Afferent Renal Nerve Research Articles

Current Status and Future Perspectives of Renal Denervation.

Catheter-based renal denervation (RDN) therapy, a new procedure that uses radiofrequency ablation to interrupt efferent and afferent renal sympathetic nerve fibers, is a complementary or alternative treatment to antihypertensive medications for optimal control of blood pressure (BP). Although several single-arm early proof-of-concept studies showed significant BP reduction, the largest sham-controlled study using the first-generation RDN device (SYMPLICITY HTN-3) failed to significantly reduce BP in patients with resistant hypertension who were taking the guideline-based combination of antihypertensive medications. Since then, new devices and techniques have been developed to improve the efficacy and safety of RDN procedures. Sham-controlled trials using second-generation RDN devices (radiofrequency- and ultrasound-based) have provided solid evidence for their BP-lowering efficacy with and without the use of concomitant antihypertensive medication. Moreover, the safety profile of RDN in several registries and clinical trials appears to be excellent. This review summarizes the current evidence for RDN and discusses its current issues, future trials, Asian perspectives, and potential roles in both hypertension and other morbidities.

The organ-specific nitric oxide synthase activity in the interaction with sympathetic nerve activity: a hypothesis.

The sympathetic nerve activity (SNA) is augmented in hypertension. SNA is regulated by neuronal nitric oxide synthase (nNOS) or endothelial nitric oxide synthase (eNOS) activity in hypothalamic paraventricular nuclei (PVN) and/or brainstem rostral ventrolateral medulla. High nNOS or eNOS activity within these brain regions lowers the SNA, whereas low cerebral nNOS and/or eNOS activity causes SNA augmentation. We hypothesize that the decreased cerebral nNOS/eNOS activity, which allows the enhancement of SNA, leads to the augmentation of renal eNOS/nNOS activity. Similarly, when the cerebral nNOS/eNOS activity is increased and SNA is suppressed, the renal eNOS/nNOS activity is suppressed as well. The activation of endothelial alpha(2)-adrenoceptors, may be a possible mechanism involved in the proposed regulation. Another possible mechanism might be based on nitric oxide, which acts as a neurotransmitter that tonically activates afferent renal nerves, leading to a decreased nNOS activity in PVN. Furthermore, the importance of the renal nNOS/eNOSactivity during renal denervation is discussed. In conclusion, the presented hypothesis describes the dual organ-specific role of eNOS/nNOS activity in blood pressure regulation and suggests possible connection between cerebral NOS and renal NOS via activation or inhibition of SNA, which is an innovative idea in the concept of pathophysiology of hypertension.

Renal Sensory Activity Regulates the γ-Aminobutyric Acidergic Inputs to the Paraventricular Nucleus of the Hypothalamus in Goldblatt Hypertension.

Renal sensory activity is centrally integrated within brain nuclei involved in the control of cardiovascular function, suggesting that renal afferents regulate basal and reflex sympathetic vasomotor activity. Evidence has shown that renal deafferentation (DAx) evokes a hypotensive and sympathoinhibitory effect in experimental models of cardiovascular diseases; however, the underlying mechanisms involved in this phenomenon need to be clarified, especially those related to central aspects. We aimed to investigate the role of renal afferents in the control of γ-aminobutyric acid (GABA)ergic inputs to the paraventricular nucleus (PVN) of the hypothalamus in renovascular hypertensive (2K1C) rats and their influence in the regulation of cardiovascular function. Hypertension was induced by clipping the left renal artery. After 4 weeks, renal DAx was performed by exposing the left renal nerve to a 33 mM capsaicin solution for 15 min. After 2 weeks of DAx, microinjection of muscimol into the PVN was performed in order to evaluate the influence of GABAergic activity in the PVN and its contribution to the control of renal sympathetic nerve activity (rSNA) and blood pressure (BP). Muscimol microinjected into the PVN triggered a higher drop in BP and rSNA in the 2K1C rats and renal DAx mitigated these responses. These results suggest that renal afferents are involved in the GABAergic changes found in the PVN of 2K1C rats. Although the functional significance of this phenomenon needs to be clarified, it is reasonable to speculate that GABAergic alterations occur to mitigate microglia activation-induced sympathoexcitation in the PVN of 2K1C rats.

The involvement of renal afferents in the maintenance of cardiorenal diseases.

Elevated sympathetic vasomotor activity is a common feature of cardiorenal diseases. Therefore, the sympathetic nervous system is an important therapeutic target, particularly the fibers innervating the kidneys. In fact, renal denervation has been applied clinically and shown promising results in patients with hypertension and chronic kidney disease. However, the underlying mechanisms involved in the cardiorenal protection induced by renal denervation have not yet been fully clarified. This mini-review highlights historical and recent aspects related to the role of renal sensory fibers in the control of cardiorenal function under normal conditions and in experimental models of cardiovascular disease. Results have demonstrated that alterations in renal sensory function participate in the maintenance of elevated sympathetic vasomotor activity and cardiorenal changes; as such, renal sensory fibers may be a potential therapeutic target for the treatment of cardiorenal diseases. Although it has not yet been applied in clinical practice, selective afferent renal denervation may be promising, since such an approach maintains efferent activity and can provide more refined control of renal function compared with total renal denervation. However, more studies are needed to understand the mechanisms by which renal afferents partially contribute to such changes, in addition to the need to evaluate the safety and advantages of the approach for application in the clinical practice.

Renal Denervation to Treat Heart Failure.

