Renal Deafferentation Prevents Progression of Hypertension and Changes to Sympathetic Reflexes in a Rabbit Model of Chronic Kidney Disease.

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Leptin and Melanocortin Signaling Mediates Hypertension in Offspring From Female Rabbits Fed a High-Fat Diet During Gestation and Lactation.

Maternal high-fat diet in rabbits leads to hypertension and elevated renal sympathetic nerve activity (RSNA) in adult offspring but whether this is due to adiposity or maternal programming is unclear. We gave intracerebroventricular (ICV) and ventromedial hypothalamus (VMH) administration of leptin-receptor antagonist, α-melanocyte-stimulating hormone (αMSH), melanocortin-receptor antagonist (SHU9119), or insulin-receptor (InsR) antagonist to conscious adult offspring from mothers fed a high-fat diet (mHFD), control diet (mCD), or mCD offspring fed HFD for 10d (mCD10d, to deposit equivalent fat but not during development). mHFD and mCD10d rabbits had higher mean arterial pressure (MAP, +6.4 mmHg, +12.1 mmHg, p < 0.001) and RSNA (+2.3 nu, +3.2 nu, p < 0.01) than mCD, but all had similar plasma leptin. VMH leptin-receptor antagonist reduced MAP (-8.0 ± 3.0 mmHg, p < 0.001) in mCD10d but not in mHFD or mCD group. Intracerebroventricular leptin-receptor antagonist reduced MAP only in mHFD rabbits (p < 0.05). Intracerebroventricular SHU9119 reduced MAP and RSNA in mHFD but only reduced MAP in the mCD10d group. VMH αMSH increased RSNA (+85%, p < 0.001) in mHFD rabbits but ICV αMSH increased RSNA in both mHFD and mCD10d rabbits (+45%, +51%, respectively, p < 0.001). The InsR antagonist had no effect by either route on MAP or RSNA. Hypothalamic leptin receptor and brain-derived neurotrophic factor (BDNF) mRNA were greater in mHFD compared with mCD rabbits and mCD10d rabbits. In conclusion, the higher MAP in mHFD and mCD10d offspring was likely due to greater central leptin signaling at distinct sites within the hypothalamus while enhanced melanocortin contribution was common to both groups suggesting that residual body fat was mainly responsible. However, the effects of SHU9119 and αMSH on RSNA pathways only in mHFD suggest a maternal HFD may program sympatho-excitatory capacity in these offspring and that this may involve increased leptin receptor and BDNF expression.

Role of Mineralocorticoid and Angiotensin Type 1 Receptors in the Paraventricular Nucleus in Angiotensin-Induced Hypertension.

The hypothalamic paraventricular nucleus (PVN) is an important site where an interaction between circulating angiotensin (Ang) and mineralocorticoid receptor (MR) activity may modify sympathetic nerve activity (SNA) to influence long-term elevation of blood pressure. We examined in conscious Ang II-treated rabbits, the effects on blood pressure and tonic and reflex renal SNA (RSNA) of microinjecting into the PVN either RU28318 to block MR, losartan to block Ang (AT1) receptors or muscimol to inhibit GABA A receptor agonist actions. Male rabbits received a moderate dose of Ang II (24 ng/kg/min subcutaneously) for 3 months (n = 13) or sham treatment (n = 13). At 3 months, blood pressure increased by +19% in the Ang II group compared to 10% in the sham (P = 0.022) but RSNA was similar. RU28318 lowered blood pressure in both Ang II and shams but had a greater effect on RSNA and heart rate in the Ang II-treated group (P < 0.05). Losartan also lowered RSNA, while muscimol produced sympatho-excitation in both groups. In Ang II-treated rabbits, RU28318 attenuated the blood pressure increase following chemoreceptor stimulation but did not affect responses to air jet stress. In contrast losartan and muscimol reduced blood pressure and RSNA responses to both hypoxia and air jet. While neither RU28318 nor losartan changed the RSNA baroreflex, RU28318 augmented the range of the heart rate baroreflex by 10% in Ang II-treated rabbits. Muscimol, however, augmented the RSNA baroreflex by 11% in sham animals and none of the treatments altered baroreflex sensitivity. In conclusion, 3 months of moderate Ang II treatment promotes activation of reflex RSNA principally via MR activation in the PVN, rather than via activation of AT1 receptors. However, the onset of hypertension is independent of both. Interestingly, the sympatho-excitatory effects of muscimol in both groups suggest that overall, the PVN regulates a tonic sympatho-inhibitory influence on blood pressure control.

