Investigation of vagal sensory neurons in mice using optical vagal stimulation and tracheal neuroanatomy.

In rats and guinea pigs, sensory innervation of the airways is derived largely from the vagus nerve, with the extrapulmonary airways innervated by Wnt1+ jugular neurons and the intrapulmonary airways and lungs by Phox2b+ nodose neurons; however, our knowledge of airway innervation in mice is limited. We used genetically targeted expression of enhanced yellow fluorescent protein-channelrhodopsin-2 (EYFP-ChR2) in Wnt1+ or Phox2b+ tissues to characterize jugular and nodose-mediated physiological responses and airway innervation in mice. With optical stimulation, Phox2b+ vagal fibers modulated cardiorespiratory function in a frequency-dependent manner while right Wnt1+ vagal fibers induced a small increase in respiratory rate. Mouse tracheae contained sparse Phox2b-EYFP fibers but dense networks of Wnt1-EYFP fibers. Retrograde tracing from the airways showed limited tracheal innervation by the jugular sensory neurons, distinct from other species. These differences in physiology and vagal sensory distribution have important implications when using mice for studying airway neurobiology.

Selective efferent vagal stimulation in heart failure.

Patients diagnosed with heart failure have high rates of mortality and morbidity. Based on promising preclinical studies, vagal nerve stimulation has been trialled in these patients using whole nerve electrical stimulation, but the results have been mixed. This is, at least in part, due to an inability to selectively recruit the activity of specific fibres within the vagus with whole nerve electrical stimulation, as well as not knowing which the 'therapeutic' fibres are. This symposium review focuses on a population of cardiac-projecting efferent vagal fibres with cell bodies located within the dorsal motor nucleus of the vagus nerve and a new method of selectively targeting these projections as a potential treatment in heart failure. NEW FINDINGS: What is the topic of this review? Selective efferent vagal stimulation in heart failure. What advances does it highlight? Selectively targeting a population of cardiac-projecting efferent vagal fibres with cell bodies within the dorsal motor nucleus of the vagus using optogenetics slows the progression of heart failure in rats.

Associations Between Systemic and Cerebral Inflammation in an Ovine Model of Cardiopulmonary Bypass.

BACKGROUND: Intraoperative inflammation may contribute to postoperative neurocognitive disorders after cardiac surgery requiring cardiopulmonary bypass (CPB). However, the relative contributions of general anesthesia (GA), surgical site injury, and CPB are unclear. METHODS: In adult female sheep, we investigated (1) the temporal profile of proinflammatory and anti-inflammatory cytokines and (2) the extent of microglia activation across major cerebral cortical regions during GA and surgical trauma with and without CPB (N = 5/group). Sheep were studied while conscious, during GA and surgical trauma, with and without CPB. RESULTS: Plasma tumor necrosis factor-alpha (mean [95% confidence intervals], 3.7 [2.5-4.9] vs 1.6 [0.8-2.3] ng/mL; P = .0004) and interleukin-6 levels (4.4 [3.0-5.8] vs 1.6 [0.8-2.3] ng/mL; P = .029) were significantly higher at 1.5 hours, with a further increase in interleukin-6 at 3 hours (7.0 [3.7-10.3] vs 1.8 [1.1-2.6] ng/mL; P < .0001) in animals undergoing CPB compared with those that did not. Although cerebral oxygen saturation was preserved throughout CPB, there was pronounced neuroinflammation as characterized by greater microglia circularity within the frontal cortex of sheep that underwent CPB compared with those that did not (0.34 [0.32-0.37] vs 0.30 [0.29-0.32]; P = .029). Moreover, microglia had fewer branches within the parietal (7.7 [6.5-8.9] vs 10.9 [9.4-12.5]; P = .001) and temporal (7.8 [7.2-8.3] vs 9.9 [8.2-11.7]; P = .020) cortices in sheep that underwent CPB compared with those that did not. CONCLUSIONS: CPB enhanced the release of proinflammatory cytokines beyond that initiated by GA and surgical trauma. This systemic inflammation was associated with microglial activation across 3 major cerebral cortical regions, with a phagocytic microglia phenotype within the frontal cortex, and an inflammatory microglia phenotype within the parietal and temporal cortices. These data provide direct histopathological evidence of CPB-induced neuroinflammation in a large animal model and provide further mechanistic data on how CPB-induced cerebral inflammation might drive postoperative neurocognitive disorders in humans.