Heart failure (HF) is a global pandemic with a poor prognosis after hospitalization. Despite HF syndrome complexities, evidence of significant sympathetic overactivity in the manifestation and progression of HF is universally accepted. Confirmation of this dogma is observed in guideline-directed use of neurohormonal pharmacotherapies as a standard of care in HF. Despite reductions in morbidity and mortality, a growing patient population is resistant to these medications, while off-target side effects lead to dismal patient adherence to lifelong drug regimens. Novel therapeutic strategies, devoid of these limitations, are necessary to attenuate the progression of HF pathophysiology while continuing to reduce morbidity and mortality. Renal denervation is an endovascular procedure, whereby the ablation of renal nerves results in reduced renal afferent and efferent sympathetic nerve activity in the kidney and globally. In this review, we discuss the current state of preclinical and clinical research related to renal sympathetic denervation to treat HF.

Neurogenic tachykinin mechanisms in experimental nephritis of rats

We demonstrated earlier that renal afferent pathways combine very likely “classical” neural signal transduction to the central nervous system and a substance P (SP)–dependent mechanism to control sympathetic activity. SP content of afferent sensory neurons is known to mediate neurogenic inflammation upon release. We tested the hypothesis that alterations in SP-dependent mechanisms of renal innervation contribute to experimental nephritis. Nephritis was induced by OX-7 antibodies in rats, 6 days later instrumented for recording of blood pressure (BP), heart rate (HR), drug administration, and intrarenal administration (IRA) of the TRPV1 agonist capsaicin to stimulate afferent renal nerve pathways containing SP and electrodes for renal sympathetic nerve activity (RSNA). The presence of the SP receptor NK-1 on renal immune cells was assessed by FACS. IRA capsaicin decreased RSNA from 62.4 ± 5.1 to 21.6 ± 1.5 mV s (*p < 0.05) in controls, a response impaired in nephritis. Suppressed RSNA transiently but completely recovered after systemic administration of a neurokinin 1 (NK1-R) blocker. NK-1 receptors occurred mainly on CD11+ dendritic cells (DCs). An enhanced frequency of CD11c+NK1R+ cell, NK-1 receptor+ macrophages, and DCs was assessed in nephritis. Administration of the NK-1R antagonist aprepitant during nephritis reduced CD11c+NK1R+ cells, macrophage infiltration, renal expression of chemokines, and markers of sclerosis. Hence, SP promoted renal inflammation by weakening sympathoinhibitory mechanisms, while at the same time, substance SP released intrarenally from afferent nerve fibers aggravated immunological processes i.e. by the recruitment of DCs.

Afferent renal innervation in anti-Thy1.1 nephritis in rats

Afferent renal nerves exhibit a dual function controlling central sympathetic outflow via afferent electrical activity and influencing intrarenal immunological processes by releasing peptides such as calcitonin gene-related peptide (CGRP). We tested the hypothesis that increased afferent and efferent renal nerve activity occur with augmented release of CGRP in anti-Thy1.1 nephritis, in which enhanced CGRP release exacerbates inflammation. Nephritis was induced in Sprague-Dawley rats by intravenous injection of OX-7 antibody (1.75 mg/kg), and animals were investigated neurophysiologically, electrophysiologically, and pathomorphologically 6 days later. Nephritic rats exhibited proteinuria (169.3 ± 10.2 mg/24 h) with increased efferent renal nerve activity (14.7 ± 0.9 bursts/s vs. control 11.5 ± 0.9 bursts/s, n = 11, P < 0.05). However, afferent renal nerve activity (in spikes/s) decreased in nephritis (8.0 ± 1.8 Hz vs. control 27.4 ± 4.1 Hz, n = 11, P < 0.05). In patch-clamp recordings, neurons with renal afferents from nephritic rats showed a lower frequency of high activity following electrical stimulation (43.4% vs. 66.4% in controls, P < 0.05). In vitro assays showed that renal tissue from nephritic rats exhibited increased CGRP release via spontaneous (14 ± 3 pg/mL vs. 6.8 ± 2.8 pg/ml in controls, n = 7, P < 0.05) and stimulated mechanisms. In nephritic animals, marked infiltration of macrophages in the interstitium (26 ± 4 cells/mm2) and glomeruli (3.7 ± 0.6 cells/glomerular cross-section) occurred. Pretreatment with the CGRP receptor antagonist CGRP8-37 reduced proteinuria, infiltration, and proliferation. In nephritic rats, it can be speculated that afferent renal nerves lose their ability to properly control efferent sympathetic nerve activity while influencing renal inflammation through increased CGRP release.

Effect of Renal Denervation on Cardiac Function and Inflammatory Factors in Heart Failure After Myocardial Infarction

