groups of animals that could not be detected by the traditional method of integrated RSNA analysis. Furthermore, this method has the potential to give further insights into RNSA patterns and synchronization to afferent regulatory input form the kidney itself or other organs.

Abstract P410: A Novel Method For The Analysis Of Renal Sympathetic Nerve Activity From Multi-fiber Nerve Recordings

Introduction: Measurement of renal sympathetic nerve activity (RSNA) via bipolar electrodes from multi-fiber preparations in various species has been used for decades in the context of autonomic regulation research. Traditionally, raw neurograms are usually integrated over short time intervals and this approach works perfectly for the analysis of dynamic RSNA changes due to acute intervention. However, comparability of baseline activities between groups is limited. We present a novel analysis method, based on the raw neurogram sampled at rate of 25 kHz. Material and methods: For RSNA-burst analysis a software based programmable algorithm was used. Continuous activities longer than three single spikes (i.e., >8ms) were defined as bursts, followed by silent periods with some single spikes. Approximately 10.000 bursts/rat were analyzed and burst amplitude, burst duration, burst area (i.e., duration integral), as well as the burst frequencies were analyzed. Results: In rats with myocardial infarction (n=11) we found no difference in integrated baseline RSNA as compared to controls ((n=10). However, the new method revealed higher higher burst rate per cardiac cycle [CC] (2.65 ± 0.39 vs. 1.61 ± 0.38 bursts/CC; P<0.001) and burst frequency (15.09 ± 2.42 vs. 8.52 ±1.54 Hz; P<0.001) compared to healthy controls, indicating increased RSNA. Conclusion: Our new method of RSNA baseline analysis was able to detect subtle differences between groups of animals that could not be detected by the traditional method of integrated RSNA analysis. Furthermore, this method has the potential to give further insights into RNSA patterns and synchronization to afferent regulatory input form the kidney itself or other organs.

Cervical spinal hemisection effects on spinal tissue oxygenation and long-term facilitation of phrenic, renal and splanchnic sympathetic nerve activity

HypothesesModerate acute intermittent hypoxia (mAIH) elicits plasticity in both respiratory (phrenic long-term facilitation; pLTF) and sympathetic nerve activity (sympLTF) in rats. Although mAIH produces pLTF in normal rats, inconsistent results are reported after cervical spinal cord injury (cSCI), possibly due to greater spinal tissue hypoxia below the injury site. There are no reports concerning cSCI effects on sympLTF. Since mAIH is being explored as a therapeutic modality to restore respiratory and non-respiratory movements in humans with chronic SCI, both effects are important. To understand cSCI effects on mAIH-induced pLTF and sympLTF, partial or complete C2 spinal hemisections (C2Hx) were performed and, 2 weeks later, we assessed: 1) ipsilateral cervical spinal tissue oxygen tension; 2) ipsilateral & contralateral pLTF; and 3) ipsilateral sympLTF in splanchnic and renal sympathetic nerves. MethodsMale Sprague-Dawley rats were studied intact, or after partial (single slice) or complete C2Hx (slice with ∼1 mm aspiration). Two weeks post-C2Hx, rats were anesthetized and prepared for recordings of bilateral phrenic nerve activity and spinal tissue oxygen pressure (PtO2). Splanchnic and renal sympathetic nerve activity was recorded in intact and complete C2Hx rats. ResultsSpinal PtO2 near phrenic motor neurons was decreased after C2Hx, an effect most prominent with complete vs. partial injuries; baseline PtO2 was positively correlated with mean arterial pressure. Complete C2Hx impaired ipsilateral but not contralateral pLTF; with partial C2Hx, ipsilateral pLTF was unaffected. In intact rats, mAIH elicited splanchnic and renal sympLTF. Complete C2Hx had minimal impact on baseline ipsilateral splanchnic or renal sympathetic nerve activity and renal, but not splanchnic, sympLTF remained intact. ConclusionGreater tissue hypoxia likely impairs pLTF and splanchnic sympLTF post-C2Hx, although renal sympLTF remains intact. Increased sympathetic nerve activity post-mAIH may have therapeutic benefits in individuals living with chronic SCI since anticipated elevations in systemic blood pressure may mitigate hypotension characteristic of people living with SCI.

Overexpression of Growth Differentiation Factor 15 Reduces Blood Pressure and Produces Anti‐neuroinflammatory Effects in Mice with Diet‐induced Obesity

The growth differentiation factor 15 (GDF15) is a stress-responsive cytokine, which belongs to the super family of transforming growth factor beta (TGFb). In the past decade, GDF15 has been implicated in multiple biological processes and diseases, including anti-inflammation, anti-obesity and anti-diabetes effects. However, the role of GDF15 in sympathetic regulation of blood pressure has not been reported in the cardiovascular disease state. The purpose of the study was to investigate the effects of GDF15 on the blood pressure and central nervous system neuroinflammatory responses in diet-induced obese mice. NAG-Tg+ transgenic mice (TG) with overexpression of GDF15 and wild-type (WT) littermates were fed on normal chow or high-fat diet (HFD) for 16 weeks. During the experimental period, arterial pressure was recorded by radiotelemetry in conscious mice. WT mice with HFD had higher mean arterial pressure (MAP) compared with the normal diet fed WT mice (WT HFD 110±4 mmHg verse WT 93±4 mmHg). Compared with WT mice with HFD, GDF15 overexpression significantly reduced MAP (TG HFD: 99±3 mmHg verse WT HFD: 110±4 mmHg) accompanied by the decreased urinary norepinephrine excretion and baseline renal sympathetic nerve activity (TG HFD: 38.9±4.1% max verse WT HFD: 61.2±5.6% max). GDF15 TG mice with HFD had lower expressions of pro-inflammatory cytokines interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-a), interleukin 1β (IL-1β), C1q/TNF-related protein-1 (CTRP1) and reduced immune cell infiltration in the hypothalamus and brain stem tissue. A downregulation in GDF15 receptor (GFRAL) expression was detected in brain stem medullary nuclei in WT HFD mice, which was significantly upregulated in GDF15 TG mice with HFD. These data suggest that GDF15 overexpression exerts a potential protective effect on hypertension in obese mice through attenuating sympathetic outflow. The mechanism for this effect involves anti-inflammatory and neuronal protective properties of GDF15 in the central nervous system.