Contribution of the Renal Nerves to Hypertension in a Rabbit Model of Chronic Kidney Disease.

Overactivity of the sympathetic nervous system and high blood pressure are implicated in the development and progression of chronic kidney disease (CKD) and independently predict cardiovascular events in end-stage renal disease. To assess the role of renal nerves, we determined whether renal denervation (RDN) altered the hypertension and sympathoexcitation associated with a rabbit model of CKD. The model involves glomerular layer lesioning and uninephrectomy, resulting in renal function reduced by one-third and diuresis. After 3-week CKD, blood pressure was 13±2 mm Hg higher than at baseline (P<0.001), and compared with sham control rabbits, renal sympathetic nerve activity was 1.2±0.5 normalized units greater (P=0.01). The depressor response to ganglion blockade was also +8.0±3 mm Hg greater, but total norepinephrine spillover was 8.7±3.7 ng/min lower (both P<0.05). RDN CKD rabbits only increased blood pressure by 8.0±1.5 mm Hg. Renal sympathetic activity, the response to ganglion blockade and diuresis were similar to sham denervated rabbits (non-CKD). CKD rabbits had intact renal sympathetic baroreflex gain and range, as well as normal sympathetic responses to airjet stress. However, hypoxia-induced sympathoexcitation was reduced by -9±0.4 normalized units. RDN did not alter the sympathetic response to hypoxia or airjet stress. CKD increased oxidative stress markers Nox5 and MCP-1 (monocyte chemoattractant protein-1) in the kidney, but RDN had no effect on these measures. Thus, RDN is an effective treatment for hypertension in this model of CKD without further impairing renal function or altering the normal sympathetic reflex responses to various environmental stimuli.

Differential sympathetic response to lesion-induced chronic kidney disease in rabbits.

Chronic kidney disease (CKD) is associated with greater sympathetic nerve activity but it is unclear if this is a kidney-specific response or due to generalized stimulation of sympathetic nervous system activity. To determine this, we used a rabbit model of CKD in which quantitative comparisons with control rabbits could be made of kidney sympathetic nerve activity and whole-body norepinephrine spillover. Rabbits either had surgery to lesion 5/6th of the cortex of one kidney by electro-lesioning and two weeks later removal of the contralateral kidney, or sham lesioning and sham nephrectomy. After three weeks, the blood pressure was statistically significantly 20% higher in conscious rabbits with CKD compared to rabbits with a sham operation, but their heart rate was similar. Strikingly, kidney nerve activity was 37% greater than in controls, with greater burst height and frequency. Total norepinephrine spillover was statistically significantly lower by 34%, and kidney baroreflex curves were shifted to the right in rabbits with CKD. Plasma creatinine and urine output were elevated by 38% and 131%, respectively, and the glomerular filtration rate was 37% lower than in sham-operated animals (all statistically significant). Kidney gene expression of fibronectin, transforming growth factor-ß, monocyte chemotactic protein1, Nox4 and Nox5 was two- to eight-fold greater in rabbits with CKD than in control rabbits. Overall, the glomerular layer lesioning model in conscious rabbits produced a moderate, stable degree of CKD characterized by elevated blood pressure and increased kidney sympathetic nerve activity. Thus, our findings, together with that of a reduction in total norepinephrine spillover, suggest that kidney denervation, rather than generalized sympatholytic treatments, may represent a preferable management for CKD associated hypertension.

Empagliflozin modulates renal sympathetic and heart rate baroreflexes in a rabbit model of diabetes.

AIMS/HYPOTHESIS: We determined whether empagliflozin altered renal sympathetic nerve activity (RSNA) and baroreflexes in a diabetes model in conscious rabbits. METHODS: Diabetes was induced by alloxan, and RSNA, mean arterial pressure (MAP) and heart rate were measured before and after 1 week of treatment with empagliflozin, insulin, the diuretic acetazolamide or the ACE inhibitor perindopril, or no treatment, in conscious rabbits. RESULTS: Four weeks after alloxan administration, blood glucose was threefold and MAP 9% higher than non-diabetic controls (p < 0.05). One week of treatment with empagliflozin produced a stable fall in blood glucose (-43%) and increased water intake (+49%) but did not change RSNA, MAP or heart rate compared with untreated diabetic rabbits. The maximum RSNA to hypotension was augmented by 75% (p < 0.01) in diabetic rabbits but the heart rate baroreflex was unaltered. Empagliflozin and acetazolamide reduced the augmentation of the RSNA baroreflex (p < 0.05) to be similar to the non-diabetic group. Noradrenaline (norepinephrine) spillover was similar in untreated diabetic and non-diabetic rabbits but twofold greater in empagliflozin- and acetazolamide-treated rabbits (p < 0.05). CONCLUSIONS/INTERPRETATION: As empagliflozin can restore diabetes-induced augmented sympathetic reflexes, this may be beneficial in diabetic patients. A similar action of the diuretic acetazolamide suggests that the mechanism may involve increased sodium and water excretion. Graphical abstract.