Heart failure (HF) affects around 100 million people and is a staggering burden for health care system worldwide. Rapid and sustained activation of inflammatory response is an important feature of HF after myocardial infarction. Sympathetic overactivation is also an important factor in the occurrence and progression of HF. The beneficial effect of renal denervation (RDN) has been demonstrated in HF. In the current study, we hypothesized that RDN improves cardiac function in HF canine models due to acute myocardial infarction (AMI) and reduced inflammation might be involved. Twenty-four beagles were randomized into the control (n = 8), HF (n = 8), and HF + RDN group (n = 8). The HF model after AMI was established by embolization the anterior descending distal artery with anhydrous ethanol in the HF and HF + RDN group. Bilateral renal artery ablation was performed in the HF + RDN group. Cardiac function, serum creatine kinase, creatine kinase-MB and NT-Pro BNP level, and expression of inflammation-related proteins in myocardial were examined. Because the paraventricular nucleus of the hypothalamus might be involved in inflammation-induced central neural excitation in HF and plays an important role in regulating extracellular fluid volume and sympathetic activity, expression of inflammation-related proteins in hypothalamus was also examined. AMI and post-AMI HF model was created successfully. Compared with the HF group, dogs in the HF + RDN group showed better cardiac function 4 weeks after AMI: lower left ventricular end-diastolic pressure, left ventricular end-diastolic dimension, and left ventricular end-systolic dimension and higher LEVF and left ventricular systolic pressure (P < 0.05 for all) were observed in the HF + RDN group. In addition, dogs in the HF + RDN group had slightly less ventricular fibrosis. Interestingly, RDN had lower expression of inflammation-related proteins including interleukin-6, tumor necrosis factors-α, nuclear factor κB, and monocyte chemotactic protein 1 (P < 0.05 for all) in both myocardial tissue and hypothalamus. RDN can improve cardiac function in dogs with HF after myocardial infarction. Our results suggested that RDN might affect cytokine-induced central neural excitation in HF and later affect sympathetic activity. Our results suggested a potential beneficial mechanism of RDN independent of mechanism involving renal afferent and efferent sympathetic nerves.

TRPV1 Activation Prevents Renal Ischemia-Reperfusion Injury-Induced Increase in Salt Sensitivity by Suppressing Renal Sympathetic Nerve Activity.

: BackgroundSalt sensitivity is increased following renal Ischemia-Reperfusion (I/R) injury. We tested the hypothesis that high salt intake induced increase in Renal Sympathetic Nerve Activity (RSNA) after renal I/R can be prevented by activation of Transient Receptor Potential Vanilloid 1 (TRPV1).MethodsRats were fed a 0.4% NaCl diet for 5 weeks after renal I/R, followed by a 4% NaCl diet for 4 more weeks in four groups: sham, I/R, I/R +High Dose Capsaicin (HDC), and I/R+Low Dose Capsaicin (LDC). The low (1mg/kg) or high (100mg/kg) dose of capsaicin was injected subcutaneously before I/R to activate or desensitize TRPV1, respectively.ResultsSystolic blood pressure was gradually elevated after fed on a high-salt diet in the I/R and I/R+HDC groups but not in the I/R+LDC group, with a greater increase in the I/R+HDC group. Renal function was impaired in the I/R group and was further deteriorated in the I/R+HDC group but was unchanged in the I/R+LDC group. At the end of high salt treatment, afferent renal nerve activity in response to unilateral intra-pelvic administration of capsaicin was decreased in the I/R group and was further suppressed in the I/R+HDC group but was unchanged in the I/R+LDC group. RSNA in response to intrathecal administration of muscimol, a selective agonist of GABA-A receptors, was augmented in the I/R group and further intensified in the I/R+HDC group but was unchanged in the I/R+LDC group. Similarly, urinary norepinephrine levels were increased in the I/R group and were further elevated in the I/R+HDC group but unchanged in the I/R+LDC group.ConclusionThese data suggest that TRPV1 activation prevents renal I/R injury-induced increase in salt sensitivity by suppressing RSNA.

GLP-1 mediated diuresis and natriuresis are blunted in heart failure and restored by selective afferent renal denervation

BackgroundGlucagon-like peptide-1 (GLP-1) induces diuresis and natriuresis. Previously we have shown that GLP-1 activates afferent renal nerve to increase efferent renal sympathetic nerve activity that negates the diuresis and natriuresis as a negative feedback mechanism in normal rats. However, renal effects of GLP-1 in heart failure (HF) has not been elucidated. The present study was designed to assess GLP-1-induced diuresis and natriuresis in rats with HF and its interactions with renal nerve activity.MethodsHF was induced in rats by coronary artery ligation. The direct recording of afferent renal nerve activity (ARNA) with intrapelvic injection of GLP-1 and total renal sympathetic nerve activity (RSNA) with intravenous infusion of GLP-1 were performed. GLP-1 receptor expression in renal pelvis, densely innervated by afferent renal nerve, was assessed by real-time PCR and western blot analysis. In separate group of rats after coronary artery ligation selective afferent renal denervation (A-RDN) was performed by periaxonal application of capsaicin, then intravenous infusion of GLP-1-induced diuresis and natriuresis were evaluated.ResultsIn HF, compared to sham-operated control; (1) response of increase in ARNA to intrapelvic injection of GLP-1 was enhanced (3.7 ± 0.4 vs. 2.0 ± 0.4 µV s), (2) GLP-1 receptor expression was increased in renal pelvis, (3) response of increase in RSNA to intravenous infusion of GLP-1 was enhanced (132 ± 30% vs. 70 ± 16% of the baseline level), and (4) diuretic and natriuretic responses to intravenous infusion of GLP-1 were blunted (urine flow 53.4 ± 4.3 vs. 78.6 ± 4.4 µl/min/gkw, sodium excretion 7.4 ± 0.8 vs. 10.9 ± 1.0 µEq/min/gkw). A-RDN induced significant increases in diuretic and natriuretic responses to GLP-1 in HF (urine flow 96.0 ± 1.9 vs. 53.4 ± 4.3 µl/min/gkw, sodium excretion 13.6 ± 1.4 vs. 7.4 ± 0.8 µEq/min/gkw).ConclusionsThe excessive activation of neural circuitry involving afferent and efferent renal nerves suppresses diuretic and natriuretic responses to GLP-1 in HF. These pathophysiological responses to GLP-1 might be involved in the interaction between incretin-based medicines and established HF condition. RDN restores diuretic and natriuretic effects of GLP-1 and thus has potential beneficial therapeutic implication for diabetic HF patients.