CHARACTERIZING THE ROLE OF THE DORSOMEDIAL HYPOTHALAMIC NUCLEUS IN THE CARDIOGENIC SYMPATHETIC AFFERENT REFLEX IN RATS

Myocardial ischemia causes the production and release of metabolites such as bradykinin (BK), which stimulates cardiac spinal sensory afferents causing chest pain and an increase in sympathetic activity. This sympathetic reflex, which increases heart rate and blood pressure, is referred to as the cardiogenic sympathetic afferent reflex (CSAR). During myocardial ischemia, this sympathetic reflex activity is important for the maintenance of blood pressure and perfusion of vital organs. However, the increased sympathetic activity may also be detrimental because it further increases the oxygen demand of the ischemic myocardium. While the brain stem nuclei, such as the nucleus tractus solitarius and rostral ventrolateral medulla have been shown to play an important role in the CSAR, the role of other supramedullary nuclei and the sources that generate sympathetic activity in the CSAR are still not clear. We have previously characterized the role of the paraventricular nucleus (PVN) of the hypothalamus in the CSAR and shown that acute chemical inhibition of the PVN attenuates the CSAR. However, we also have shown that the CSAR recovers in rats after chronic ibotenic acid lesion of the PVN suggesting that other supramedullary sites are involved. The dorsomedial hypothalamic nucleus (DMH) is another important hypothalamic site that plays an important role in cardiovascular sympathetic regulation. However, little is known regarding the role of the DMH in the CSAR. In this study, we tested the hypothesis that “inhibition of the DMH attenuates the CSAR in rats”. Renal sympathetic nerve activity (RSNA) and blood pressure were recorded in anesthetized rats during epicardial application of BK (10 μg/ml). Bilateral microinjection of muscimol, a GABAA agonist (0.5 nmol in 50 nl), was used to inhibit the DMH. Vehicle injection into the DMH had no significant effect (n=6, P > 0.05) on blood pressure or RSNA. In vehicle‐injected rats, epicardial BK application significantly increased (P < 0.05) RSNA to 204.4 ±35.08% of baseline and mean arterial pressure from 103.4 ±2.02 mmHg to 118.1 ±4.73 mmHg. In a sperate group of rats (n=8) muscimol injection into the DMH significantly (P < 0.05) reduced basal blood pressure from 98.0 ±4.66 mmHg to 77.1 ±7.33 mmHg but had no significant effect (P > 0.05) on baseline RSNA. In this group of rats, epicardial BK application significantly increased (P < 0.05) RSNA to 193.9 ±28.00% of baseline and increased mean arterial pressure from 77.1 ±7.33 mmHg to 89.29 ±8.48 mmHg. Whereas muscimol injection into the DMH appears to attenuate the overall magnitude of the blood pressure response it had no effect on the RSNA response to epicardial bradykinin. These results suggest that in the presence of intact spinal and medullary circuits inhibition of DMH does not play an important role in the generation of sympathetic activity in the CSAR. Whereas inhibition of DMH does not appear to impair the CSAR there is good evidence to show that disinhibition of DMH increases basal vasomotor activity and augment reflex sympathetic responsiveness. Thus, our next objective is to determine if activation of DMH augments the CSAR and if so to determine the medullary connections of these DMH neurons.Support or Funding InformationNHLBI R15HL145645‐01

High mobility group box-1 in hypothalamic paraventricular nuclei attenuates sympathetic tone in rats at post-myocardial infarction.

Inflammation is associated with increased sympathetic drive in cardiovascular diseases. The paraventricular nucleus (PVN) of the hypothalamus is a key regulator of sympathetic nerve activity at post-myocardial infarction (MI). High mobility group box-1 (HMGB1) exhibits inflammatory cytokine like activity in the extracellular space. Inflammation is associated with increased sympathetic drive in cardiovscular diseases. However, the role of HMGB1 in sympathetic nerve activity at post-MI remains unknown. The aim of the present study is to determine the role and mechanism of HMGB1 in the PVN, in terms of sympathetic activity and arrhythmia after MI. Sprague-Dawley rats underwent left anterior descending coronary artery ligation to induce MI. Anti-HMGB1 polyclonal antibody or control IgG was bilaterally microinjected into the PVN (5 μL every second day for seven consecutive days). Then, renal sympathetic nerve activity (RSNA) was recorded. The association between ventricular arrhythmias (VAs) and MI was evaluated using programmed electrophysiological stimulation. After performing electrophysiological experiments in vivo, immunohistochemistry was used to detect the distribution of HMGB1, while Western blot was used to detect the expression of HMGB1 and p-ERK in the PVN of MI rats. HMGB1 and p-ERK were upregulated in the PVN in rats at post-MI. Moreover, bilateral PVN microinjection of anti-HMGB1 polyclonal antibody reversed the expression of HMGB1 and p-ERK, and consequently decreased the baseline RSNA and inducible VAs, when compared to those in sham rats. These results suggest that MI causes the translocation of HMGB1 in the PVN, which leads to sympathetic overactivation through the ERK1/2 signaling pathway. The bilateral PVN microinjection of anti-HMGB1 antibody can be an effective therapy for MI-induced arrhythmia.

