Afferent Renal Nerve Activity Research Articles

IL-1R Mediated Activation of Renal Sensory Nerves in DOCA-Salt Hypertension.

Clinical trials of renal denervation for the treatment of hypertension have shown a variety of off-target improvements in conditions associated with sympathetic overactivity. This may be due to the ablation of sympathoexcitatory afferent renal nerves, which are overactive under conditions of renal inflammation. Renal IL (interleukin)-1β is elevated in the deoxycorticosterone acetate-salt model of hypertension, and its activity may be responsible for the elevation in afferent renal nerve activity and arterial pressure. Continuous blood pressure recording of deoxycorticosterone acetate-salt mice with IL-1R (IL-1 receptor) knockout or antagonism was used individually and combined with afferent renal denervation (ARDN) to assess mechanistic overlap. Protein quantification and histological analysis of kidneys were performed to characterize renal inflammation. ARDN attenuated deoxycorticosterone acetate-salt hypertension (-20±2-Δmm Hg mean arterial pressure [MAP] relative to control at study end) to a similar degree as total renal denervation (-21±2-Δmm Hg MAP), IL-1R knockout (-16±4-Δmm Hg MAP), or IL-1R antagonism (-20±3-Δmm Hg MAP). The combination of ARDN with knockout (-18±2-Δmm Hg MAP) or antagonism (-19±4-Δmm Hg MAP) did not attenuate hypertension any further than ARDN alone. IL-1R antagonism was found to have an acute depressor effect (-15±3-Δmm Hg MAP, day 10) in animals with intact renal nerves but not those with ARDN. These findings suggest that IL-1R signaling is partially responsible for the elevated afferent renal nerve activity, which stimulates central sympathetic outflow to drive deoxycorticosterone acetate-salt hypertension.

Renal Afferent Nerve Activity and Association with Neurohumoral Dysregulation in a Model of Autosomal Recessive Polycystic Kidney Disease

Polycystic kidney disease (PKD) is a rare, genetic kidney disease characterized by cyst growth that leads to kidney failure. Renal nerves play an important role in the development and maintenance of hypertension and other renal diseases. Afferent (sensory) nerves convey information to the central nervous system in response to intrarenal stimuli. Our laboratory demonstrated that afferent renal nerve activity (ARNA) is elevated in 10-week-old PCK rats compared to non-cystic, age-matched Sprague-Dawley (SD) controls. Furthermore, ARNA positively correlated with cystic index (CI; a measure of cyst growth). To further elucidate the relationship between renal nerve activity, age, and cyst progression, we aimed to quantify both ARNA and renal sympathetic nerve activity (RSNA) in PCK and SD rats. We hypothesized that ARNA and RSNA would be greater in PCK than SD. Furthermore, arginine vasopressin (AVP) is elevated in PKD and increases cyst growth. There is evidence that activation of the afferent renal nerves can contribute to secretion of AVP. Thus, we propose that elevated ARNA increases AVP secretion, thereby increasing cyst growth. We also hypothesized that ARNA would positively correlate with age, CI, and copeptin (an indirect marker of AVP) in young PCK rats. To test our first hypothesis, we conducted direct renal nerve recordings in 10- to 12-week-old PCK (n=16;11M/5F) and 10-week-old SD (n=9;7M/2F) rats. For our second hypothesis, we conducted nerve recordings in 4- to 10-week-old PCK rats to evaluate the correlation of nerve activity with age, cystic index, and copeptin in animals ages 4- to 12-weeks-old (n=28;21M/7F). Multiunit nerve recording was performed under inactin anesthesia. As we previously published, a renal nerve bundle is isolated, placed on an iridium bipolar electrode, and encased in silicone. Resting RSNA was recorded following 10-minute stabilization period, then the nerve bundle was severed proximal to the electrode to isolate ARNA. ARNA recordings were normalized to maximal ARNA evoked by intrarenal 50 μM capsaicin. Comparisons between PCK and SD were analyzed by Student’s t-test (p<.05). Linear regression analyses were used to assess variable correlation (p<.05). Data presented as mean ± SEM. In contrast to our first hypothesis, RSNA did not differ between 10- to 12-week-old SD and PCK rats, whether measured via burst frequency (SD 6.92 ± 0.82; PCK *6.97 ± 0.34 Hz) or bursts/heart beat (SD 1.36±0.16; PCK 1.28 ± 0.06). ARNA was calculated as a percentage of maximal ARNA using either integrated ARNA (ARNAInt) or spikes/s (ARNASpikes). Baseline ARNAInt was greater in PCK rats (29.14±3.35%) than SD (13.46±2.08%, n=4), whereas PCK ARNASpikes trended (p=.06) higher than SD controls (SD 5.57±2.21; PCK 13.90±2.21%). In correlative analysis, RSNA trended (p=.05) to correlate with age in the PCK rat. However, there was no relationship between RSNA and (1) CI (p=0.29) or (2) copeptin (p=0.15). Similarly, there was no correlation detected between ARNA and age (ARNAInt: p=0.41; ARNASpikes: p=0.16) or serum copeptin (ARNAInt: p=0.96; ARNASpikes: p=0.20). ARNAInt trended (p=.07) to correlate with CI, but ARNASpikes did not correlate with CI (p=0.48). Overall, these data partially support our hypotheses. RSNA did not differ between SD and PCK rats, but ARNAInt was increased in PCK rats vs. SD. We failed to detect a correlation between either ARNA or RSNA and age, CI, or copeptin in PCK rats though, ARNAInt trended to correlate with CI. As ARNA does not appear to be correlated with serum copeptin, further studies are required to uncover the relationship between afferent renal nerves and cystogenesis in this model of PKD. PKD Foundation Research Award 1022534 (CB) NIDDK/PKD RRC Pilot and Feasibility Award U24DK126110 (CB) AHA 21PRE0000216068 (MG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The Positive Effect of Renal Denervation Persists even Through Regrowth of Afferent Axons Improving Renal Afferent Nerve Activity During High Sodium Intake in Rats