Neural suppression of miRNA-181a in the kidney elevates renin expression and exacerbates hypertension in Schlager mice.

BPH/2J mice are a genetic model of hypertension with overactivity of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS). BPH/2J display higher renal renin mRNA and low levels of its negative regulator microRNA-181a (miR-181a). We hypothesise that high renal SNS activity may reduce miR-181a expression, which contributes to elevated RAS activity and hypertension in BPH/2J. Our aim was to determine whether in vivo administration of a renal-specific miR-181a mimic or whether renal denervation could increase renal miR-181a abundance to reduce renal renin mRNA, RAS activity and hypertension in BPH/2J mice. Blood pressure (BP) in BPH/2J and normotensive BPN/3J mice was measured via radiotelemetry probes. Mice were administered miR-181a mimic or a negative control (1-25 nmol, i.v., n = 6-10) with BP measured for 48 h after each dose or they underwent renal denervation or sham surgery (n = 7-9). Injection of 5-25 nmol miR-181a mimic reduced BP in BPH/2J mice after 36-48 h (-5.3 ± 1.8, -6.1 ± 1.9 mmHg, respectively, P < 0.016). Treatment resulted in lower renal renin and inflammatory marker (TLR4) mRNA levels in BPH/2J. The mimic abolished the hypotensive effect of blocking the RAS with enalaprilat (P < 0.01). No differences between mimic or vehicle were observed in BPN/3J mice except for a higher level of renal angiotensinogen in the mimic-treated mice. Renal miR-181a levels that were lower in sham BPH/2J mice were greater following renal denervation and were thus similar to those of BPN/3J. Our findings suggest that the reduced renal miR-181a may partially contribute to the elevated BP in BPH/2J mice, through an interaction between the renal sympathetic nerves and miR-181a regulation of the RAS.

Effect of the novel histamine H4 receptor antagonist SENS-111 on spontaneous nystagmus in a rat model of acute unilateral vestibular loss.

BACKGROUND AND PURPOSE: Histamine H4 receptors are expressed in the peripheral vestibular system, and their selective inhibition improves vertigo symptoms in rats with unilateral vestibular lesions. The effects of SENS-111, a selective oral H4 receptor antagonist with high affinity to both animal and human receptors, on vertigo symptoms was evaluated in a translational in vivo model of unilateral vestibular loss. EXPERIMENTAL APPROACH: Pharmacokinetics of SENS-111 in rats was determined to aid dose selection for efficacy testing. Vestibular lesions were induced in rats by unilateral transtympanic injection of kainic acid. The effect of SENS-111 (10 or 20 mg·kg-1 ) on spontaneous nystagmus was evaluated compared with placebo vehicle using video-nystagmography, and the effective dose was compared with those of similar drugs used clinically, as single agents or combined with SENS-111. KEY RESULTS: Doses were selected for plasma exposure were consistent with published phase 1 results from healthy volunteers. SENS-111 of 10 mg·kg-1 gave a 21-22% reduction in nystagmus at 1 hr post-administration, whereas a loss of efficacy was seen with 20 mg·kg-1 . Compared with SENS-111, meclizine and methylprednisolone had minimal effects on nystagmus as single agents, and meclizine abolished the effect of SENS-111 when combined with SENS-111. All evaluated drugs were well tolerated. CONCLUSIONS AND IMPLICATIONS: The exposure-efficacy relationship for improved spontaneous nystagmus seen with SENS-111 in this in vivo model is consistent with phase 1 clinical results and provides preclinical support for pharmacokinetic/pharmacodynamic modelling and selection of effective clinical drug concentrations. LINKED ARTICLES: This article is part of a themed section on New Uses for 21st Century. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v177.3/issuetoc.

Mechanisms Responsible for Genetic Hypertension in Schlager BPH/2 Mice.