Central angiotensin II-Protein inhibitor of neuronal nitric oxide synthase (PIN) axis contribute to neurogenic hypertension

Activation of renin-angiotensin- system, nitric oxide (NO•) bioavailability and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of many cardiovascular diseases, including hypertension. Previously we have shown increased protein expression of PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with heart failure. To elucidate the molecular mechanism by which Angiotensin II (Ang II) increases PIN expression, we used Sprague-Dawley rats (250–300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 0.5 μl/h) or saline as vehicle (Veh) for 14 days through osmotic mini-pumps and NG108-15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline renal sympathetic nerve activity and mean arterial pressure. Ang II infusion increased the expression of PIN (1.24 ± 0.04* Ang II vs. 0.65 ± 0.07 Veh) with a concomitant 50% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1.00 ± 0.03 Veh) in the PVN. Substrate-dependent ligase assay in cells transfected with pCMV-(HA-Ub)8 vector revealed a reduction of HA-Ub-PIN conjugates after Ang II and a proteasome inhibitor, Lactacystin (LC), treatment (4.5 ± 0.7* LC Ang II vs. 9.2 ± 2.5 LC). TUBE (Tandem Ubiquitin-Binding Entities) assay showed decrease PIN-Ub conjugates in Ang II-treated cells (0.82 ± 0.12* LC Ang II vs. 1.21 ± 0.06 LC) while AT1R blocker, Losartan (Los) treatment diminished the Ang II-mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.16 ± 0.04* LC Ang II Los). Taken together, our studies suggest that increased central levels of Ang II contribute to the enhanced expression of PIN leading to reduced expression of the dimeric form of nNOS, thus diminishing the inhibitory action of NO• on pre-autonomic neurons in the PVN resulting in increased sympathetic outflow.

Upregulating Nrf2 in the RVLM ameliorates sympatho-excitation in mice with chronic heart failure

Nuclear factor E2-related factor 2 (Nrf2) is a key transcription factor that maintains redox homeostasis by governing a broad array of antioxidant genes in response to oxidant stress. We hypothesized that overexpression of Nrf2 in the rostral ventrolateral medulla (RVLM) ameliorates sympatho-excitation in mice with coronary artery ligation-induced chronic heart failure (CHF). To address this, we overexpressed Nrf2 in the RVLM using an HIV-CamKIIa-Nrf2 lenti virus in C57BL/6 mice. In addition, we used a Lenti-Cre virus in Keap1flox/flox mice to upregulate Nrf2 non-selectively in the RVLM. Arterial blood pressure (AP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were recorded under conscious and anesthetized conditions, respectively. Protein expression was assayed using western blotting and immunofluorescence staining. We found that (1) Nrf2 and two target proteins, NQO1 and HO-1 in the RVLM were significantly lower in CHF compared to Sham mice. Nrf2 viral transfection of the RVLM upregulated Nrf2 protein. (2) Urinary NE excretion in CHF mice was markedly attenuated following Nrf2 upregulation (812 ± 133 vs 1120 ± 271 ng/24hr mean.±SE, *p < 0.05, n = 8/group). (3) In the conscious state, CHF mice overexpressing Nrf2 exhibited an enhancement in spontaneous baroreflex gain and in phenylephrine-induced baroreflex control of HR. (4) Acute experiments under anesthetisa revealed a significant decrease in basal RSNA (44.0 ± 6.5 vs 64.7 ± 8.3% of Max. *P < 0.05 n = 8/group) and enhancement in baroreflex sensitivity (Maximal gain −1.8 ± 0.3 vs 1.1 ± 0.2 of mmHg. **p < 0.01. n = 6/group) in CHF mice that were virally transfected with Nrf2 compared with CHF mice transfected with Lenti-GFP. Finally, Lenti-Cre viral overexpression of Nrf2 in Keap1flox/flox mice reduced Keap1 protein and increased Nrf2, NQO1, and HO-1 in the RVLM of Sham and CHF mice. CHF-Cre mice exhibited a significant decrease in baseline RSNA and plasma NE concentration (8.9 ± 1.1 vs 12.7 ± 0.9 ng/mL *P < 0.05 n = 6/group) as compared with CHF-GFP mice. Based on the above data, we conclude that upregulating Nrf2 selectively in the RVLM attenuates sympatho-excitation in CHF mice. Nrf2 may be an important central target for autonomic modulation in cardiovascular disease and during stress.