The Positive Effect of Renal Denervation Persists even Through Regrowth of Afferent Axons Improving Renal Afferent Nerve Activity During High Sodium Intake in Rats

Abstract 057: The Positive Effect Of Renal Denervation Persists Even Through Regrowth Of Afferent Axons Improving Renal Afferent Nerve Activity During High Sodium Intake In Rats

Introduction: Previous work of ours suggests that pathologically decreased sensitivity of renal afferent neurons (reduced number of highly active tonic neurons) due to high salt diet is normalized 1 wk after renal denervation (DNX). Now we tested the hypothesis that normalized sensitivity of renal neurons persists after DNX even through regrowth of afferent axons after 12 wk. This morphological regrowth of afferent nerves 12wk after renal DNX (postDNX) was shown by us previously. Material and methods: 6 male Sprague Dawley (SD) rats were put on high salt diet (HS; 8% NaCl) for 10 days. In another group of 12 rats on high salt diet (HS) left kidneys were denervated (postDNX) 12 weeks prior to examination. Rats on standard diet were used as controls. Harvested dorsal root ganglion neurons (DRG Th11-L2) with renal afferents were investigated in primary neuronal cell culture using current clamp mode to assess action potential generation during current injection and to characterize neurons as tonic highly active and phasic less active neurons. Results: In renal neurons from rats on HS the relation of tonic to phasic neurons shifted towards less active phasic units (62% tonic neurons in controls [n=72 out of 114] vs. 42% [n=37 out of 86] on HS, (p<0.05, z-test)). Denervation of the left kidney in rats on high salt diet (HS-DNX) led to a recovery of afferent renal DRG neurons after 1 wk; (40% tonic neurons [n=32 out of 80] on HS vs. 72% tonic neurons [n=58 out of 81] on HS+DNX)(p<0.05, z-test)). Even 12 weeks after renal denervation this regained electrophysiological property of tonic firing persisted (43% tonic neurons [n=35 out of 82] on HS vs. 69% tonic neurons [n=46 of 67]on HS-postDNX, p<0.05, z-test)). Conclusion: The reduced proportion of highly active tonic neurons increased 1 wk after renal denervation to control levels. Even 12 weeks after renal denervation, this effect of renal denervation persisted, and the sensitivity of renal neurons to electrical stimuli did not change despite high-salt diet. Hence, the positive effects of renal denervation persisted even through the regrowth of renal afferent axons to the kidney.