Afferent renal nerve input is involved in blood pressure elevation through activation of angiotensin receptor type 1 in hypothalamic paraventricular nucleus in stroke‐prone spontaneously hypertensive rats

BackgroundRenal nerves contain both sympathetic efferent fibers and sensory afferent fibers. Activation of efferent renal nerves increases renin release and renal sodium reabsorption, causing hypertension. However, previous studies suggested that the contribution of afferent renal nerves to blood pressure (BP) regulation may differ among the animal models of hypertension. Activation of brain renin‐angiotensin system, specifically angiotensin receptor type 1 (AT1R), in hypothalamic paraventricular nucleus (PVN), which receives afferent renal input, is crucially involved in BP elevation. Therefore, the aim of this study was to examine whether the afferent renal input contributes to BP elevation through AT1R activation in the PVN in stroke‐prone spontaneously hypertensive rats (SHRSP).MethodsIn chronic experiment, total renal denervation (TRDN), selective afferent renal denervation (ARDN), or sham surgery was performed in male SHRSP at 10 weeks of age, which is the developing phase of hypertension. ARDN was produced by wrapping renal artery and vein with capsaicin‐soaked gauze. Systolic BP was measured by tail‐cuff method at baseline and 2 and 6 days after surgery. In acute experiment, we evaluated the effect of AT1R blockade with losartan in the PVN on pressor response to afferent renal nerve stimulation by intrapelvic infusion of 10 μM capsaicin under anesthesia in male Wistar‐Kyoto rats (WKY) and SHRSP at 10 weeks of age.ResultsSystolic BP was sequentially increased at day 2 and day 6 in sham‐operated SHRSP ([n=4], baseline, 188.3±9.2 mmHg; day 2, 192.1±8.1 mmHg; day 6, 201.1±3.9 mmHg). In both ARDN‐SHRSP and TRDN‐SHRSP, systolic BP was decreased from baseline at day 2 after surgery (ARDN [n=4], baseline, 187.3±10.3 mmHg; day 2, 170.5±5.2 mmHg; p=0.085; TRDN [n=4], baseline, 186.4±5.9 mmHg; day2 145.1±4.2 mmHg; p=0.003), and the decrease in systolic BP was smaller in ARDN‐SHRSP than in TRDN‐SHRSP (ARDN, −16.7±13.2 mmHg; TRDN, ‐ 41.4±10.0 mmHg; p=0.03). Although systolic BP was increased at day 6 compared to day 2 in ARDN‐SHRSP and TRDN‐SHRSP (ARDN, 197.8±14.9 mmHg; TRDN, 185.8±19.3 mmHg), the BP change from baseline to day 6 was smaller in ARDN‐SHRSP and TRDN‐SHRSP compared with sham‐operated SHRSP. We validated TRDN and ARDN by immunohistochemistry and western blotting: in the renal pelvis, both CGRP and tyrosine hydroxylase (a marker for afferent and efferent renal nerves, respectively) were abolished in TRDN‐SHRSP, and CGRP was selectively abolished in ARDN‐SHRSP. In acute experiment, BP elevation induced by afferent renal nerve stimulation was greater in SHRSP than in WKY (Δmean BP, SHRSP [n=4],12.2±4.1 mmHg; WKY [n=4], 6.6±5.4 mmHg; p=0.07). Prior microinjection of losartan into the PVN attenuated the pressor response to afferent renal nerve stimulation in SHRSP (Δmean BP, 1.1±4.4 mmHg, p=0.04 vs before losartan), but not in WKY (Δmean BP, 3.2±6.7 mmHg, p&gt;0.10 vs before losartan).ConclusionNot only efferent renal nerve activation but afferent renal nerve input contributes to development of hypertension in SHRSP. The AT1R activation in the PVN may be involved in its mechanism.

Renal Afferent Nerve Activation and Arterial Pressure Control: Role of Inflammatory Cytokines in Hypertension