Combined V1a and V1b Receptor Activation Within the Paraventricular Nucleus Contributes to Sympathoexcitation and Hypertension in Rats Exposed to Chronic Mild Unpredictable Stress

The paraventricular nucleus (PVN) in the brain has been recognized as a major integrative center for neurohumoral regulation which receives a broad array of afferent inputs. As such, the function of the PVN becomes even more important in times of physical or physiological stress. Two distinct neuronal populations we are interested in include the magnocellular and parvocellular neurons. Specifically, we are interested in deciphering how intranuclearly released vasopressin (AVP) from the magnocellular neurons acts upon the parvocellular pre‐sympathetic neurons and glia that are richly endowed with V1a and V1b receptors in a rat model of depression. It has been demonstrated that V1a receptor activation depolarizes PVN pre‐sympathetic neurons and initiates an action potential, which puts forth a signaling cascade that increases sympathetic nerve activity, arterial pressure and heart rate. Additionally, we previously published that renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP) and heart rate (HR) responses to AVP microinjected in the PVN is significantly attenuated when the rats were pretreated with V1b receptor antagonist nelivaptan. In this study, we set out to identify differences in baseline RSNA, MAP and HR between rats exposed to the chronic mild unpredictable stress protocol (CMS) and their normal counterparts, as well as their responses to AVP microinjected into the PVN in the presence of the V1a receptor antagonist, Manning compound, and the V1b receptor inhibitor, nelivaptan. We observed that baseline MAP is higher in CMS rats (110 ± 2 mmHg vs. 115 ± 1 mmHg; P &lt; 0.05). Furthermore, in control rats, combined inhibition of AVP with nelivaptan and Manning compound resulted in near complete abolition of the rise in MAP and baroreflex‐mediated decreases in HR and RSNA. CMS rats exhibited exaggerated baseline RSNA, 43.6 ± 9.97 μV.ms compared with control rats, 8.04 ± 3.24 μV.ms (P &lt; 0.025). PVN AVP injection increased RSNA by 52.1 ± 3.3 %baseline in controls and was blocked by the dual blockade. RSNA did not increase significantly after PVN AVP in CMS rats and V1a receptor and V1b receptor blockade did not change this response. CMS rats displayed higher baseline RSNA 43.6 ± 9.9 μV.ms vs control rats 8.0 ± 3.9 μV.ms P &lt; 0.025). Moreover, dual blockade significantly decreased RSNA from baseline values only in CMS rats: −12.6 ± 0.4 μV.ms compared with control rats −1.1 ± 0.6 μV.ms (P &lt; 0.05). These findings are consistent with the concept that arterial pressure and RSNA are increased during chronic stress via dendritic release of AVP within the PVN thereby contributing to sympathoexcitation and hypertension.Support or Funding InformationDepartment of Veterans Affairs Merit Award to NFRThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract 106: Central Angiotensin II Mediates Neurogenic Hypertension Through Post-Translational Regulation of Neuronal Nitric Oxide Synthase in the Paraventricular Nucleus of the Hypothalamus

Activation of renin-angiotensin- system, nitric oxide (NO) bioavailability and subsequent sympathoexcitation plays a pivotal role in the pathogenesis of many cardiovascular diseases including hypertension. Previously we have shown Ang II enhances PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) mediated ubiquitination of nNOS in the PVN of rats with heart failure. To further elucidate the mechanism by which Ang II increases sympathetic outflow by regulating PIN mediated nNOS ubiquitination, we used Sprague-Dawley rats (250-300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 14days, 0.5μl/h) through osmotic mini-pumps and NG108-15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion increased baseline mean arterial pressure (126 ± 9* vs. 84 ± 4 mmHg) and renal sympathetic nerve activity (20.5 ± 2.3* vs. 6.4 ± 1.9 % of Max. Activity). Ang II infusion increased the expression of PIN (1.36 ± 0.04* Ang II vs. 0.81 ± 0.02 Veh) with a concomitant 52% decrease in dimeric nNOS and PIN-Ub conjugates (0.73 ± 0.04* Ang II vs. 1 ± 0.03 Veh) in the PVN. Further, Ang II-mediated increase in PIN expression(0.67±0.06* CHX AngII vs. 0.40±0.08 CHX 0h) was independent of CHX (protein synthesis inhibitor) mediated decrease in PIN expression (0.41±0.06* CHX AngII vs. 0.19±0.04 CHX 4h) suggesting Ang II-mediated post-translational stabilization of PIN. Substrate-dependent ligase assay in cells transfected with pCMV-(HA-Ub)8 vector revealed a reduction of HA-Ub-PIN conjugates after Ang II and proteasome inhibitor lactacystin (LC) treatment (4.5 ± 0.6* LC Ang II vs. 9.2 ± 2.2 LC). TUBE (Tandem Ubiquitin-Binding Entities) assay showed decrease PIN-Ub conjugates in Ang II-treated cells (0.82 ± 0.12* LC Ang II vs. 1.23 ± 0.05 LC) while AT1R blocker Losartan(Los) treatment diminishes the Ang II-mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.14 ± 0.04* LC AngII Los). Taken together, our studies suggest that increased central levels of Ang II contribute to the enhanced expression of PIN leading to reduced expression of the dimeric form of nNOS, thus diminishing the inhibitory action of NO on pre-autonomic neurons in the PVN resulting in an increase in sympathetic outflow.