Deletion of the Transient Receptor Potential Vanilloid 1 Channel Attenuates Sympathoexcitation and Hypertension and Improves Glomerular Filtration Rate in 2-Kidney-1-Clip Rats.

Renal denervation lowers arterial blood pressure in both clinical populations and multiple experimental models of hypertension. This therapeutic effect is partly attributed to the removal of overactive renal sensory nerves. The TRPV1 (transient receptor potential vanilloid 1) channel is highly expressed in renal sensory nerves and detects changes in noxious and mechanosensitive stimuli, pH, and chemokines. However, the extent to which TRPV1 channels contribute to 2-kidney-1-clip (2K1C) renovascular hypertension has not been tested. We generated a novel Trpv1-/- (TRPV1 knockout) rat using CRISPR/Cas9 and 26-bp deletion in exon 3 and induced 2K1C hypertension. The majority (85%) of rat renal sensory neurons retrogradely labeled from the kidney were TRPV1-positive. Trpv1-/- rats lacked TRPV1 immunofluorescence in the dorsal root ganglia, had a delayed tail-flick response to hot but not cold water, and lacked an afferent renal nerve activity response to intrarenal infusion of the TRPV1 agonist capsaicin. Interestingly, 2K1C hypertension was significantly attenuated in male Trpv1-/- versus wild-type rats. 2K1C hypertension significantly increased the depressor response to ganglionic blockade, total renal nerve activity (efferent and afferent), and afferent renal nerve activity in wild-type rats, but these responses were attenuated in male Trpv1-/- rats. 2K1C hypertension was attenuated in female rats with no differences between female strains. Finally, glomerular filtration rate was reduced by 2K1C in wild-type rats but improved in Trpv1-/- rats. These findings suggest that renovascular hypertension requires activation of the TRPV1 channel to elevate renal afferent and sympathetic nerve activity, reduce glomerular filtration rate, and increase arterial blood pressure.

Elevated renal afferent nerve activity in a rat model of endothelin B receptor deficiency.

Renal nerves have been an attractive target for interventions aimed at lowering blood pressure; however, the specific roles of renal afferent (sensory) versus efferent sympathetic nerves in mediating hypertension are poorly characterized. A number of studies have suggested that a sympathoexcitatory signal conveyed by renal afferents elicits increases in blood pressure, whereas other studies identified sympathoinhibitory afferent pathways. These sympathoinhibitory pathways have been identified as protective against salt-sensitive increases in blood pressure through endothelin B (ETB) receptor activation. We hypothesized that ETB-deficient (ETB-def) rats, which are devoid of functional ETB receptors except in adrenergic tissues, lack appropriate sympathoinhibition and have lower renal afferent nerve activity following a high-salt diet compared with transgenic controls. We found that isolated renal pelvises from high salt-fed ETB-def animals lack a response to a physiological stimulus, prostaglandin E2, compared with transgenic controls but respond equally to a noxious stimulus, capsaicin. Surprisingly, we observed elevated renal afferent nerve activity in intact ETB-def rats compared with transgenic controls under both normal- and high-salt diets. ETB-def rats have been previously shown to have heightened global sympathetic tone, and we also observed higher total renal sympathetic nerve activity in ETB-def rats compared with transgenic controls under both normal- and high-salt diets. These data indicate that ETB receptors are integral mediators of the sympathoinhibitory renal afferent reflex (renorenal reflex), and, in a genetic rat model of ETB deficiency, the preponderance of sympathoexcitatory renal afferent nerve activity prevails and may contribute to hypertension.NEW & NOTEWORTHY Here, we found that endothelin B receptors are an important contributor to renal afferent nerve responsiveness to a high-salt diet. Rats lacking endothelin B receptors have increased afferent nerve activity that is not responsive to a high-salt diet.