Renal, Cardiac, and Autonomic Effects of Catheter-Based Renal Denervation in Ovine Heart Failure.

[Figure: see text].

Selective optogenetic stimulation of efferent fibers in the vagus nerve of a large mammal.

BACKGROUND: Electrical stimulation applied to individual organs, peripheral nerves, or specific brain regions has been used to treat a range of medical conditions. In cardiovascular disease, autonomic dysfunction contributes to the disease progression and electrical stimulation of the vagus nerve has been pursued as a treatment for the purpose of restoring the autonomic balance. However, this approach lacks selectivity in activating function- and organ-specific vagal fibers and, despite promising results of many preclinical studies, has so far failed to translate into a clinical treatment of cardiovascular disease. OBJECTIVE: Here we report a successful application of optogenetics for selective stimulation of vagal efferent activity in a large animal model (sheep). METHODS AND RESULTS: Twelve weeks after viral transduction of a subset of vagal motoneurons, strong axonal membrane expression of the excitatory light-sensitive ion channel ChIEF was achieved in the efferent projections innervating thoracic organs and reaching beyond the level of the diaphragm. Blue laser or LED light (>10 mW mm-2; 1 ms pulses) applied to the cervical vagus triggered precisely timed, strong bursts of efferent activity with evoked action potentials propagating at speeds of ∼6 m s-1. CONCLUSIONS: These findings demonstrate that in species with a large, multi-fascicled vagus nerve, it is possible to stimulate a specific sub-population of efferent fibers using light at a site remote from the vector delivery, marking an important step towards eventual clinical use of optogenetic technology for autonomic neuromodulation.

Systemic administration of pentoxifylline attenuates the development of hypertension in renovascular hypertensive rats.

There is evidence to suggest that hypertension involves a chronic low-grade systemic inflammatory response; however, the underlying mechanisms are unclear. To further understand the role of inflammation in hypertension, we used a rat renovascular model of hypertension in which we administered the TNF-α synthesis inhibitor pentoxifylline (PTX, 30 mg/kg/day) in the drinking water for 60 days. In conscious rats, PTX administration significantly attenuated the development of hypertension (systolic blood pressure, PTX: 145 ± 8 vs. vehicle (Veh): 235 ± 11 mmHg, after 38 days of treatment, P < 0.05, N = 5/group). This attenuation in hypertension was coupled with a decrease in the low-frequency spectra of systolic blood pressure variability (PTX: 1.23 ± 0.2 vs Veh: 3.05 ± 0.8 arbitrary units, P < 0.05, N = 5/group). Furthermore, systemic PTX administration decreased c-Fos expression within the hypothalamic paraventricular nucleus (PTX: 17 ± 4 vs. Veh: 70 ± 13 cells, P < 0.01, N = 5, PVN) and increased the total number of microglial branches (PTX: 2129 ± 242 vs. Veh: 1415 ± 227 branches, P < 0.05, N = 4/group). Acute central injection of PTX (20 µg) under urethane anesthesia caused a small transient decrease in blood pressure but did not change renal sympathetic nerve activity. Surprisingly, we found no detectable basal levels of plasma TNF-α in either PTX- or vehicle-treated animals. These results suggest that inflammation plays a role in renovascular hypertension and that PTX might act both peripherally and centrally to prevent hypertension.

Transcriptome Analysis Reveals Downregulation of Urocortin Expression in the Hypothalamo-Neurohypophysial System of Spontaneously Hypertensive Rats.