Central Angiotensin II regulates Protein Inhibitor of Neuronal Nitric Oxide Synthase through post‐translational mechanisms in the Paraventricular Nucleus resulting in increased Sympathetic outflow

Increased activation of Renin‐Angiotensin System in the paraventricular nucleus (PVN) of the hypothalamus has been associated with the increased sympathetic outflow observed in hypertension and chronic heart failure (CHF). Previously we have shown increased protein expression of the PIN (a protein inhibitor of nNOS: neuronal nitric oxide synthase, known to dissociate nNOS dimers into monomers) with concomitantly reduced levels of catalytically active dimers of nNOS in the PVN of rats with CHF. However, PIN transcripts levels remain unchanged. To elucidate the molecular mechanism of increased PIN expression post‐transcriptionally, we used Sprague‐Dawley rats (250–300 g) subjected to intracerebroventricular infusion of Ang II (20 ng/min, 14 days, 0.5 μl/h) through osmotic mini‐pumps and NG108‐15 hybrid neuronal cell line treated with Ang II as an in vitro model. Ang II infusion significantly increased baseline mean arterial pressure (126 ± 9 vs. 84 ± 4 mmHg) and baseline renal sympathetic nerve activity (20.5 ± 2.3 vs. 6.4 ± 1.9 % of Max activity). Ang II infusion increased the expression of the PIN (1.36 ± 0.04* Ang II vs. 0.81 ± 0.02 Veh) a with concomitant decrease in PIN‐Ub conjugates (0.73 ± 0.04* Ang II vs. 1 ± 0.03 Veh) in the PVN. Further, PIN translation was inhibited in vitro using protein synthesis inhibitor cycloheximide (CHX) for 0–4h after 20h of pretreatment with Ang II. Ang II‐mediated increase in PIN expression(0.67±0.06* CHX AngII vs. 0.40±0.08 CHX 0h) was independent of CHX mediated decrease in PIN expression (0.41±0.06* CHX AngII vs. 0.19±0.04 CHX 4h) suggesting Ang II‐mediated post‐translational stabilization of PIN. Ubiquitination assay in cells transfected with pCMV‐(HA‐Ub) 8 vector revealed a reduction of HA‐Ub‐PIN conjugates after Ang II and proteasome inhibitor lactacystin (LC) treatment (4.5 ± 0.6* LC Ang II vs. 9.2 ± 2.2 LC). TUBE (Tandem Ubiquitin‐Binding Entities) assay showed decrease PIN‐Ub conjugates in Ang II‐treated cells (0.82 ± 0.12* LC Ang II vs. 1.23 ± 0.05 LC) while AT1R blocker Losartan(Los) treatment diminishes the Ang II‐mediated stabilization of PIN (1.21 ± 0.07 LC Los vs. 1.14 ± 0.04* LC AngII Los). These results suggest that PIN is targeted for rapid degradation by the ubiquitin‐proteasome and Ang II via AT1R delays the rate of degradation resulting in accumulation of PIN. It of interest to note that previously we observed that there was a decreased accumulation of PIN‐Ub conjugates in the PVN of CHF rats compared to Sham (0.63 ± 0.05* CHF vs. 1.00 ± 0.10 Sham). Taken together, our studies suggest that increased central levels of Ang II contribute to the upregulation of PIN expression in the PVN via decreasing the ubiquitination which may be involved in reducing the expression of dimeric nNOS of pre‐autonomic neurons in the PVN resulting in the increased sympathetic outflow.Support or Funding InformationSupported by AHA ‐ 14SDG19980007 and NIH grants HL124104 &amp; HL62222.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Fructose Overconsumption since Early Age on Renal Function and Sympathetic Nerve Activity

