Renal and hypothalamic inflammation in renovascular hypertension: role of afferent renal nerves.

Renal denervation (RDN) is a potential therapy for drug-resistant hypertension. However, whether its effects are mediated by ablation of efferent or afferent renal nerves is not clear. Previous studies have implicated that renal inflammation and the sympathetic nervous system are driven by the activation of afferent and efferent renal nerves. RDN attenuated the renal inflammation and sympathetic activity in some animal models of hypertension. In the 2 kidney,1 clip (2K1C) model of renovascular hypertension, RDN also decreased sympathetic activity; however, mechanisms underlying renal and central inflammation are still unclear. We tested the hypothesis that the mechanisms by which total RDN (TRDN; efferent + afferent) and afferent-specific RDN (ARDN) reduce arterial pressure in 2K1C rats are the same. Male Sprague-Dawley rats were instrumented with telemeters to measure mean arterial pressure (MAP), and after 7 days, a clip was placed on the left renal artery. Rats underwent TRDN, ARDN, or sham surgery of the clipped kidney and MAP was measured for 6 wk. Weekly measurements of water intake (WI), urine output (UO), and urinary copeptin were conducted, and urine was analyzed for cytokines/chemokines. Neurogenic pressor activity (NPA) was assessed at the end of the protocol calculated by the depressor response after intraperitoneal injection of hexamethonium. Rats were euthanized and the hypothalamus and kidneys removed for measurement of cytokine content. MAP, NPA, WI, and urinary copeptin were significantly increased in 2K1C-sham rats, and these responses were abolished by both TRDN and ARDN. 2K1C-sham rats presented with renal and hypothalamic inflammation and these responses were largely mitigated by TRDN and ARDN. We conclude that RDN attenuates 2K1C hypertension primarily by ablation of afferent renal nerves which disrupts bidirectional renal neural-immune pathways.NEW & NOTEWORTHY Hypertension resulting from reduced perfusion of the kidney is dependent on renal sensory nerves, which are linked to inflammation in the kidney and hypothalamus. Afferent renal nerves are required for chronic increases in both water intake and vasopressin release observed following renal artery stenosis. Findings from this study suggest an important role of renal sensory nerves that has previously been underestimated in the pathogenesis of 2K1C hypertension.

Precision Targeted Ablation of Fine Neurovascular Structures In Vivo Using Dual-mode Ultrasound Arrays.

Carotid bodies (CBs) are chemoreceptors that monitor and register changes in the blood, including the levels of oxygen, carbon dioxide, and pH, and regulate breathing. Enhanced activity of CBs was shown to correlate with a significant elevation in the blood pressure of patients with hypertension. CB removal or denervation were previously shown to reduce hypertension. Here we demonstrate the feasibility of a dual-mode ultrasound array (DMUA) system to safely ablate the CB in vivo in a spontaneously hypertensive rat (SHR) model of hypertension. DMUA imaging was used for guiding and monitoring focused ultrasound (FUS) energy delivered to the target region. In particular, 3D imaging was used to identify the carotid bifurcation for targeting the CBs. Intermittent, high frame rate imaging during image-guided FUS (IgFUS) delivery was used for monitoring the lesion formation. DMUA imaging provided feedback for closed-loop control (CLC) of the lesion formation process to avoid overexposure. The procedure was tolerated well in over 100 SHR and normotensive rats that received unilateral and bilateral treatments. The measured mean arterial pressure (MAP) exhibited measurable deviation from baseline 2-4 weeks post IgFUS treatment. The results suggest that the direct unilateral FUS treatment of the CB might be sufficient to reduce the blood pressure in hypertensive rats and justify further investigation in large animals and eventually in human patients.

Renal Inflammation in DOCA-Salt Hypertension.