Neural control of the kidney as well as aberrant renal nerve signaling remain a primary focus of research in cardiovascular and renal disease, including hypertension. Recent preclinical reports employing targeted afferent renal nerve ablation have confirmed the central role for afferent renal nerves in the development of hypertension, including studies in the DOCA‐salt rat. Moreover, direct measurement of afferent renal nerve activity (ARNA) in DOCA‐salt rats revealed elevated tonic ARNA; however, the mechanisms contributing to the increased ARNA, and in turn hypertension, remain unaddressed. Renal inflammation is postulated to be a primary contributor to the aberrant ARNA in this model, due to the elevated pro‐inflammatory cytokines (IL‐1β, IL‐6, TNFα, MCP‐1, GRO/KC) present in the DOCA‐salt kidneys. Therefore, we aimed to directly test the role of these cytokines in modulating ARNA and concomitant cardiovascular responses in normotensive and DOCA‐salt rats. We hypothesized intrarenal inflammatory cytokine administration would increase ARNA and arterial pressure (AP) in both treatment groups.To interrogate this hypothesis, unilateral nephrectomized male Sprague Dawley rats (300–350g; n=16) were administered 100mg DOCA (s.c.) or silicone vehicle (n=8/group), followed by 3vweeks of high salt diet administered through drinking water (0.9% NaCl). Each animal then underwent an acute surgical preparation under isoflurane anesthesia (2%) to record AP, heart rate (HR), and ARNA responses to randomized intrarenal injection (10μL; 0.1 μg/μL) of the following cytokines: IL‐1β, IL‐6, TNFα, MCP‐1, GRO/KC, and 0.9% saline (VEH). ARNA responses were normalized to maximal afferent activation (%Amax) achieved by TRPV1‐agonist (capsaicin; 50μM) injection at end of protocol. Responses between and within treatment groups were analyzed by a repeated‐measures two‐way ANOVA (α=.05). Data presented as mean ± SEM. * p&lt;.05 vs. Vehicle; # p&lt;.05 vs. Saline.Prior to stimulation, baseline ARNA (4.9 ± 1.3 vs. 13.8 ± 2.1 %Amax) and AP (106 ± 2 vs. *151 ± 5 mmHg) were elevated in DOCA rats vs. vehicle controls. Compared to VEH responses, no changes from baseline ARNA, AP or HR responses were detected following IL‐1β, TNFα, MCP‐1, or GRO/KC injection. Compared to saline vehicle, IL‐6 injection produced an increase in ARNA in both VEH (5.9 ± 1.8 vs. #24.1 ± 5.4 %Amax) and DOCA rats (14.7 ± 2.2 vs. #24.1 ± 5.4 %Amax). Interestingly, AP decreased following IL‐6 injection in parallel to ARNA stimulation in VEH (1 ± 1 vs. −6 ± 2mmHg) and DOCA (0 ± 1 vs. −11 ± 4mmHg) compared to saline. No differences in responses between DOCA and VEH were detected.To summarize, IL‐6 injection increased ARNA in both VEH and DOCA supported our hypothesis; however, the corresponding depressor responses did not. Together, we conclude IL‐6 is a direct activator of ARNA independent of DOCA. Several studies have previously reported a depressor response following ARNA activation under anesthesia, so the central processing of ARNA may be altered under anesthesia compared to the conscious state, which is a limitation of this study. Current dose‐response studies and conscious preparations are underway to elucidate this important cardio‐renal‐neural axis in blood pressure homeostasis.Support or Funding InformationNIH R00HL141650 (CTB); R01HL116476 (JWO)

High Sodium Diet – Role in Renal Sympathodepression

Afferent renal nerve pathways are likely involved in the development of salt sensitive hypertension. We recently reported that intrarenal NaCl elicited a long‐lasting sympatho‐depression via a neuro‐humoral TRPV1 dependent and tachykinin mediated renal afferent nerve pathway. We now wanted to test the hypothesis that high sodium intake impairs this afferent sympatho‐depressory mechanism.Respective groups were put on tap water, 0.9 % saline for drinking or chow containing 8% NaCl. Cultured dorsal root ganglion neurons (DRG Th11‐L2) of rats with renal afferents were investigated in current clamp mode to assess action potential generation during current injection. Rats were equipped with femoral catheters for blood pressure (BP) &amp; heart rate (HR) assessment, drug application, a renal arterial catheter for intrarenal administration (IRA) of NaCl boil (10 % NaCl, 10 μl) or Capsaicin (CAP 3.3, 6.6, 10, 33*10‐7 M, 10 μl) and a bipolar electrode for renal sympathetic nerve activity (RSNA) recordings; eventually an intravenous (iv) bolus of the NK1‐receptor blocker RP67580 (10*10‐3M, 15 μl) was administered. Results are mean±SEM.In neurons from rats on high salt diet, but not on 0.9 % saline or tap water, the relation of tonic highly active neurons to less active neurons shifted towards less active units (62% tonic neurons in a control group vs. 40% on high salt diet and 63% on saline, significant, p&lt;0.05, z‐test, mean +/−SEM). However, cultured renal neurons from rats on 0.9% saline or on high salt diet exhibited increased action potential production upon stimulation (Controls 13,3+/11,03 APs/600ms vs. saline 19,8+/−2,33 APs/600ms vs. high salt diet 22,2+/−4,54 APs/600ms, significant, &lt;0.05, t−test, mean+/−SEM). 10% NaCl boli IRA induced decreases of RSNA from baseline 4.1±0.6 μV*sec to 2.2±0.8 μV*sec (10% NaCl, p&lt;0.05) impaired in rats on 8% NaCl chow. (Suppressed RSNA unmasked in all groups by an i.v. NK1‐inhibitor).In rats on high salt diet highly active tonic neurons with renal afferents in vitro decreased at the expense of less active phasic neurons in spite of tonic neurons producing more action potentials upon stimulation. The sodium inducible long‐lasting sympatho‐depression via a neuro‐humoral tachykinin mediated afferent renal nerve pathway got eventually impaired.

Total and Afferent Renal Denervation Blunts Hypertension and Central and Renal Inflammation in the Developmental Phase of 2‐Kidney, 1‐Clip Hypertension