Increased consumption of ready‐to‐eat foods and beverages that contain high fructose content may contribute to the onset of cardiovascular and renal diseases. Moreover, fructose overload in diet since prenatal period may increase the risk of the offspring to diseases later in life. Thus, the objective of this study was to evaluate the effects of increased consumption of fructose since prenatal period until adulthood on: blood pressure, renal function and renal sympathetic nerve activity of the offspring at adulthood.MethodsFemales Wistar rats were randomly assigned to groups: Control (C) and Fructose (F). The C group received food and drinking water ad libitum, and F group received food and D‐fructose solution (20%) for drinking ad libitum. In F, the offer of D‐fructose solution begun 1 week before mating and continued during pregnancy and lactation. After birth, the progeny was limited to 8 offspring and designated as Control Offspring (CO) and Fructose Offspring (FO). At 21st day of life, male offspring were placed in collective cages and received the same treatment of the dams. The experiments were performed at 4 months old rats. Direct measurement of systolic arterial blood pressure and mean arterial blood pressure (SAP and MAP), and the registration of the renal sympathetic nerve activity (RSNA) were performed in 6 animals of each group (CO and FO). The renal function parameters were evaluated in other 6 rats of each group. Renal parameters evaluated: glomerular filtration rate (GFR ‐ inulin clearance), renal plasma flow (RPF ‐ PAH clearance), protein excretion (EProt), urine osmolarity (Uosm), urinary excretion/24 h (V), sodium (ENa+) and potassium (EK+) excretion. The study was approved by the Ethical Research Committee of the Universidade Federal de São Paulo (protocol: 2757270117).ResultsCompared with CO group, FO presented higher values of SAP (FO: 165 ± 5 vs CO: 141 ± 6 mmHg) and MAP (FO: 130 ± 5; CO: 110 ± 4 mmHg). FO had decreased GFR (FO: 5.1 ± 0.7; CO: 8.4 ± 0.5 ml/min/kg), Uosm (FO: 305 ± 60; CO: 1522 ± 81 mosm/l) and potassium excretion (FO: 182 ± 18; CO: 302 ± 28 mM/24 h). The urine excretion and protein excretion were increased in FO compared with CO group (V: FO: 52 ± 1; CO: 14 ± 0.7 ml/24 h; EProt: FO: 10.3 ± 1; CO: 6.4 ± 1 mg/24 h). There were no significant differences between the groups regarding the baseline RSNA (FO: 59 ± 4; CO: 71 ± 14 spikes/s), RPF (FO: 15.0 ± 2; CO: 16.2 ± 1 ml/min/kg) and sodium excretion (FO: 91.2 ± 15; CO: 85.7 ± 15 mM/24 h). Values are presented as mean ± sem, p ≤ 0.05.ConclusionsOur data show that increased consumption of fructose since early age leads to increase in blood pressure and alterations in renal function in the offspring (reduced: GFR, urine osmolarity and potassium excretion; and increased protein excretion).Support or Funding InformationCAPESThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Selective Nrf2 Gene Deletion in the Rostral Ventrolateral Medulla Evokes Hypertension and Sympathoexcitation in Mice.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a master transcriptional regulator of redox homeostasis that impacts antioxidant gene expression. Central oxidative stress and reduced antioxidant enzyme expression in the rostral ventrolateral medulla (RVLM) contributed to sympathoexcitation in chronic heart failure. In the current study, we hypothesized that deletion of Nrf2 in the RVLM would increase sympathetic drive and blood pressure. Experiments were performed in Nrf2-floxed mice treated with microinjection of lentiviral-Cre-GFP or lentiviral-GFP into the RVLM. Two weeks after viral administration, Nrf2 message, protein, oxidative stress, cardiovascular function, and sympathetic outflow were evaluated. We found that (1) Nrf2 mRNA and protein in the RVLM were significantly lower in Cre mice compared with control GFP mice. Nrf2-targeted antioxidant enzymes were downregulated, whereas reactive oxygen species were elevated. (2) Blood pressure measurements indicated that Cre mice displayed a significant increase in blood pressure (mean arterial pressure, 123.7±3.8 versus 100.2±2.2 mm Hg; P<0.05, n=6), elevated urinary norepinephrine (NE) concentration (456.4±16.9 versus 356.5±19.9 ng/mL; P<0.05, n=6), and decreased spontaneous baroreflex gain (up sequences, 1.66±0.17 versus 3.61±0.22 ms/mm Hg; P<0.05, n=6; down sequences, 1.89±0.12 versus 2.98±0.19 ms/mm Hg; P<0.05, n=6). (3) Cre mice displayed elevated baseline renal sympathetic nerve activity and impaired inducible baroreflex function. These data suggest that Nrf2 gene deletion in the RVLM elevates blood pressure, increases sympathetic outflow, and impairs baroreflex function potentially by impaired antioxidant enzyme expression.

Evidence that remodeling of insular cortex neurovascular unit contributes to hypertension-related sympathoexcitation.

The intermediate region of the posterior insular cortex (intermediate IC) mediates sympathoexcitatory responses to the heart and kidneys. Previous studies support hypertension‐evoked changes to the structure and function of neurons, blood vessels, astrocytes and microglia, disrupting the organization of the neurovascular unit (NVU). In this study, we evaluated the functional and anatomical integrity of the NVU at the intermediate IC in the spontaneously hypertensive rat (SHR) and its control the Wistar‐Kyoto (WKY). Under urethane anesthesia, NMDA microinjection (0.2 mmol/L/100 nL) was performed at the intermediate IC with simultaneous recording of renal sympathetic nerve activity (RSNA), heart rate (HR) and mean arterial pressure (MAP). Alterations in NVU structure were investigated by immunofluorescence for NMDA receptors (NR1), blood vessels (70 kDa FITC‐dextran), astrocytes (GFAP), and microglia (Iba1). Injections of NMDA into intermediate IC of SHR evoked higher amplitude responses of RSNA, MAP, and HR. On the other hand, NMDA receptor blockade decreased baseline RSNA, MAP and HR in SHR, with no changes in WKY. Immunofluorescence data from SHR intermediate IC showed increased NMDA receptor density, contributing to the SHR enhanced sympathetic responses, and increased in vascular density (increased number of branches and endpoints, reduced average branch length), suggesting angiogenesis. Additionally, IC from SHR presented increased GFAP immunoreactivity and contact between astrocyte processes and blood vessels. In SHR, IC microglia skeleton analysis supports their activation (reduced number of branches, junctions, endpoints and process length), suggesting an inflammatory process in this region. These findings indicate that neurogenic hypertension in SHR is accompanied by marked alterations to the NVU within the IC and enhanced NMDA‐mediated sympathoexcitatory responses likely contributors of the maintenance of hypertension.

The role of brain angiotensin II (type 2) receptors and nitric oxide in the renal sympathoinhibitory response to acute volume expansion in conscious rats.