Recent reports indicate that, in addition to treating hypertension, renal denervation (RDN) also mitigates renal inflammation. However, because RDN decreases renal perfusion pressure, it is unclear whether these effects are because of the direct effects of RDN on inflammatory signaling or secondary to decreased arterial pressure (AP). Therefore, this study was conducted to elucidate the contribution of renal nerves to renal inflammation in the deoxycorticosterone (DOCA)-salt rat, a model in which RDN decreases AP and abolishes renal inflammation. In Experiment 1, we assessed the temporal changes in renal inflammation by measuring renal cytokines and AP in DOCA-salt rats. Uninephrectomized (1K) adult male Sprague Dawley rats that received surgical RDN or sham (Sham) were administered DOCA (100 mg, SC) and 0.9% saline for 21 days. AP was measured by radiotelemetry, and urinary cytokine excretion was measured repeatedly. In Experiment 2, the contribution of renal nerves in renal inflammation was assessed in a 2-kidney DOCA-salt rat to control for renal perfusion pressure. DOCA-salt treatment was administered after unilateral (U-)RDN. In Experiment 1, DOCA-salt-induced increases in AP and renal inflammation (assessed by urinary cytokines) were attenuated by RDN versus Sham. In Experiment 2, GRO/KC (growth-related oncogene/keratinocyte chemoattractant), MCP (monocyte chemoattractant protein)-1, and macrophage infiltration were lower in the denervated kidney versus the contralateral Sham kidney. No differences in T-cell infiltration were observed. Together, these data support the hypothesis that renal nerves mediate, in part, the development of renal inflammation in the DOCA-salt rat independent of hypertension. The mechanisms and cell-specificity mediating these effects require further investigation.

Renal Denervation Normalizes Arterial Pressure With No Effect on Glucose Metabolism or Renal Inflammation in Obese Hypertensive Mice.

Hypertension often occurs in concurrence with obesity and diabetes mellitus, commonly referred to as metabolic syndrome. Renal denervation (RDNx) lowers arterial pressure (AP) and improves glucose metabolism in drug-resistant hypertensive patients with high body mass index. In addition, RDNx has been shown to reduce renal inflammation in the mouse model of angiotensin II hypertension. The present study tested the hypothesis that RDNx reduces AP and renal inflammation and improves glucose metabolism in obesity-induced hypertension. Eight-week-old C57BL/6J mice were fed either a low-fat diet (10 kcal%) or a high-fat diet (45 kcal%) for 10 weeks. Body weight, food intake, fasting blood glucose, and glucose metabolism (glucose tolerance test) were measured. In a parallel study, radiotelemeters were implanted in mice for AP measurement. High fat-fed C57BL/6J mice exhibited an inflammatory and metabolic syndrome phenotype, including increased fat mass, increased AP, and hyperglycemia compared with low-fat diet mice. RDNx, but not Sham surgery, normalized AP in high-fat diet mice (115.8±1.5 mm Hg in sham versus 96.6±6.7 mm Hg in RDNx). RDNx had no significant effect on AP in low-fat diet mice. Also, RDNx had no significant effect on glucose metabolism or renal inflammation as measured by the number of CD8, CD4, and T helper cells or levels of inflammatory cytokines in the kidneys. These results indicate that although renal nerves play a role in obesity-induced hypertension, they do not contribute to impaired glucose metabolism or renal inflammation in this model.

Differential role of afferent and efferent renal nerves in the maintenance of early- and late-phase Dahl S hypertension.

Clinical data suggest that renal denervation (RDNX) may be an effective treatment for human hypertension; however, it is unclear whether this therapeutic effect is due to ablation of afferent or efferent renal nerves. We have previously shown that RDNX lowers arterial pressure in hypertensive Dahl salt-sensitive (S) rats to a similar degree observed in clinical trials. In addition, we have recently developed a method for selective ablation of afferent renal nerves (renal-CAP). In the present study, we tested the hypothesis that the antihypertensive effect of RDNX in the Dahl S rat is due to ablation of afferent renal nerves by comparing the effect of complete RDNX to renal-CAP during two phases of hypertension in the Dahl S rat. In the early phase, rats underwent treatment after 3 wk of high-NaCl feeding when mean arterial pressure (MAP) was ∼ 140 mmHg. In the late phase, rats underwent treatment after 9 wk of high NaCl feeding, when MAP was ∼ 170 mmHg. RDNX reduced MAP ∼ 10 mmHg compared with sham surgery in both the early and late phase, whereas renal-CAP had no antihypertensive effect. These results suggest that, in the Dahl S rat, the antihypertensive effect of RDNX is not dependent on pretreatment arterial pressure, nor is it due to ablation of afferent renal nerves.

Inhibition of dipeptidyl peptidase 8/9 impairs preadipocyte differentiation.