The chronically increased blood pressure characteristic of essential hypertension represents an insidious and cumulative risk for cardiovascular disease. Essential hypertension is a multifactorial condition, with no known specific aetiology but a strong genetic component. The Spontaneously Hypertensive rat (SHR) shares many characteristics of human essential hypertension, and as such is a commonly used experimental model. The mammalian hypothalamo-neurohypophyseal system (HNS) plays a pivotal role in the regulation of blood pressure, volume and osmolality. In order to better understand the possible role of the HNS in hypertension, we have used microarray analysis to reveal differential regulation of genes in the HNS of the SHR compared to a control normotensive strain, the Wistar Kyoto rat (WKY). These results were validated by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). One of the genes identified and validated as being downregulated in SHR compared to WKY was that encoding the neuropeptide urocortin (Ucn). Immunohistochemical analyses revealed Ucn to be highly expressed within magnocellular neurons of the PVN and SON, with pronounced localisation in dendritic projections containing oxytocin and vasopressin. When Ucn was overexpressed in the PVN of the SHR by in vivo lentiviral mediated gene transfer, blood pressure was unaffected but there were significant, transient reductions in the VLF spectra of systolic blood pressure consistent with an action on autonomic balance. We suggest that Ucn may act, possibly via dendritic release, to subtly regulate neurohumoral aspects of arterial pressure control.

Chemoattraction and Recruitment of Activated Immune Cells, Central Autonomic Control, and Blood Pressure Regulation.

Inflammatory mediators play a critical role in the regulation of sympathetic outflow to cardiovascular organs in hypertension. Emerging evidence highlights the involvement of immune cells in the regulation of blood pressure. However, it is still unclear how these immune cells are activated and recruited to key autonomic brain regions to regulate sympathetic outflow to cardiovascular organs. Chemokines such as C-C motif chemokine ligand 2 (CCL2), and pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-α) and interleukin 1 beta (IL-1ß), are upregulated both peripherally and centrally in hypertension. More specifically, they are upregulated in key autonomic brain regions that control sympathetic activity and blood pressure such as the paraventricular nucleus of the hypothalamus and the rostral ventrolateral medulla. Furthermore, this upregulation of inflammatory mediators is associated with the infiltration of immune cells to these brain areas. Thus, expression of pro-inflammatory chemokines and cytokines is a potential mechanism promoting invasion of immune cells into key autonomic brain regions. In pathophysiological conditions, this can result in abnormal activation of brain circuits that control sympathetic nerve activity to cardiovascular organs and ultimately in increases in blood pressure. In this review, we discuss emerging evidence that helps explain how immune cells are chemoattracted to autonomic nuclei and contribute to changes in sympathetic outflow and blood pressure.

Stretchable Low Impedance Electrodes for Bioelectronic Recording from Small Peripheral Nerves.

Monitoring of bioelectric signals in peripheral sympathetic nerves of small animal models is crucial to gain understanding of how the autonomic nervous system controls specific body functions related to disease states. Advances in minimally-invasive electrodes for such recordings in chronic conditions rely on electrode materials that show low-impedance ionic/electronic interfaces and elastic mechanical properties compliant with the soft and fragile nerve strands. Here we report a highly stretchable low-impedance electrode realized by microcracked gold films as metallic conductors covered with stretchable conducting polymer composite to facilitate ion-to-electron exchange. The conducting polymer composite based on poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) obtains its adhesive, low-impedance properties by controlling thickness, plasticizer content and deposition conditions. Atomic Force Microscopy measurements under strain show that the optimized conducting polymer coating is compliant with the micro-crack mechanics of the underlying Au-layer, necessary to absorb the tensile deformation when the electrodes are stretched. We demonstrate functionality of the stretchable electrodes by performing high quality recordings of renal sympathetic nerve activity under chronic conditions in rats.

Blunted diuretic and natriuretic responses to acute sodium loading early after catheter-based renal denervation in normotensive sheep.