Afferent renal nerve activity response to IL-6 and IL-1ß antagonism in the DOCA-salt rat model of hypertension

Hypertension is the highest risk factor for cardiovascular disease, which remains the leading cause of death worldwide. Though hypertension is a multifaceted disease, there is an increasingly clear role for renal nerves in hypertension progression – both sympathetic and afferent. Moreover, afferent renal nerve activity (ARNA) is reportedly elevated in several models of hypertension and hypothesized to be a major contributor to the disease etiology. Yet, the underlying stimulus for this increased activity is unknown. Previous studies from our lab highlighted a direct correlation between ARNA and renal proinflammatory cytokine content in hypertensive rats, specifically IL-6 and IL-1ß. Therefore, we aimed to acutely disrupt IL-6 and IL-1ß activity to elucidate the direct effects on ARNA in hypertension. We hypothesized that antagonism of IL-6 and IL-1ß will decrease ARNA in hypertensive rats. To address this hypothesis, we employed the deoxycorticosterone acetate (DOCA)-salt model of hypertension. Briefly, uninephrectomized male Sprague Dawley rats (275-325g, n=3) were administered DOCA (100 mg, s.c) and saline drinking water (0.9% NaCl) for three weeks. Next, rats were surgically prepared for renal nerve activity measurement and administration of cytokine antagonists. Under anesthesia (Urethane, 1800 mg/kg/h, IV), a renal nerve bundle of the left kidney was hooked with a bipolar electrode. Renal sympathetic nerve activity (RSNA) was rectified and integrated to identify burst rate. To isolate the ARNA, the proximal end of the nerve bundle was cut and nerve activity was rectified and integrated over 50 ms. Basal ARNA was normalized to peak ARNA response to 5 μl capsaicin (10 μg/μl). To suppress IL-6 and IL-1ß intrarenally, 10 μl of IL-6 antibody (0.1 μg/μl) and 10 μl of IL-1ß antibody (0.1 μg/μl) were injected into the renal artery. Vehicle (saline) injections were used as control. ARNA was recorded for 10 mins after injections and expressed as percentage of baseline (%ARNABase). Data were analyzed by repeated measures one-way ANOVA with Dunnett post hoc test vs. vehicle (*p<.05) and presented as mean±SEM. At time of acute surgical preparation, mean arterial blood pressure was 152±1mmHg. Basal RSNA was 5.4±0.6bursts/s and 1.5±0.26bursts/beat. Resting ARNA was 9.5±2.8%ARNAMAX. Compared to the response to vehicle administration (84.4±8.0 %ARNABase), IL-6 antibody decreased ARNA to *49.8±6.9 %ARNABase. No difference in IL-1ß antibody response (53.6±15.4 %ARNABase, P>.05) was detected compared to vehicle. These preliminary results partially support our hypothesis that antibody-mediated suppression of renal IL-6 acutely lowers ARNA in hypertensive DOCA-salt rats. This highlights that increased ARNA in the hypertension may be driven in part by direct stimulation from proinflammatory cytokines such as IL-6. Further studies are underway to increase sample size, include female cohorts, and measure the effects of antibody treatment in normotensive controls. R00HL141650 (CTB), Doris Duke Charitable Foundation (CTB) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Activation of TRPV1-Expressing Renal Sensory Nerves of Rats with N-Oleoyldopamine Attenuates High-Fat-Diet-Induced Impairment of Renal Function.