Adipocytes are the primary cells in adipose tissue, and adipocyte dysfunction causes lipodystrophy, obesity and diabetes. The dipeptidyl peptidase (DPP) 4 family includes four enzymes, DPP4, DPP8, DPP9 and fibroblast activation protein (FAP). DPP4 family inhibitors have been used for the treatment of type 2 diabetes patients, but their role in adipocyte formation are poorly understood. Here we demonstrate that the DPP8/9 selective inhibitor 1G244 blocks adipogenesis in preadipocyte 3T3-L1 and 3T3-F422A, while DPP4 and FAP inhibitors have no effect. In addition, knockdown of DPP8 or DPP9 significantly impairs adipocyte differentiation in preadipocytes. We further uncovered that blocking the expression or activities of DPP8 and DPP9 attenuates PPARγ2 induction during preadipocyte differentiation. Addition of PPARγ agonist thiazolidinediones (TZDs), or ectopic expression of PPARγ2, is able to rescue the adipogenic defect caused by DPP8/9 inhibition in preadipocytes. These results indicate the importance of DPP8 and DPP9 on adipogenesis.

Role of cardiac sympathetic nerves in blood pressure regulation.

Stellate ganglionectomy (SGx) was used to assess the contribution of cardiac sympathetic nerves to neurogenic hypertension in deoxycorticosterone (DOCA)-salt treated rats. Experiments were conducted in two substrains of Sprague-Dawley (SD) rats since previous studies reported bradycardia in Charles River-SD (CR-SD) rats and tachycardia in SASCO-SD (SA-SD) rats with DOCA treatment suggesting different underlying neural mechanisms. Uninephrectomized male rats underwent SGx or SHAM surgery and were instrumented for telemetric monitoring of mean arterial pressure (MAP) and heart rate (HR). After recovery, 0.9% saline solution and DOCA (50mg) were administered. Baseline MAP (Days 0-5 average) after SGx in CR-SD rats (96±2mmHg; n=7) was not significantly different (p=0.08) than CR-SD SHAM rats (103±3mmHg; n=9); however, there was a significantly lower HR during the baseline period (377±7 vs. 432±7bpm, p<0.05) in SGx rats. In SA-SD rats baseline MAP was not different between SGx and SHAM rats and HR was lower in SGx rats (428±8 vs. 371±5bpm, p<0.05). After DOCA treatment in both substrains, MAP and HR were elevated similarly in SHAM and SGx groups showing minimal impact in both groups of SGx on hypertension development. However, overall MAP in SA-SD SHAM rats reached a significantly higher level (155±10mmHg vs 135±5mmHg, p<0.05) than that observed in CR-SD SHAM rats demonstrating that the magnitude of hypertensive response to DOCA-salt treatment varies between substrains. In conclusion, removal of cardiac sympathetic nerves did not alter the development or maintenance of DOCA-salt hypertension in SD rats.

OVLT lesion decreases basal arterial pressure and the chronic hypertensive response to AngII in rats on a high-salt diet.

We have reported that lesion of the organum vasculosum of the lamina terminalis (OVLT) has no effect on basal levels of mean arterial pressure (MAP) but abolishes the hypertensive effects of angiotensin II (AngII) in rats consuming a normal-salt diet. These results suggest that the OVLT does not contribute to regulation of MAP under conditions of normal salt intake, but it is an important brain site for the hypertensive actions of AngII. The OVLT has been proposed as a major sodium sensor in the brain and the hypertensive effects of AngII are exacerbated by high-salt intake. Therefore, the objective of this study was to investigate the role of the OVLT during AngII-induced hypertension in rats fed a high-salt diet. Male Sprague-Dawley rats underwent sham (Sham; n = 9) or OVLT lesion (OVLTx; n = 8) surgery and were placed on a high-salt (2% NaCl) diet. MAP was measured by radio telemetry during three control days, 10 days of AngII infusion (10 ng/kg/min, i.v.), and a 3-day recovery period. MAP was significantly lower in OVLTx (97 ± 2 mmHg) compared to Sham (106 ± 1 mmHg) rats during the control period (P < 0.05). Moreover, the chronic pressor response to AngII was markedly attenuated in OVLTx rats. MAP increased 58 ± 3 mmHg in Sham rats by Day 10 of AngII compared to a 40 ± 7 mmHg increase in OVLTx rats (P < 0.05). We conclude that (1) the OVLT regulates the basal levels of MAP in rats consuming a high-salt and (2) the OVLT is an important brain site of action for the pathogenesis of AngII-salt hypertension in the rat. Supported by HL076312.