The study was performed to investigate the role of angiotensin II type 2 (AT2) receptors and nitric oxide in the renal sympathoinhibitory response to volume expansion (VEP). Conscious rats were subjected to volume expansion (VEP) [0.25% body weight/min saline for 10 min intravenously (i.v.)] following intracerebroventricular (i.c.v.) infusion of either saline or angiotensin II (Ang II), or a combination of Ang II with either losartan, PD123319, or N-nitro-L-arginine methyl ester (L-NAME). Intracerebroventricular losartan, PD123319, or L-NAME did not change baseline mean arterial pressure, heart rate, or renal sympathetic nerve activity (RSNA). However, i.c.v. Ang II increased mean arterial pressure and decreased heart rate and RSNA baselines (113 ± 2 vs. 107 ± 2 mmHg, 365 ± 7 vs. 379 ± 5 beats/min, 1.03 ± 0.13 vs. 1.29 ± 0.15 μV.s, respectively, all P < 0.05). During i.c.v. saline infusion, VEP decreased RSNA by 27 ± 2% (P < 0.05) after 10 min and the magnitude of this response was unchanged during i.c.v. infusion of Ang II, losartan, or PD123319 but was decreased by L-NAME compared with that obtained with i.c.v. saline (14 ± 3 vs. 30 ± 5%, P < 0.05). i.c.v. Ang II in combination with losartan enhanced (41 ± 3 vs. 29 ± 5%) but with PD123319 decreased (15 ± 2 vs. 28 ± 4%, P < 0.05) the renal sympathoinhibition compared with Ang II alone. The renal sympathoinhibitory response was enhanced (43 ± 5 vs. 29 ± 1%, P < 0.05) by i.c.v. infusion of an AT2 agonist, CGP42112 the magnitude of which was unchanged when combined with L-NAME. The sympathoinhibitory response to VEP following Ang II plus L-NAME was similar to Ang II alone. These findings suggest that activation of central AT2 receptors enhances the renal sympathoinhibitory response to VEP but this effect is not dependent on nitric oxide.

Central Angiotensin-II Increases Blood Pressure and Sympathetic Outflow via Rho Kinase Activation in Conscious Rabbits.

Elevated sympathetic tone and activation of the renin-angiotensin system are pathophysiologic hallmarks of hypertension, and the interactions between these systems are particularly deleterious. The importance of Rho kinase as a mediator of the effects of angiotensin-II (AngII) in the periphery is clear, but the role of Rho kinase in sympathoexcitation caused by central AngII is not well established. We hypothesized that AngII mediates its effects in the brain by the activation of the RhoA/Rho kinase pathway. Chronically instrumented, conscious rabbits received the following intracerebroventricular infusion treatments for 2 weeks via osmotic minipump: AngII, Rho kinase inhibitor Fasudil, AngII plus Fasudil, or a vehicle control. AngII increased mean arterial pressure over the course of the infusion, and this effect was prevented by the coadministration of Fasudil. AngII increased cardiac and vascular sympathetic outflow as quantified by the heart rate response to metoprolol and the depressor effect of hexamethonium; coadministration of Fasudil abolished both of these effects. AngII increased baseline renal sympathetic nerve activity in conscious animals and impaired baroreflex control of sympathetic nerve activity; again Fasudil coinfusion prevented these effects. Each of these end points showed a statistically significant interaction between AngII and Fasudil. Quantitative immunofluorescence of brain slices confirmed that Rho kinase activity was increased by AngII and decreased by Fasudil. Taken together, these data indicate that hypertension, elevated sympathetic outflow, and baroreflex dysfunction caused by central AngII are mediated by Rho kinase activation and suggest that Rho kinase inhibition may be an important therapeutic target in sympathoexcitatory cardiovascular diseases.

Chronic Mild Stress Increases Baseline Renal Sympathetic Nerve Activity and the Response to Acute Stressors in Conscious Rats

Depression is a non‐traditional risk factor for cardiovascular morbidity and mortality. Increased sympathetic tone is also recognized as deleterious to cardiovascular health. Chronic mild unpredictable stress paradigm (CMS) is a validated model of depression that has been associated with cardiovascular dysfunction in rats. We hypothesized that rats exposed to CMS will have higher arterial pressure and renal sympathetic nerve activity (RSNA) and enhanced responses to acute stressors. Male Sprague Dawley rats were equipped with telemetry transmitters for hemodynamic monitoring and subjected to 4 weeks of CMS or standard housing (control). During the last week, rats were equipped with telemetry transmitters for renal nerve recording. Baseline RSNA was obtained a minimum of 3 days after completion of CMS protocol and the rats were subjected to air jet stress and nasopharygeal reflex testing. Baseline arterial pressure was higher in CMS rats. Baseline RSNA was 0.80 ± 0.05 mV.s in control vs 1.20 ± 0.14 mV.sec in CMS rats (P &lt; 0.05). Peak responses to either air jet stress or nasopharyngeal stimulation were significantly higher in the CMS rats. Although the latencies of the responses were shorter and the duration of the responses were longer in the CMS group, these did not achieve significance. The area under the curve (AUC = latency × duration) for air jet stress was 82% greater for air jet stress and 362% greater for nasopharygeal stimulation in the CMS vs control rats (P &lt; 0.05 for both vs control). These results support the concept that rats subjected to a chronic unpredictable stress paradigm that models depression display higher basal sympathetic nerve activity and a more robust response to acute stress. This enhanced sympathetic tone and neuroexcitability may contribute to increased cardiovascular risk.Support or Funding InformationVA Merit Grant to NFR

Evidence that Combined Aliskiren and L‐arginine Treatment Reverses Renovascular Hypertension in an Animal Model