Enhanced renal sympathetic nerve activity (RSNA) contributes to obesity-induced renal disease, while the role of afferent renal nerve activity (ARNA) is not fully understood. The present study tested the hypothesis that activating the transient receptor potential vanilloid 1 (TRPV1) channel in afferent renal nerves suppresses RSNA and prevents renal dysfunction and hypertension in obese rats. N-oleoyldopamine (OLDA, 1 ng/kg, daily) was administrated intrathecally (T8-L3) via an indwelled catheter to chronically activate, TRPV1-positive afferent renal nerves in rats fed a chow diet or high-fat diet (HFD) for 8 weeks. HFD intake significantly increased the body weight, impaired glucose and insulin tolerance, decreased creatinine clearance, and elevated systolic blood pressure in rats compared with the levels of the chow-fed rats (all p < 0.05). An intrathecal OLDA treatment for 8 weeks did not affect the fasting glucose level, glucose tolerance, and insulin tolerance in rats fed either chow or HFD. As expected, the chronic OLDA treatment significantly increased the levels of plasma calcitonin gene-related peptide and substance P and ARNA in the HFD-fed rats (all p < 0.05). Interestingly, the OLDA treatment decreased the urinary norepinephrine level and RSNA in rats fed HFD (both p < 0.05). Importantly, the OLDA treatment attenuated HFD-induced decreases in creatinine clearance and urinary Na+ excretion and increases in the plasma urea level, urinary albumin level, and systolic blood pressure at the end of an 8-week treatment (all p < 0.05). Taken together, the intrathecal administration of OLDA ameliorates the enhancement of RSNA, renal dysfunction, and hypertension in obese rats. These findings shed light on the roles of TRPV1-positive renal afferent nerves in obesity-related renal dysfunction and hypertension.

Abstract 057: Macula Densa Stimulation Acutely Increases Afferent Renal Nerve Activity And Blood Pressure

Macula densa (MD) cells in the kidney are known to regulate renal hemodynamics and the renin-angiotensin system, and indirectly systemic blood pressure (BP). However, neuron-like differentiation and activity as non-traditional MD functions are emerging. This study addressed the hypothesis that MD cells function similarly to peripheral ganglia, form neuronal networks that are part of the peripheral (renal) nervous system and synapse with renal afferent/efferent nerves as a new pathway for the direct control of sympathetic tone and BP. Single cell MD Ca 2+ signaling was analyzed in vivo using intravital multiphoton microscopy (MPM) of MD-G6 mice that selectively express the ratiometric Ca 2+ reporter GCaMP6f/tdTomato in MD cells. Afferent renal nerve activity (ARNA) was isolated and measured via recording electrodes. Systemic BP was monitored via the cannulated carotid artery. Optogenetic MD stimulation was induced by blue light (470nm) exposure of the whole left kidney of MD-Ai27 mice that selectively express the depolarizing channelrhodopsin ChR2 in MD cells. Pacemaker-like regular Ca 2+ oscillations (4-fold elevations in baseline Ca 2+ and frequency of 0.03 Hz) and their cell-to-cell propagation via long, axon-like processes were exclusively confined to the MD area. Bulk and scRNA seq and MD transcriptome analysis identified the high expression of genes related to synaptic transmission that was validated by immunohistochemistry. Acute intra-renal infusion of arginine-vasopressin (AVP) produced 3-fold increase in MD Ca 2+ firing frequency (via the MD-specific AVPR1a) and a dose-dependent increase in ARNA and BP (saline: -1±1; 0.01ug/mL: 2±1; 0.1ug/mL: 7±2; 0.25ug/mL: 7±3; 0.5ug/mL: 10±3; 1.0ug/mL: 23±13). IV administration of AVP (0.25ug/mL) did not significantly alter ARNA (108±3%, n=3) but produced a significant increase in arterial blood pressure (40±6mmHg, n=3). Blue light exposure of MD-Ai27 mouse kidneys acutely and reversibly increased systemic BP by 22±5mmHg (P&lt;0.05, n=5). In summary, MD cells send interoceptive signals to the brain via renal sensory afferent nerves to directly control BP. Targeting of novel MD mechanisms may result in exciting new therapeutic opportunities for renal and cardiovascular diseases and hypertension.

Sympathoexcitatory responses to renal chemosensitive stimuli are exaggerated at nighttime in rats.