Reversal of genetic salt-sensitive hypertension by targeted sympathetic ablation.

The sympathetic nervous system plays an important role in some forms of human hypertension as well as the Dahl salt-sensitive rat model of hypertension; however, the sympathetic targets involved remain unclear. To address this, we examined the role of the renal and splanchnic sympathetic nerves in Dahl hypertension by performing sham surgery (n=10) or targeted sympathetic ablation of the renal nerves (renal denervation, n=11), the splanchnic nerves (celiac ganglionectomy, n=11), or both renal and splanchnic nerves (n=11) in hypertensive Dahl rats. Mean arterial pressure increased from ≈120 mm Hg, while on a 0.1% sodium chloride diet, to ≈140 mm Hg after being fed a 4.0% sodium chloride diet for 3 weeks. At that point, rats underwent sham or targeted sympathetic ablation. Four weeks after treatment, mean arterial pressure was lower in renal denervated (150.4±10.4) and celiac ganglionectomized (147.0±6.1) rats compared with sham rats (165.0±3.7) and even lower in rats that underwent both ablations (128.4±6.6). There were no differences in heart rate or fluid balance between sham and renal denervated rats; however, rats that underwent either celiac ganglionectomy or both ablations exhibited marked tachycardia as well as sodium and water retention after treatment. These data suggest that targeted sympathetic ablation is an effective treatment for established hypertension in the Dahl rat and that the kidneys and the splanchnic vascular bed are both independently important targets of the sympathetic nervous system in this model.

The role of the subfornical organ in angiotensin II-salt hypertension in the rat.

Hypertension caused by chronic infusion of angiotensin II (Ang II) in experimental animals is dependent, in part, on increased activity of the sympathetic nervous system. This chronic sympathoexcitatory response is amplified by a high-salt diet, suggesting an interaction of circulating Ang II and dietary salt on sympathetic regulatory pathways in the brain. The present study tested the hypothesis that the subfornical organ (SFO), a forebrain circumventricular organ known to be activated by circulating Ang II, is crucial to the pathogenesis of hypertension induced by chronic Ang II administration in rats on a high-salt diet (Ang II-salt model). Rats were randomly selected to undergo either subfornical organ lesion (SFOx) or sham surgery (Sham) and then placed on a high-salt (2% NaCl) diet. One week later, rats were instrumented for radiotelemetric measurement of mean arterial pressure (MAP) and heart rate (HR) and placed in metabolic cages to measure sodium and water balance. Baseline MAP was slightly (but not statistically) lower in SFOx compared with Sham rats during the 5 day control period. During the subsequent 10 days of Ang II administration, MAP was statistically lower in SFOx rats. However, when MAP responses to Ang II were analysed by comparing the change from the 5 day baseline period, only on the fifth day of Ang II was MAP significantly different between groups. There were no differences between groups for water or sodium balance throughout the protocol. We conclude that, although the SFO is required for the complete expression of Ang II-salt hypertension in the rat, other brain sites are also involved.

The effect of cardiac sympathetic denervation through bilateral stellate ganglionectomy on electrical properties of the heart.

The role of the cardiac sympathetic nerve activity in various cardiac diseases is typically evaluated using ß-adrenergic receptor antagonists. However, these antagonists induce global denervation effects not only in the cardiovascular system, but also in the brain and kidney. The objective of this study was to detect the electrophysiological property changes due to 8 days of cardiac sympathetic denervation and investigate the possible mechanisms underlying these changes using a more cardiac-specific bilateral stellate ganglionectomy (SGX) rat model. High-resolution optical mapping using a voltage-sensitive dye was performed in isolated Langendorff-perfused sham and SGX hearts, which were paced at progressively reduced basic cycle lengths under several different conditions: control, pretreatment with isoproterenol, and administration of atenolol and esmolol. Several electrophysiological parameters were recorded during periodic pacing and ventricular fibrillation (VF). Our results demonstrate that cardiac sympathetic denervation by bilateral SGX shortens action potential duration (APD) and flattens the APD restitution curve, but does not significantly affect spatial dispersion of APD. We found that, although the vulnerability of sham and SGX hearts to VF is similar, the dynamics of VF are different. The maximum dominant frequency is higher, and the spatial distribution of VF is more complex in the SGX heart, resulting in different mechanisms of VF. We demonstrated that ß(1)-adrenergic receptors are downregulated in the SGX compared with sham hearts. In addition, our data suggest that the mechanism of cardiac sympathetic denervation by SGX surgery is more similar to the administration of ß-blocker esmolol than atenolol.