Catheter-based renal denervation (RDN) was introduced as a treatment for resistant hypertension. There remain critical questions regarding the physiological mechanisms underlying the hypotensive effects of catheter-based RDN. Previous studies indicate that surgical denervation reduces renin and the natriuretic response to saline loading; however, the effects on these variables of catheter-based RDN, which does not yield complete denervation, are largely unknown. The aim of this study was to investigate the effects of catheter-based RDN on glomerular-associated renin and regulation of fluid and sodium homeostasis in response to physiological challenges. First, immunohistochemical staining for renin was performed in normotensive sheep (n = 6) and sheep at 1 wk (n = 6), 5.5 mo (n = 5), and 11 mo (n = 5) after unilateral RDN using the same catheter used in patients (Symplicity). Following catheter-based RDN (1 wk), renin-positive glomeruli were significantly reduced compared with sham animals (P < 0.005). This was sustained until 5.5 mo postdenervation. To determine whether the reduction in renin after 1 wk had physiological effects, in a separate cohort, Merino ewes were administered high and low saline loads before and 1 wk after bilateral RDN (n = 9) or sham procedure (n = 8). After RDN (1 wk), the diuretic response to a low saline load was significantly reduced (P < 0.05), and both the diuretic and natriuretic responses to a high saline load were significantly attenuated (P < 0.05). In conclusion, these findings indicate that catheter-based RDN acutely alters the ability of the kidney to regulate fluid and electrolyte balance. Further studies are required to determine the long-term effects of catheter-based RDN on renal sodium and water homeostasis.

From sensory circumventricular organs to cerebral cortex: Neural pathways controlling thirst and hunger.

Much progress has been made during the past 30 years with respect to elucidating the neural and endocrine pathways by which bodily needs for water and energy are brought to conscious awareness through the generation of thirst and hunger. One way that circulating hormones influence thirst and hunger is by acting on neurones within sensory circumventricular organs (CVOs). This is possible because the subfornical organ and organum vasculosum of the lamina terminalis (OVLT), the sensory CVOs in the forebrain, and the area postrema in the hindbrain lack a normal blood-brain barrier such that neurones within them are exposed to blood-borne agents. The neural signals generated by hormonal action in these sensory CVOs are relayed to several sites in the cerebral cortex to stimulate or inhibit thirst or hunger. The subfornical organ and OVLT respond to circulating angiotensin II, relaxin and hypertonicity to drive thirst-related neural pathways, whereas circulating amylin, leptin and possibly glucagon-like peptide-1 act at the area postrema to influence neural pathways inhibiting food intake. As a result of investigations using functional brain imaging techniques, the insula and anterior cingulate cortex, as well as several other cortical sites, have been implicated in the conscious perception of thirst and hunger in humans. Viral tracing techniques show that the anterior cingulate cortex and insula receive neural inputs from thirst-related neurones in the subfornical organ and OVLT, with hunger-related neurones in the area postrema having polysynaptic efferent connections to these cortical regions. For thirst, initially, the median preoptic nucleus and, subsequently, the thalamic paraventricular nucleus and lateral hypothalamus have been identified as likely sites of synaptic links in pathways from the subfornical organ and OVLT to the cortex. The challenge remains to identify the links in the neural pathways that relay signals originating in sensory CVOs to cortical sites subserving either thirst or hunger.

Glycinergic neurotransmission in the rostral ventrolateral medulla controls the time course of baroreflex-mediated sympathoinhibition.