The circadian cycle impacts sympathetic nerve activity (SNA), cardiovascular hemodynamics, and renal function. Activation of renal sensory nerves by chemosensory and mechanosensory stimuli reflexively changes efferent SNA and arterial blood pressure (ABP) to maintain homeostasis. However, it is unclear to what extent circadian cycle influences reflex SNA and ABP responses to renal sensory stimuli. Renal, splanchnic, and lumbar SNA and ABP responses to intrarenal arterial infusion of bradykinin or capsaicin and elevated renal pelvic pressure were measured in male and female Sprague-Dawley rats during nighttime (wakeful/active phase) and daytime (inactive phase). Intrarenal arterial bradykinin infusion significantly increased efferent renal SNA, splanchnic SNA, and ABP but not lumbar SNA. Responses were greater during nighttime versus daytime. Similarly, intrarenal arterial capsaicin infusion significantly increased renal SNA and splanchnic SNA, and responses were again greater during nighttime. Elevated renal pelvic pressure increased renal SNA and splanchnic SNA; however, responses did not differ between daytime and nighttime. Finally, afferent renal nerve activity responses to bradykinin were not different between daytime and nighttime. Thus, renal chemokines elicit greater sympathoexcitatory responses at nighttime that cannot be attributed to differences in afferent renal nerve activity. Collectively, these data suggest that the circadian cycle alters the excitability of central autonomic networks to alter baseline SNA and ABP as well as the magnitude of visceral reflexes.NEW & NOTEWORTHY The current study discovers that the circadian cycle influences sympathetic and hemodynamic responses to activation of renal chemosensitive sensory fibers. Sympathetic responses to intrarenal bradykinin or capsaicin infusion were exaggerated during nighttime (active period), but mechanosensitive responses to elevated renal pelvic pressure were not. Importantly, renal afferent nerve responses were not different between nighttime and daytime. These data suggest that the circadian cycle modulates sympathetic responses to visceral afferent activation.

Hypertension Precedes Changes in Afferent Renal Nerve Activity and Renal Dysfunction in DOCA‐Salt Rat Model

Hypertension (HTN) is a major risk factor for cardiovascular disease, which is a leading cause of morbidity and mortality worldwide. Though the etiology of HTN is multifaceted, one important contributor is aberrant autonomic signaling, both to (sympathetic nerve activity; SNA) and from (afferent renal nerve activity; ARNA) the kidney. ARNA is reportedly elevated in several models of HTN, and ablation of the renal afferent nerves can lower arterial pressure. It remains unknown, however, when this increase occurs in comparison to the cardiovascular and renal function in the hypertensive paradigm. The aim of the study was to determine whether ARNA mirrors the increase in arterial pressure and decline in kidney function in a preclinical model of salt‐sensitive HTN. We hypothesized that increased ARNA would increase in parallel to arterial pressure (AP) and the decline in renal function in the DOCA‐salt rat model.To test the hypothesis, 20 adult male Sprague Dawley rats (275‐325 g) underwent a unilateral nephrectomy followed by two‐weeks for recovery. Next, deoxycorticosterone acetate (DOCA; 100mg, s.c.) was implanted, and henceforth animals were maintained on 0.9% saline drinking water ad libitum. Cardiovascular and renal measurements were collected weekly. Mean systolic blood pressure (SBP) were measured weekly by tail‐cuff, and renal function was estimated weekly by serum creatinine (Cr). Animals were randomly assigned to undergo multiunit ARNA recording on protocol day (D) 0, D7, D14, or D21. ARNA recordings were performed under urethane anesthesia (1500mg/kg, IP) as previously described. ARNA was isolated by sectioning the proximal end of the nerve bundle, and recorded for 10 minutes following preparation. Activity was rectified and integrated over 50ms and normalized to maximal ARNA achieved by intrapelvic 50µM capsaicin. Data were analyzed by one‐way repeated‐measures ANOVA with Bonferroni post‐hoc test (α=0.05). Data presented as mean±SEM.ARNA (presented as %ARNAmax) is increased (p&lt;0.05) at D21 and D14 vs. baseline, but no detectable changes were observed at D7. Mean SBP progressively increased at D7, D14, and D21 vs. all prior weeks. Serum creatinine increased (p&lt;.05) at D14 and D21 vs. baseline, indicating a time‐dependent decline in kidney function. Necropsy data demonstrate kidney weight was increased (p&lt;.05) only on D21 compared to baseline, and no differences were detected at D7 and D14 vs. D0. In contrast, heart weights were greater (p&lt;.05) on D14 and D21 vs. baseline.Collectively, these data partially support our hypothesis that ARNA increases in parallel with increasing AP in the DOCA‐salt model; however, AP appears to increased independent of detectable changes in both ARNA and renal dysfunction. Interestingly, renal dysfunction and ARNA were detected at similar times (D14), potentially highlighting to a closer physiological relationship between ARNA and renal function. The onset of HTN prior to changes in ARNA and renal dysfunction delineate these variables from the early stages of this HTN model; however, we can not rule on the contributions of ARNA and renal dysfunction as the model progresses. Further studies on the timing of ARNA activators such as renal inflammation or fibrosis are underway to further elucidate the connection of renal function decline and ARNA.