Hypertension (HTN) is a multifactorial disease that affects one third of the population of western countries. Although 95% of cases are of unknown origin (i.e., primary HTN), it is well established that renal ischemia results in secondary renovascular HTN. Experimental renovascular HTN is induced in rats by placing a silver clip on the left renal artery creating a stenosis, which decreases renal perfusion pressure and increases renin release from the clipped kidney (2K1C‐HTN). Previous studies indicate a role of renal nerves in 2K1C‐HTN since renal denervation (RDN) attenuates HTN in 2K1C‐rats. Moreover, activation of the sympathetic nervous system in this model is associated with inflammation in the hypothalamus, a key site for regulation of sympathetic activity and an area that also receives input from afferent renal nerves. In the present study, we tested the hypothesis that renal nerves modulate the levels of hypothalamic and renal proinflammatory cytokines and mean arterial pressure (MAP) during the development of 2K1C hypertension in rats. Male Sprague‐Dawley rats (150–180 g, n=5–8/group) were instrumented with telemeters to measure MAP. One week later a clip was placed on the left renal artery of the rats to generate 2K1C HTN. In the same surgery rats also underwent either total or afferent RDN (2K1C‐TRDN or 2K1C‐ARDN) of the clipped kidney. In sham rats (2K1C‐sham) the left renal artery was exposed but not denervated. MAP was continuously measured for the following 6 weeks after which rats were euthanized and brains and kidneys removed for measurement of hypothalamic and renal cytokine content. MAP at the end of the study was 160±6 mmHg in 2K1C‐sham rats. In contrast, MAP was significantly lower in both 2K1C‐TRDN (135±7 mmHg) and 2K1C‐ARDN (133±7 mmHg) rats. TRDN and ARDN statistically reduced hypothalamic protein levels of TNF‐α (0.24±0.01 and 0.2±0.01 pg/mg, respectively) compared to 2K1C‐sham rats (0.3±0.02 pg/mg). Similarly, ARDN statistically reduced hypothalamic IL1β, IL‐2 in 2K1C rats. TRDN trended to the same effect but this was not statistically significant. Renal inflammatory cytokines/chemokines (MCP‐1, IL‐6, TNF‐α) were increased in clipped compared with non‐clipped kidney in 2K1C‐rats. Moreover, ARDN and TRDN statistically reduced this inflammatory response. These results suggest that RDN decreases hypothalamic and renal inflammation in 2K1C hypertensive rats and this may be one mechanism by which RDN decreases MAP in the developmental phase of the 2K‐1C hypertension model.Support or Funding InformationNIH R01 HL116476, FAPESP (2018/11221‐1) and PROPE/FUNDUNESP

Effects of Sex and Anesthetics on Renal Sensory Nerve Responses

The anti‐hypertensive effects of renal denervation are partly attributed to denervation of renal sensory nerves, which detect changes in intrarenal pressure and nociceptive chemokines to reflexively alter sympathetic nerve activity, renal function, and blood pressure. To date, the majority of experiments have assessed renal sensory responses using either anesthesia and exclusively male animals. The purpose of the present study was to evaluate the impact of anesthesia and sex on in vivo renal afferent nerve activity (ARNA) to chemo‐ and mechano‐sensitive stimuli. Male and female Sprague‐Dawley rats (250–400g) were assessed by multifiber nerve recordings in unanesthetized decerebrate preparations, Inactin, isoflurane, or urethane anesthesia. Baseline ARNA was not different across preparations or between sexes (n=6–8/group): decerebration (M: 20±6Hz, F: 20±6Hz), Inactin (M:12±5Hz, F: 16±8 Hz), and isoflurane (M: 23±6Hz, F: 29±6Hz). However, ARNA was significantly lower in urethane‐anesthetized male rats (M: 3±1Hz, F: 29±6Hz; p&lt;0.05).To evaluate chemosensitive responses, increasing doses of capsaicin [0 – 50 μM], (50μL over 15 s) were infused into the renal artery via a suprarenal arterial catheter, which produced concentration‐dependent increase in ARNA in all groups (n=4–6/group). ARNA was most sensitive in decerebrate and Inactin vs isoflurane or urethane groups. In male rats, the threshold concentration of capsaicin to evoke a response was 1μM decerebrate (117.8±29.2Hz, p=0.02) &lt; 10μM Inactin (137.0±61.0, p=0.0002) &lt; 30μM isoflurane (102.2±37.8, p=0.02) = 30μM urethane (96.5±35.9, p=0.02). In female rats, the threshold was 1μM Inactin (117.2±37.5Hz, p=0.03) &lt; 10μM decerebrate (184.8±41.6, p=0.03) = 10μ isoflurane (93.5±10.4, p=0.02) &lt; 30μM urethane (158.8±47.6, p=0.02).To evaluate mechanosensitive responses, renal artery occlusion (30s) increased ARNA equally in all groups. However, a significant sex effect was observed using Isoflurane (M: 9±4Hz, F: 33±3Hz; p&lt;0.01). Lastly, 5‐mmHg stepwise increases in renal pelvic pressure (0–30mmHg, 30s) produced linear increases in ARNA in all groups. Using linear regression to test the magnitude (y‐intercept) and sensitivity (slope) of ARNA we found significant differences in the magnitude of males (n=6–8/group): decerebrate, 6.5±5.3Hz; Inactin, 15.7±3.4 Hz; isoflurane, 3.8±3.2Hz; urethane, 0.2±1.1Hz; p&lt;0.0001) and females (n=4–7/group): decerebrate, 8.8±10.5Hz; inactin, 9.5±6.0Hz; isoflurane, 14.2±8.6Hz; urethane, 0.2±4.5Hz; p&lt;0.0001). Between sex comparisons resulted in blunted male sensitivity to urethane (M: slope=0.1±0.1; F: slope=0.7±0.23, p&lt;0.001), and elevated female sensitivity to isoflurane (M: slope=0.1±0.2, F: slope=1.1±0.4, p&lt;0.03). Decerebrate and inactin anesthesia were similarly sensitive between sexes.In conclusion, ARNA to chemo‐ and mechano‐sensitive stimuli were similar between an unanesthetized‐decerebrate preparation vs Inactin anesthesia. Isoflurane and urethane attenuated ARNA to capsaicin and differentially altered ARNA to increased renal pelvic pressure. Sex did not consistently impact ARNA per se, but sex differences were linked to the use of isoflurane and urethane. These findings suggest decerebration or Inactin anesthesia should be employed to study renal afferent nerve function.Support or Funding InformationNIH Grants: R01 HL145875