KEY POINTS: To maintain appropriate blood flow to various tissues of the body under a variety of physiological states, autonomic nervous system reflexes regulate regional sympathetic nerve activity and arterial blood pressure. Our data obtained in anaesthetized rats revealed that glycine released in the rostral ventrolateral medulla (RVLM) plays a critical role in maintaining arterial baroreflex sympathoinhibition. Manipulation of brainstem nuclei with known inputs to the RVLM (nucleus tractus solitarius and caudal VLM) unmasked tonic glycinergic inhibition in the RVLM. Whole-cell, patch clamp recordings demonstrate that both GABA and glycine inhibit RVLM neurons. Potentiation of neurotransmitter release from the active synaptic inputs in the RVLM produced saturation of GABAergic inhibition and emergence of glycinergic inhibition. Our data suggest that GABA controls threshold excitability, wherreas glycine increases the strength of inhibition under conditions of increased synaptic activity within the RVLM. ABSTRACT: The arterial baroreflex is a rapid negative-feedback system that compensates changes in blood pressure by adjusting the output of presympathetic neurons in the rostral ventrolateral medulla (RVLM). GABAergic projections from the caudal VLM (CVLM) provide a primary inhibitory input to presympathetic RVLM neurons. Although glycine-dependent regulation of RVLM neurons has been proposed, its role in determining RVLM excitability is ill-defined. The present study aimed to determine the physiological role of glycinergic neurotransmission in baroreflex function, identify the mechanisms for glycine release, and evaluate co-inhibition of RVLM neurons by GABA and glycine. Microinjection of the glycine receptor antagonist strychnine (4 mm, 100 nL) into the RVLM decreased the duration of baroreflex-mediated inhibition of renal sympathetic nerve activity (control = 12 ± 1 min; RVLM-strychnine = 5.1 ± 1 min), suggesting that RVLM glycine plays a critical role in regulating the time course of sympathoinhibition. Blockade of output from the nucleus tractus solitarius and/or disinhibition of the CVLM unmasked tonic glycinergic inhibition of the RVLM. To evaluate cellular mechanisms, RVLM neurons were retrogradely labelled (prior injection of pseudorabies virus PRV-152) and whole-cell, patch clamp recordings were obtained in brainstem slices. Under steady-state conditions GABAergic inhibition of RVLM neurons predominated and glycine contributed less than 25% of the overall inhibition. By contrast, stimulation of synaptic inputs in the RVLM decreased GABAergic inhibition to 53%; and increased glycinergic inhibition to 47%. Thus, under conditions of increased synaptic activity in the RVLM, glycinergic inhibition is recruited to strengthen sympathoinhibition.

In renovascular hypertension, TNF-α type-1 receptors in the area postrema mediate increases in cardiac and renal sympathetic nerve activity and blood pressure.

AIMS: Neuroinflammation is a common feature in renovascular, obesity-related, and angiotensin II mediated hypertension. There is evidence that increased release of the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α) contributes to the development of the hypertension, but the underlying neural mechanisms are unclear. Here, we investigated whether TNF-α stimulates neurons in the area postrema (AP), a circumventricular organ, to elicit sympathetic excitation, and increases in blood pressure (BP). METHODS AND RESULTS: In rats with renovascular hypertension, AP neurons that expressed TNF-α type-1 receptor (TNFR1) remained constantly activated (expressed c-Fos) and injection of TNFR1 neutralizing antibody into the AP returned BP (systolic: ∼151 mmHg) to normotensive levels (systolic: ∼108 mmHg). Nanoinjection of TNF-α (100 pg/50 nL) into the AP of anaesthetized normotensive rats increased BP (∼16 mmHg) and sympathetic nerve activity, predominantly to the heart (∼53%), but also to the kidneys (∼35%). These responses were abolished by prior injection of a TNFR1 neutralizing antibody (1 ng/50 nL) within the same site. TNFR1 were expressed in the somata of neurons activated by TNF-α that were retrogradely labelled from the rostral ventrolateral medulla (RVLM). CONCLUSION: These findings indicate that in renovascular hypertension, blocking TNFR1 receptors in the AP significantly reduces BP, while activation of TNFR1 expressing neurons in the AP by TNF-α increases BP in normotensive rats. This is mediated, in part, by projections to the RVLM and an increase in both cardiac and renal sympathetic nerve activity. These findings support the notion that proinflammatory cytokines and neuroinflammation are important pathological mechanisms in the development and maintenance of hypertension.

Anti-inflammatory reflex action of splanchnic sympathetic nerves is distributed across abdominal organs.