Renovascular hypertension is characterized by increased renal sympathetic activity, angiotensin II, and by endothelial dysfunction. The purpose of this study was to determine the role of renal sympathetic nerve activity (RSNA) in mediating the anti-hypertensive effects of aliskiren (ALSK) and L-arginine (L-ARG) in a rat renovascular hypertension model. Hypertension was induced by clipping the right renal artery, and the following 5 groups were divided: Sham operated; 2-kidney, 1-clip (2K1C); 2K1C plus ALSK; 2K1C plus L-ARG; and 2K1C plus ALSK+ L-ARG. The systolic blood pressure (SBP) of 2K1C rats increased from 114.4±5.2 to 204±12.7 mmHg, (P< 0.05) and was only reduced by ALSK+L-ARG treatment (138.4±4.37 mmHg). The 2K1C hypertension increased the baseline RSNA (SHAM: 62.4±6.39 vs 2K1C: 97.4±8.43%). L-ARG or ALSK+L-ARG treatment significantly decreased baseline RSNA (2K1C L-ARG:70.7±2.39; 2K1C ALSK+L-ARG: 69.3±4.23%), but ALSK treatment alone did not (2K1C ALSK: 84.2±2.5%). Urinary water, Na+, Cl- and urea excretion were similar in the 2K1C L-ARG, 2K1C ALSK+L-ARG and SHAM groups. The combination of ALSK+L-ARG restored urine flow and increased the glomerular filtration rate. The nNOS expression in the non clipped kidney was significantly increased in 2K1C ALSK+L-ARG rats. In conclusion, combined ALSK+L-ARG treatment normalizes SBP and prevents renal dysfunction in 2K1C hypertensive rats, an effect that can be attributed to reduced RSNA.

Renal Sympathetic Nerve Activity – Controlled by Renal Afferent Sympathoexcitatory or ‐Inhibitory Nerves ?

Renal sympathetic nerve activity (RSNA) is important in hypertension, volume disorders or renal disease. It is unclear if increases of RSNA in disease are due to sympathoexcitatory or impaired sympathoinhibitory renal afferent nerves. We present data from nephritic rats suggesting the latter.Nephritis due to OX7‐antibodies. Methohexital anesthetized nephritic rats &amp; controls instrumented to stimulate renal afferent nerve activity (ARNA) in order to influence RSNA: ipsilateral renal arterial catheter for intrarenal administration (IRA) of the TRPV1 agonist capsaicin to stimulate ARNA (CAP 6.6*10‐7M) and induce release of the tachykinin receptor agonist SP from renal afferents; contralateral stainless steel electrode for RSNA recording; before and after IRA CAP the tachykinin‐receptor blocker RP67580 was given. Baseline RSNA &amp; ARNA were assessed. Some nephritic rats pretreated with tachykinin receptor antagonists to prove increased SP effects.IRA CAP decreased RSNA from 67.5±12.0 µV*sec to 14.8±4.2 µV*sec (p&lt;0.05) over 60 minutes while in nephritis RSNA suppression was abolished. Suppressed RSNA in controls was transiently reversed by the tachykinin inhibitor. Under resting conditions RSNA was higher, ARNA lower in nephritis as compared to controls. Tachykinreceptor antagonism ameliorated damage in renal nephritis suggesting increased SP release from renal afferent nerves despite lack of the tachykinin dependant sympathoinhibition seen in controls.Our data suggest that a tachykinin dependant reno‐sympathetic reflex mechanism exerts sympathoinhibitory effects being impaired under pathophysiological circumstances.

PSD95 Coupling of NMDA Receptor Activation and Nitric Oxide Activity in the Hypothalamic PVN

PVN regulation of sympathetic nerve activity (SNA) and mean arterial pressure (MAP) strongly depends on the balance of glutamatergic excitation by NMDAR and GABAergic inhibition facilitated by nitric oxide (NO). The adaptor protein PSD95 regulates NO production by tethering nNOS to Ca2+‐permeable GluN2B‐containing NMDA receptors. To determine the importance of PSD95 in NMDA‐NO auto‐inhibitory feedback in PVN, lentivirus encoding PSD95 shRNA or scrambled shRNA (control) was injected into PVN (400 nL/side) of male rats. Rats were anesthetized 3‐4 weeks later and NMDA was injected into PVN. Consistent with diminished NMDA‐mediated stimulation of NO in LV‐PSD95‐shRNA rats, PVN NMDA evoked greater increases of renal SNA (PSD95 shRNA: 80±15% vs control: 28±5%) and MAP (PSD95 shRNA: 13±3 mmHg vs control: 10±1 mmHg) compared to controls. Whereas the non‐selective NOS inhibitor L‐NAME in PVN had no effect on baseline renal SNA or MAP, it reduced PVN NO as measured in controls by amperometry. During L‐NAME suppression of NO in controls, PVN NMDA caused greater increases of renal SNA (NMDA: 28±5% vs NMDA+L‐NAME: 70±12%) and MAP (NMDA: 10±1 mmHg vs NMDA+L‐NAME: 21±1 mmHg). Consistent with reduced capacity for NMDA stimulation of NO in LV‐PSD95‐shRNA rats, PVN L‐NAME did not enhance renal SNA (NMDA: 62±16% vs NMDA+L‐NAME: 59±13%) or MAP (NMDA: 13±1 mmHg vs NMDA+L‐NAME: 13±1 mmHg) responses to NMDA. Results indicate that PVN PSD95 couples NMDAR activation to NO production, which restrains NMDAR driven responses. In the context of neurogenic cardiovascular diseases, decreased PVN PSD95 expression/function could contribute to loss of NO inhibition and to exaggerated glutamatergic/NMDAR driven SNA. HL 088052 &amp; 102310 (GMT), T32 07446 (MEB)