SPARC ‐ The Functional Anatomy of Murine Myelinated Renal Afferents

Perhaps because they are a small minority of the total population of renal afferents, myelinated renal afferents (MRA ) have been the least studied. Yet, they are promising targets for neuromodulation because of their low thresholds for electrical stimulation. We are studying the calibers, branching patterns, terminal morphologies, and intrarenal targets of MRA. We identify the myelin sheaths and axons of MRA using antibodies to myelin protein zero and heavy‐chain neurofilament, respectively. The diameters of a majority of MRA range between 1 and 4 u. The diameters of a small minority range between 6 and 10u. Thus, the diameters of MRA are distinctly bimodal. Unmyelinated terminal segments of MRA arise from the myelinated segments of axons at two sites. First, an unmyelinated segment of axon projects from the end of each MRA. Second, unmyelinated segments arise as axon collaterals at nodes of Ranvier along the course of the myelinated axon. Most unmyelinated segments end without any terminal specialization. However, some terminate in small “bulbs, that are approximately twice the diameter of the axon. We have not observed receptor‐related, connective tissue specializations surrounding either type of termination. The distal myelinated portions of MRA are frequently tortuous, sometimes forming hairpin turns and loops. These complex features result in spatial concentrations of nodes of Ranvier, concentrations of unmyelinated collaterals, and concentrations of receptors. A marked narrowing at the origin of each unmyelinated segment suggests the presence of a spike‐initiation zone. The majority of MRA terminate deep in the renal cortex, most often in the extracellular space and frequently within bundles of connective tissue. Some terminations closely appose, but never penetrate, the renal tubular system. However, a small number of MRA appear to penetrate the walls of small arteries and venules. Terminations of MRA show no consistent relationship to glomeruli. The functions of MRA are, thus far, uncertain. Their terminations in the extracellular space would permit them to respond to changes in the composition of the extracellular fluid. However, their associations with connective tissue in the extracellular space could also provide them with mechanoreceptor properties. Their presence in the walls of the vasculature could mediate either chemoreceptor or mechanoreceptor functions. The distinctly bimodal distribution of the diameters of MRA suggests that they may participate in more than one physiological function. From a clinical perspective, the differences in stimulation thresholds afforded by those differences in axon caliber could facilitate selective neuromodulation of multiple physiological processes.Support or Funding InformationSupported by SPARC Award 1U01DK116320‐02, JW Osborn, Jr., PI

Glucagon‐like peptide‐1 mediated renal sympathetic activation in obese rats: role of intrarenal mechanisms

Accumulated evidence indicates that obesity is associated with enhanced sympathetic activation. Chronic sympathetic activation increases the risk of cardiovascular complications and subsequent mortality. The mechanisms linking obesity with sympathetic activation are complex and not completely understood. Gut produced hormone glucagon‐like peptide‐1 (GLP‐1) affects cardiovascular function via regulating blood pressure and sympathetic nerve activity. The aim of the study was to determine the role of intrarenal GLP‐1 mediated renal sympathetic activation in obese rats. In anesthetic Sprague Dawley rats, renal intra‐pelvic infusion of GLP‐1 receptor agonist exendin‐4 increased afferent renal sympathetic nerve activity (A‐RSNA), efferent renal sympathetic nerve activity (T‐RSNA), arterial pressure and heart rate. All the sympathetic and hemodynamic responses to the exendin‐4 infusion were significantly enhanced in high fat diet (HFD, 42% of calories from fat for 12 weeks) induced obese rats. The natriuretic and diuretic effects mediated by intrarenal exendin‐4 infusion was significantly blunted in the obese rats. Furthermore, ablation of renal sympathetic nerves significantly improved the diuretic and natriuretic responses to exendin‐4 infusion in obese rats. We also found significant altered GLP‐1 level and GLP‐1 receptor expression in the kidney of obese rats. The results suggest that altered intrarenal GLP‐1 mediated mechanisms may contribute to the development of obesity‐related high sympathetic activation and obesity hypertension.Support or Funding InformationThe study was supported by the author’s faculty start‐up fund from Sanford School of Medicine of the University of South Dakota Department of Basic Biomedical Sciences.