The splanchnic anti-inflammatory pathway has been proposed as the efferent arm of the inflammatory reflex. Although much evidence points to the spleen as the principal target organ where sympathetic nerves inhibit immune function, a systematic study to locate the target organ(s) of the splanchnic anti-inflammatory pathway has not yet been made. In anesthetized rats made endotoxemic with lipopolysaccharide (LPS, 60 µg/kg iv), plasma levels of tumor necrosis factor-α (TNF-α) were measured in animals with cut (SplancX) or sham-cut (Sham) splanchnic nerves. We confirm here that disengagement of the splanchnic anti-inflammatory pathway in SplancX rats (17.01 ± 0.95 ng/ml, mean ± SE) strongly enhances LPS-induced plasma TNF-α levels compared with Sham rats (3.76 ± 0.95 ng/ml). In paired experiments, the responses of SplancX and Sham animals were compared after the single or combined removal of organs innervated by the splanchnic nerves. Removal of target organ(s) where the splanchnic nerves inhibit systemic inflammation should abolish any difference in LPS-induced plasma TNF-α levels between Sham and SplancX rats. Any secondary effects of extirpating organs should apply to both groups. Surprisingly, removal of the spleen and/or the adrenal glands did not prevent the reflex splanchnic anti-inflammatory action nor did the following removals: spleen + adrenals + intestine; spleen + intestine + stomach and pancreas; or spleen + intestine + stomach and pancreas + liver. Only when spleen, adrenals, intestine, stomach, pancreas, and liver were all removed did the difference between SplancX and Sham animals disappear. We conclude that the reflex anti-inflammatory action of the splanchnic nerves is distributed widely across abdominal organs.

Increased cardiac sympathetic nerve activity in ovine heart failure is reduced by lesion of the area postrema, but not lamina terminalis.

Increased cardiac sympathetic nerve activity (CSNA) is a key feature of heart failure (HF) and is associated with poor outcome. There is evidence that central angiotensinergic mechanisms contribute to the increased CSNA in HF, but the central sites involved are unknown. In an ovine, rapid pacing model of HF, we investigated the contribution of the lamina terminalis and area postrema to the increased CSNA and also the responses to fourth ventricular infusion of the angiotensin type 1 receptor antagonist losartan. Ablation of the area postrema or sham lesion (n = 6/group), placement of lamina terminalis lesion electrodes (n = 5), and insertion of a cannula into the fourth ventricle (n = 6) were performed when ejection fraction was ~ 50%. When ejection fraction was < 40%, recording electrodes were implanted, and after 3 days, resting CSNA and baroreflex control of CSNA were measured before and following lesion of the lamina terminalis, in groups with lesion or sham lesion of the area postrema and before and following infusion of losartan (1.0 mg/h for 5 h) into the fourth ventricle. In conscious sheep with HF, lesion of the lamina terminalis did not significantly change CSNA (91 ± 2 vs. 86 ± 3 bursts/100 heart beats), whereas CSNA was reduced in the group with lesion of the area postrema (89 ± 3 to 45 ± 10 bursts/100 heart beats, P < 0.01) and following fourth ventricular infusion of losartan (89 ± 3 to 48 ± 8 bursts/100 heartbeats, P < 0.01). These findings indicate that the area postrema and brainstem angiotensinergic mechanisms may play an important role in determining the increased CSNA in HF.

The role of the blood-brain barrier in hypertension.

NEW FINDINGS: What is the topic of this review? This review highlights the importance of the blood-brain barrier in the context of diseases involving autonomic dysfunction, such as hypertension and heart failure. What advances does it highlight? It highlights the potential role of pro-inflammatory cytokines, leucocytes and angiotensin II in disrupting the blood-brain barrier in cardiovascular diseases. Advances are highlighted in our understanding of neurovascular unit cells, astrocytes and microglia, with a specific emphasis on their pathogenic roles within the brain. The blood-brain barrier (BBB) is a crucial barrier that provides both metabolic and physical protection to an immune-privileged CNS. The BBB has been shown to be disrupted in hypertension. This review addresses the importance of the BBB in maintaining homeostasis in the context of diseases related to autonomic dysfunction, such as hypertension. We highlight the potentially important roles of the immune system and neurovascular unit in the maintenance of the BBB, whereby dysregulation may lead to autonomic dysfunction in diseases such as heart failure and hypertension. Circulating leucocytes and factors such as angiotensin II and pro-inflammatory cytokines are thought ultimately to downregulate endothelial tight junction proteins that are a crucial component of the BBB. The specific mechanisms underlying BBB disruption and their role in contributing to autonomic dysfunction are not yet fully understood but are a growing area of interest. A greater understanding of these systems and advances in our knowledge of the molecular mechanisms causing BBB disruption will allow for the development of future therapeutic interventions in the treatment of autonomic imbalance associated with diseases such as heart failure and hypertension.