Afferent Renal Nerve Activity is Proportionately Increased with Cystic Progression in a Preclinical Model of Autosomal Recessive Polycystic Kidney Disease

Polycystic kidney disease (PKD) is the most common genetic cause of kidney failure, though the mechanisms underlying cyst formation and disease progression remain unclear. Innervation of the kidney contributes to the pathology of other cardiorenal dysfunction, including hypertension and chronic kidney disease ‐ frequent complications of PKD. In previous studies, we demonstrated renal denervation (RDNX), either total or afferent‐targeted (sensory) nerve ablation, reduced cystogenesis in a rat model of autosomal recessive PKD, thus highlighting a role for renal afferent nerves in PKD renal cystogenesis. To further elucidate this novel relationship, we aimed to quantify the renal afferent nerve activity (ARNA) in the adult PCK rat compared to non‐cystic controls. We hypothesized that ARNA would be elevated in the PCK rat compared to controls, and ARNA would directly correlate with renal cystic severity.To address our hypothesis, we conducted direct afferent renal nerve recordings in 10‐week‐old PCK (n=15; 11M/4F) and age‐matched Sprague‐Dawley controls (SD; n=14; 6M/8F). Multiunit nerve recording was performed under isoflurane anesthesia by isolating and placing a renal nerve bundle on an iridium bipolar electrode, which was then encased in silicone. The nerve bundle was severed proximal to the electrode to isolate afferent renal nerve activity (ARNA). Following a 10‐minute stabilization period, resting ARNA was recorded for 10 minutes. To permit comparison between animals, resting ARNA was normalized to a maximal stimulus, achieved by renal pelvic perfusion of 50 µM capsaicin. Finally, electrical noise was isolated by sectioning the distal end of the nerve bundle. Recordings were rectified and integrated over 50 ms. ARNA was expressed as a percentage of maximal ARNA. Following nerve recording, kidneys were collected, formalin fixed, and embedded in paraffin for histologic analysis of renal cystic index (CI). Kidneys were stained with Masson’s Trichrome and analyzed using ImageJ, and CI was quantified by normalizing cystic area to the total kidney area. Data presented as mean ±SEM.Resting ARNA was higher in PCK (22.7±4.2%) rats compared to non‐cystic SD controls (7.4±1.3%; Figure 1). Overall, PCK rats had an average CI of 8.7±0.7%. No sex differences in ARNA between male and female PCK rats were detected; however, CI was higher in male PCK rats (9.5±2.3%) compared to female PCK (6.3±1.9%). Further analysis of ARNA versus cystic index revealed a direct correlation between ARNA and renal cystic severity (F=5.9; R=0.61; p&lt;0.05; Figure 2).These findings support our hypothesis that ARNA is elevated in the PCK rat. Importantly, ARNA is directly correlated with cystic progression in this model. To our knowledge, this study is the first to directly measure ARNA in this model of ARPKD, as well as to compare ARNA to cyst severity. These findings, combined with our previous studies, lead us to conclude that increased ARNA may directly contribute to the progression of PKD in this model. Presently, these data are limited to single endpoint observations. We are currently conducting additional studies to further dissect the temporal relationship between ARNA and renal cyst development. Our future studies will investigate the stimuli amplifying ARNA in a continued effort to parse the role of this novel neural‐renal axis in the pathogenesis of PKD.