It has been 45 years since Gunther Schlager used a cross breeding program in mice to develop inbred strains with high, normal, and low blood pressure (BPH/2, BPN/3, and BPL/1 respectively). Thus, it is timely to gather together the studies that have characterized and explored the mechanisms associated with the hypertension to take stock of exactly what is known and what remains to be determined. Growing evidence supports the notion that the mechanism of hypertension in BPH/2 mice is predominantly neurogenic with some of the early studies showing aberrant brain noradrenaline levels in BPH/2 compared with BPN/3. Analysis of the adrenal gland using microarray suggested an association with the activity of the sympathetic nervous system. Indeed, in support of this, there is a larger depressor response to ganglion blockade, which reduced blood pressure in BPH/2 mice to the same level as BPN/3 mice. Greater renal tyrosine hydroxylase staining and greater renal noradrenaline levels in BPH/2 mice suggest sympathetic hyperinnervation of the kidney. Renal denervation markedly reduced the blood pressure in BPH/2 but not BPN/3 mice, confirming the importance of renal sympathetic nervous activity contributing to the hypertension. Further, there is an important contribution to the hypertension from miR-181a and renal renin in this strain. BPH/2 mice also display greater neuronal activity of amygdalo-hypothalamic cardiovascular regulatory regions. Lesions of the medial nucleus of the amygdala reduced the hypertension in BPH/2 mice and abolished the strain difference in the effect of ganglion blockade, suggesting a sympathetic mechanism. Further studies suggest that aberrant GABAergic inhibition may play a role since BPH/2 mice have low GABAA receptor δ, α4 and ß2 subunit mRNA expression in the hypothalamus, which are predominantly involved in promoting tonic neuronal inhibition. Allopregnanolone, an allosteric modulator of GABAA receptors, which increase the expression of these subunits in the amygdala and hypothalamus, is shown to reduce the hypertension and sympathetic nervous system contribution in BPH/2 mice. Thus far, evidence suggests that BPH/2 mice have aberrant GABAergic inhibition, which drives neuronal overactivity within amygdalo-hypothalamic brain regions. This overactivity is responsible for the greater sympathetic contribution to the hypertension in BPH/2 mice, thus making this an ideal model of neurogenic hypertension.

Hsp22 overexpression induces myocardial hypertrophy, senescence and reduced life span through enhanced oxidative stress.

H11 kinase/Hsp22 (Hsp22) is a small heat shock protein, which, when overexpressed cardiac specifically in transgenic (TG) mice, induces stable left ventricular (LV) hypertrophy. Hsp22 also increases oxidative phosphorylation and mitochondrial reactive oxygen species (ROS) production, mechanisms mediating LV hypertrophy, senescence and reduced lifespan. Therefore, we investigated whether ROS production mediates LV hypertrophy, senescence and reduced life span in Hsp22 TG mice. Survival curves revealed that TG mice had a 48% reduction in their mean life span compared to wild type (WT) mice. This was associated with a significant increase in senescence markers, such as p16, p19 mRNA levels as well as the percentage of ß-galactosidase positive cells and telomerase activity. Oxidized (GSSG)/reduced (GSH) glutathione ratio, an indicator of oxidative stress, and ROS production from 3 major cellular sources was measured in cardiac tissue. Hearts from TG mice exhibited a decrease in GSH/GSSG ratio together with increased ROS production from all sources. To study the role of ROS, mice were treated with the antioxidant Tempol from weaning to their sacrifice. Chronic Tempol treatment abolished oxidative stress and overproduction of ROS, and reduced myocardial hypertrophy and Akt phosphorylation in TG mice. Tempol also significantly extended life span and prevented aging markers in TG mice. Taken together these results show that overexpression of Hsp22 increases oxidative stress responsible for the induction of hypertrophy and senescence and ultimately reduction in life span.

Potential Therapeutic Use of Neurosteroids for Hypertension.

The sympathetic nervous system (SNS) contribution to long-term setting of blood pressure (BP) and hence hypertension has been a continuing controversy over many decades. However, the contribution of increased sympathetic vasomotor tone to the heart, kidney, and blood vessels has been suggested as a major influence on the development of high BP which affects 30-40% of the population. This is relevant to hypertension associated with chronic stress, being overweight or obese as well to chronic kidney disease. Treatments that have attempted to block the peripheral aspects of the SNS contribution have included surgery to cut the sympathetic nerves as well as agents to block α- and ß-adrenoceptors. Other treatments, such as centrally acting drugs like clonidine, rilmenidine, or moxonidine, activate receptors within the ventrolateral medulla to reduce the vasomotor tone overall but have side effects that limit their use. None of these treatments target the cause of the enhanced sympathetic tone. Recently we have identified an antihypertensive action of the neurosteroid allopregnanolone in a mouse model of neurogenic hypertension. Allopregnanolone is known to facilitate high-affinity extra-synaptic γ-aminobutyric acid A receptors (GABAAR) through allosteric modulation and transcriptional upregulation. The antihypertensive effect was specific for increased expression of δ subunits in the amygdala and hypothalamus. This focused review examines the possibility that neurosteroids may be a novel therapeutic approach to address the neurogenic contribution to hypertension. We discuss the causes and prevalence of neurogenic hypertension, current therapeutic approaches, and the applicability of using neurosteroids as antihypertensive therapy.