A single-chain derivative of the relaxin hormone is a functionally selective agonist of the G protein-coupled receptor, RXFP1.

Human gene-2 relaxin (H2 relaxin) is a pleiotropic hormone with powerful vasodilatory and anti-fibrotic properties which has led to its clinical evaluation and provisional FDA approval as a treatment for acute heart failure. The diverse effects of H2 relaxin are mediated via its cognate G protein coupled-receptor (GPCR), Relaxin Family Peptide Receptor (RXFP1), leading to stimulation of a combination of cell signalling pathways that includes cyclic adenosine monophosphate (cAMP) and extracellular-signal-regulated kinases (ERK)1/2. However, its complex two-chain (A and B), disulfide-rich insulin-like structure is a limitation to its facile preparation, availability and affordability. Furthermore, its strong activation of cAMP signaling is likely responsible for reported detrimental tumor-promoting actions that may preclude long-term use of this drug for treating human disease. Here we report the design and synthesis of a H2 relaxin B-chain-only analogue, B7-33, which was shown to bind to RXFP1 and preferentially activate the pERK pathway over cAMP in cells that endogenously expressed RXFP1. Thus, B7-33 represents the first functionally selective agonist of the complex GPCR, RXFP1. Importantly, this small peptide agonist prevented or reversed organ fibrosis and dysfunction in three pre-clinical rodent models of heart or lung disease with similar potency to H2 relaxin. The molecular mechanism behind the strong anti-fibrotic actions of B7-33 involved its activation of RXFP1-angiotensin II type 2 receptor heterodimers that induced selective downstream signaling of pERK1/2 and the collagen-degrading enzyme, matrix metalloproteinase (MMP)-2. Furthermore, in contrast to H2 relaxin, B7-33 did not promote prostate tumor growth in vivo. Our results represent the first known example of the minimisation of a two-chain cyclic insulin-like peptide to a single-chain linear peptide that retains potent beneficial agonistic effects.

The role of the renal afferent and efferent nerve fibers in heart failure.

Renal nerves contain afferent, sensory and efferent, sympathetic nerve fibers. In heart failure (HF) there is an increase in renal sympathetic nerve activity (RSNA), which can lead to renal vasoconstriction, increased renin release and sodium retention. These changes are thought to contribute to renal dysfunction, which is predictive of poor outcome in patients with HF. In contrast, the role of the renal afferent nerves remains largely unexplored in HF. This is somewhat surprising as there are multiple triggers in HF that have the potential to increase afferent nerve activity, including increased venous pressure and reduced kidney perfusion. Some of the few studies investigating renal afferents in HF have suggested that at least the sympatho-inhibitory reno-renal reflex is blunted. In experimentally induced HF, renal denervation, both surgical and catheter-based, has been associated with some improvements in renal and cardiac function. It remains unknown whether the effects are due to removal of the efferent renal nerve fibers or afferent renal nerve fibers, or a combination of both. Here, we review the effects of HF on renal efferent and afferent nerve function and critically assess the latest evidence supporting renal denervation as a potential treatment in HF.

Osmoregulation requires brain expression of the renal Na-K-2Cl cotransporter NKCC2.

The Na-K-2Cl cotransporter 2 (NKCC2) was thought to be kidney specific. Here we show expression in the brain hypothalamo-neurohypophyseal system (HNS), wherein upregulation follows osmotic stress. The HNS controls osmotic stability through the synthesis and release of the neuropeptide hormone, arginine vasopressin (AVP). AVP travels through the bloodstream to the kidney, where it promotes water conservation. Knockdown of HNS NKCC2 elicited profound effects on fluid balance following ingestion of a high-salt solution-rats produced significantly more urine, concomitant with increases in fluid intake and plasma osmolality. Since NKCC2 is the molecular target of the loop diuretics bumetanide and furosemide, we asked about their effects on HNS function following disturbed water balance. Dehydration-evoked GABA-mediated excitation of AVP neurons was reversed by bumetanide, and furosemide blocked AVP release, both in vivo and in hypothalamic explants. Thus, NKCC2-dependent brain mechanisms that regulate osmotic stability are disrupted by loop diuretics in rats.