SPARC: Renal Denervation Attenuates DOCA‐salt Hypertension in the Mouse

Hypertension affects approximately one third of the adult population in the United States. Catheter based renal denervation (RDN) has emerged as a novel treatment for hypertension in patients. However, the mechanisms by which RDN lowers arterial pressure has not been fully determined. We hypothesize that an interaction between renal nerves (afferent and efferent) and renal inflammation contributes to hypertension. To test this hypothesis, we utilized a model of hypertension in the mouse produced by the combination of uni‐nephrectomy with deoxycorticosterone acetate (DOCA) and a high salt diet (DOCA‐salt). 10‐week‐old male C57BL6/J mice were implanted with radio‐telemetry probes in the carotid artery for measurement of arterial pressure. DOCA was administered via subcutaneous implantation of a silicon pellet containing 50 mg of DOCA. Sham mice received a drug free silicon pellet. Uni‐nephrectomy, pellet insertion, salt treatment, and RDN were all performed or started on the same day. RDN was performed through surgical ablation and peri‐axonal application of 10% phenol. Baseline arterial pressure was measured 3 days prior to induction of DOCA‐salt hypertension by radiotelemetry. Separate cohorts of mice were generated for protein quantification and histology, with urine and kidneys harvested 21 days post induction of DOCA‐salt hypertension. Cytokine protein quantification was obtained through Multiplex ELISA. All errors reported are SEM. Following 7 days of DOCA‐salt, mean arterial pressure (MAP) increased +44.8 ± 3.2 mmHg from baseline (153.1± 1.8 mmHg day 7 absolute value), comparted to −1.5 ± 5.5 mmHg in sham mice (105.6 ± 0.5 mmHg day 7 absolute value). RDN reduced this increase by more than 50% in RDN‐DOCA‐salt mice (+20.5 ± 5.0 mmHg delta, 131.6 ± 8.1 mmHg absolute). Renal and urinary cytokines and chemokines were increased in DOCA‐salt compared to sham treated mice. For example, renal MCP‐5 was 24.7 ± 7.2 pg/mg in DOCA‐salt mice compared to 5.0 ± 0.6 pg/mg in sham treated mice. Similarly, urinary MCP‐5 was significantly higher in in DOCA‐salt mice (12.4 ± 5.1 pg/mg) compared to sham mice (2.1 ± 1.1 pg/mg). IL‐1α and IL‐1β were both increased in DOCA‐salt kidneys (112.4 ± 19.2 pg/mg and 2.1 ± 0.4 pg/mg respectively) compared to sham kidneys (31.98 ± 4.4 pg/mg and 0.36 ± 0.06 pg/mg respectively). We also measured, by immunohistochemistry, an increase expression of the IL‐1 receptor in kidneys of DOCA‐salt mice compared to sham controls. We hypothesize that renal cytokines activate renal afferent nerves and ultimately elevate arterial pressure by activation of the sympathetic nervous system. Future studies will test this hypothesis by examining whether elimination of cytokine generated afferent nerve activity attenuates hypertension in DOCA‐salt mice.Support or Funding InformationNIH: R01HL116476, MnOPT: T32DK083250–01A1, SPARC: 5U01DK116320‐03

The Impact of Renal Denervation on the Progression of Heart Failure in a Canine Model Induced by Right Ventricular Rapid Pacing

Heart failure (HF) has been proposed as a potential indication of renal denervation (RDN). However, the mechanisms enabling RDN to attenuate HF are not well understood, especially the central effects of RDN. The aim of this study was to decipher the mode of operation of RDN in the treatment of HF using a canine model of right ventricular rapid pacing-induced HF. Accordingly, 24 Chinese Kunming dogs were randomly grouped to receive sham procedure (sham-operated group), bilateral RDN (RDN group), rapid pacing to induce HF (HF-control group), and bilateral RDN plus rapid pacing (RDN + HF group). Echocardiography, plasma brain natriuretic peptide (BNP), and norepinephrine (NE) concentrations of randomized dogs were measured at baseline and 4 weeks after interventions, followed by histological and molecular analyses. Twenty dogs completed the research successfully and were enrolled for data analyses. Results showed that the average optical density of renal efferent and afferent nerves were significantly lower in the RDN and RDN + HF groups than in the sham-operated group, with a significant reduction of renal NE concentration. Rapid pacing in the RDN + HF and HF-control groups, compared with the sham-operated group, induced a significant increase in left ventricular end-diastolic volume and decrease in left ventricular ejection fraction and correspondingly resulted in cardiac fibrosis and dysfunction. Cardiac fibrosis evaluated by Masson’s trichrome staining and the expression of transforming growth factor-β1 (TGF-β1) were significantly higher in the HF-control group than in the sham-operated group, which were remarkably attenuated by the application of the RDN technique in the RDN + HF group. In terms of central renin–angiotensin system (RAS), the expression of angiotensin II (AngII)/angiotensin-converting enzyme (ACE)/AngII type 1 receptor (AT1R) in the hypothalamus of dogs in the HF-control group, compared with the sham-operated group, was upregulated and that of the angiotensin-(1-7) [Ang-(1-7)]/ACE2 was downregulated. Furthermore, both of them were significantly attenuated by the RDN therapy in the RDN + HF group. In conclusion, the RDN technique could damage renal nerves and suppress the cardiac remodeling procedure in canine with HF while concomitantly attenuating the overactivity of central RAS in the hypothalamus.

Afferent innervation of the ischemic kidney contributes to renal dysfunction in renovascular hypertensive rats.

The ablation of renal nerves, by destroying both the sympathetic and afferent fibers, has been shown to be effective in lowering blood pressure in resistant hypertensive patients. However, experimental studies have reported that the removal of sympathetic fibers may lead to side effects, such as the impairment of compensatory cardiorenal responses during a hemodynamic challenge. In the present study, we evaluated the effects of the selective removal of renal afferent fibers on arterial hypertension, renal sympathetic nerve activity, and renal changes in a model of renovascular hypertension. After 4weeks of clipping the left renal artery, afferent renal denervation (ARD) was performed by exposing the left renal nerve to a 33mM capsaicin solution for 15min. After 2weeks of ARD, we found reduced MAP (~ 18%) and sympathoexcitation to both the ischemic and contralateral kidneys in the hypertensive group. Moreover, a reduction in reactive oxygen species was observed in the ischemic (76%) and contralateral (27%) kidneys in the 2K1C group. In addition, ARD normalized renal function markers and proteinuria and podocin in the contralateral kidney. Taken altogether, we show that the selective removal of afferent fibers is an effective method to reduce MAP and improve renal changes without compromising the function of renal sympathetic fibers in the 2K1C model. Renal afferent nerves may be a new target in neurogenic hypertension and renal dysfunction.