Effect of intracerebroventricular benzamil on cardiovascular and central autonomic responses to DOCA-salt treatment.

DOCA-salt treatment increases mean arterial pressure (MAP), while central infusion of benzamil attenuates this effect. The present study used c-Fos immunoreactivity to assess the role of benzamil-sensitive proteins in the brain on neural activity following chronic DOCA-salt treatment. Uninephrectomized rats were instrumented with telemetry transmitters for measurement of MAP and with an intracerebroventricular (ICV) cannula for benzamil administration. Groups included rats receiving DOCA-salt treatment alone, rats receiving DOCA-salt treatment with ICV benzamil, and appropriate controls. At study completion, MAP in vehicle-treated DOCA-salt rats reached 142 ± 4 mmHg. In contrast DOCA-salt rats receiving ICV benzamil had lower MAP (124 ± 3 mmHg). MAP in normotensive controls was 102 ± 3 mmHg. c-Fos immunoreactivity was quantified in the supraoptic nucleus (SON) and across subnuclei of the hypothalamic paraventricular nucleus (PVN), as well as other cardiovascular regulatory sites. Compared with vehicle-treated normotensive controls, c-Fos expression was increased in the SON and all subnuclei of the PVN, but not in other key autonomic nuclei, such as the rostroventrolateral medulla. Moreover, benzamil treatment decreased c-Fos immunoreactivity in the SON and in medial parvocellular and posterior magnocellular neurons of the PVN in DOCA-salt rats but not areas associated with regulation of sympathetic activity. Our results do not support the hypothesis that DOCA-salt increases neuronal activity (as indicated by c-Fos immunoreactivity) of other key regions that regulate sympathetic activity. These results suggest that ICV benzamil attenuates DOCA-salt hypertension by modulation of neuroendocrine-related PVN nuclei rather than inhibition of PVN sympathetic premotor neurons in the PVN and rostroventrolateral medulla.

Effect of stellate ganglionectomy on basal cardiovascular function and responses to beta1-adrenoceptor blockade in the rat.

Cardiac sympathetic nerve activity is an important short-term controller of cardiac function and arterial pressure. Studies also suggest that long-term increases in cardiac sympathetic nerve activity may contribute to hypertension, coronary artery disease, and cardiac remodeling in heart failure. However, our understanding of the role of cardiac sympathetic nerves in chronic models of cardiovascular disease has been limited by inadequate experimental approaches. The present study was conducted to develop a surgical method to surgically denervate the sympathetic nerves of the rat heart for long-term cardiovascular studies. We characterized the effect of cardiac sympathetic denervation on basal levels of mean arterial pressure (MAP) and heart rate (HR) and the responses to a chronic administration of atenolol, a beta1-adrenoceptor antagonist. Rats were instrumented with telemetry transmitters for continuous recording of MAP and HR. After a 4-day baseline period, the rats were subjected to bilateral stellate ganglionectomy (SGX; n=9) or sham surgery (Sham; n=8). Seven days following SGX or Sham, the rats were administered atenolol for 5 days, followed by a 7-day recovery period. Following a transient decrease, SGX had no effect on basal MAP but decreased HR compared with baseline and Sham rats. Five days of atenolol treatment decreased MAP similarly in SGX and Sham rats. Atenolol resulted in a marked bradycardia in Sham rats but had a neglible effects on HR in SGX rats. The measurement of the content of cardiac catecholamines in all cardiac chambers at the end of the study verified a successful sympathetic denervation. This study confirms that bilateral SGX is a useful method to study the contribution of cardiac sympathetic nerves on the regulation of cardiac function. Moreover, these results suggest that cardiac sympathetic nerves are relatively unimportant in maintaining the basal level of MAP or the depressor response to atenolol in conscious, unrestrained rats.