Renal nerves contribute to hypertension in Schlager BPH/2J mice.

Schlager mice (BPH/2J) are hypertensive due to a greater contribution of the sympathetic nervous system (SNS) and renin-angiotensin system (RAS). The kidneys of BPH/2J are hyper-innervated suggesting renal nerves may contribute to the hypertension. We therefore determined the effect of bilateral renal denervation (RD) on hypertension in BPH/2J. Mean arterial pressure (MAP) was measured by radiotelemetry before and for 3 weeks after RD in BPH/2J and BPN/3J. The effects of pentolinium and enalaprilat were examined to determine the contribution of the SNS and RAS, respectively. After 3 weeks, MAP was -10.9 ± 2.1 mmHg lower in RD BPH/2J compared to baseline and -2.1 ± 2.2 mmHg in sham BPH/2J (P < 0.001, n = 8-10). RD had no effect in BPN/3J (P > 0.1). The depressor response to pentolinium was greater in BPH/2J than BPN/3J, but in both cases the response in RD mice was similar to sham. Enalaprilat decreased MAP more in RD BPH/2J compared to sham (-12 vs -3 mmHg, P < 0.001) but had no effect in BPN/3J. RD reduced renal noradrenaline in both strains but more so in BPH/2J. RD reduced renin mRNA and protein, but not plasma renin in BPH/2J to levels comparable with BPN/3J mice. We conclude that renal nerves contribute to hypertension in BPH mice as RD induced a sustained fall in MAP, which was associated with a reduction of intrarenal renin expression. The lack of inhibition of the depressor effects of pentolinium and enalaprilat by RD suggests that vasoconstrictor effects of the SNS or RAS are not involved.

Neurological and histological consequences induced by in vivo cerebral oxidative stress: evidence for beneficial effects of SRT1720, a sirtuin 1 activator, and sirtuin 1-mediated neuroprotective effects of poly(ADP-ribose) polymerase inhibition.

Poly(ADP-ribose)polymerase and sirtuin 1 are both NAD(+)-dependent enzymes. In vitro oxidative stress activates poly(ADP-ribose)polymerase, decreases NAD(+) level, sirtuin 1 activity and finally leads to cell death. Poly(ADP-ribose)polymerase hyperactivation contributes to cell death. In addition, poly(ADP-ribose)polymerase inhibition restores NAD(+) level and sirtuin 1 activity in vitro. In vitro sirtuin 1 induction protects neurons from cell loss induced by oxidative stress. In this context, the role of sirtuin 1 and its involvement in beneficial effects of poly(ADP-ribose)polymerase inhibition were evaluated in vivo in a model of cerebral oxidative stress induced by intrastriatal infusion of malonate in rat. Malonate promoted a NAD(+) decrease that was not prevented by 3-aminobenzamide, a poly(ADP-ribose)polymerase inhibitor, at 4 and 24 hours. However, 3-aminobenzamide increased nuclear SIRT1 activity/expression ratio after oxidative stress. Malonate induced a neurological deficit associated with a striatal lesion. Both were reduced by 3-aminobenzamide and SRT1720, a sirtuin 1 activator, showing beneficial effects of poly(ADP-ribose)polymerase inhibition and sirtuin 1 activation on oxidative stress consequences. EX527, a sirtuin 1 inhibitor, given alone, modified neither the score nor the lesion, suggesting that endogenous sirtuin 1 was not activated during cerebral oxidative stress. However, its association with 3-aminobenzamide suppressed the neurological improvement and the lesion reduction induced by 3-aminobenzamide. The association of 3-aminobenzamide with SRT1720, the sirtuin 1 activator, did not lead to a better protection than 3-aminobenzamide alone. The present data represent the first demonstration that the sirtuin 1 activator SRT1720 is neuroprotective during in vivo cerebral oxidative stress. Furthermore sirtuin 1 activation is involved in the beneficial effects of poly(ADP-ribose)polymerase inhibition after in vivo cerebral oxidative stress.