Renal chemosensitive and mechanosensitive afferent responses are preserved in TRPV1 knockout rats

The transient receptor potential vanilloid type‐1 (TRPV1) channel is expressed in renal sensory nerves, and intrarenal administration of the TRPV1 agonist capsaicin increases renal afferent nerve activity (ARNA). Nonselective denervation of renal sensory nerves using high concentration capsaicin reduces arterial blood pressure (ABP) in experimental models of hypertension. Pharmacological blockade of TRPV1 using renal pelvic infusion of capsazepine attenuates ARNA responses to capsaicin, CGRP, and elevated pelvic pressure. Thus, TRPV1 channels mediate activation of renal sensory nerves.To test this hypothesis, we generated a novel TRPV1 knockout rat (TRPV1‐/‐) using CRISPR/Cas9 to produce a 26bp deletion in exon 3. Multifiber ARNA responses to chemical (bradykinin and capsaicin) and mechanical (elevated renal pelvic pressure) stimuli were compared in Inactin‐anesthetized male and female wild‐type and TRPV1‐/‐ littermates (250‐400g). There were no significant differences in baseline mean ABP between wild‐type and TRPV1‐/‐ rats (122±2 mmHg vs 122±3 mmHg, respectively; p=0.48, n=8‐10), or baseline ARNA between wild‐type and TRPV1‐/‐ rats (14±3 Hz vs 18±5 Hz, respectively; p=0.24, n=8‐10).Intrarenal artery infusion of capsaicin (0.1–10μM, 50μL per 15s) significantly increased ipsilateral ARNA in wild‐type but not TRPV1‐/‐ rats (Δ discharge 65±14 Hz vs 9±3 Hz at 10μM, respectively; p&lt;0.05, n=7). As a second chemosensitive stimulus, intrarenal artery infusion of bradykinin (0.1–10μM, 50μL per 15s) produced significant increases in ARNA in ­both wild‐type and TRPV1‐/‐ rats that were not significantly different between groups (Δ discharge of 10μM: 52±11 Hz vs 76±21 Hz, respectively; p=0.33, n=7). Elevated renal pelvic pressure (0–20mmHg; 30s) significantly increased ARNA in both wild‐type and TRPV1‐/‐ rats; however, ARNA responses were significantly smaller in wild‐type versus TRPV1‐/‐ rats (Δ discharge with 20mmHg: 17±3 Hz versus 29±6 Hz, respectively; p=0.03, n=8‐9).The preservation of ARNA responses in TRPV1‐/‐ rats contradicts prior studies using pharmacological TRPV1 antagonists. A final set of experiments tested whether intrapelvic capsazepine infusion (200μM, 200μl per 3 min) attenuates ARNA responses to elevated pelvic pressure (20mmHg) in both wild‐type and Trpv1‐/‐ rats. Interestingly, pelvic administration of capsazepine attenuated mechanosensitive ARNA responses in both wildtype (saline: 31±3 Hz vs capsazepine: 8±1 Hz; n=3) and TRPV1‐/‐ (saline: 20±7 Hz vs capsazepine: 6±1 Hz; n=3) rats thereby suggesting a TRPV1‐independent action of capsazepine.In conclusion, TRPV1 channels are dispensable for the activation of chemo‐ and mechanosensitive renal sensory nerves.

Contributions of afferent and sympathetic renal nerves to cystogenesis and arterial pressure regulation in a preclinical model of autosomal recessive polycystic kidney disease.

Polycystic kidney disease (PKD) is the most common inheritable cause of kidney failure, and the underlying mechanisms remain incompletely uncovered. Renal nerves contribute to hypertension and chronic kidney disease-frequent complications of PKD. There is limited evidence that renal nerves may contribute to cardiorenal dysfunction in PKD and no investigations of the role of sympathetic versus afferent nerves in PKD. Afferent renal nerve activity (ARNA) is elevated in models of renal disease and fibrosis. However, it remains unknown if this is true in PKD. We tested the hypothesis that ARNA is elevated in a preclinical model of autosomal recessive PKD and that targeted renal nerve ablation would attenuate cystogenesis and cardiorenal dysfunction. We tested this by performing total renal denervation (T-RDNx) or afferent renal denervation (A-RDNx) denervation in 4-wk-old male and female PCK rats and then quantified renal and cardiovascular responses 6 wk following treatment. Cystogenesis was attenuated with A-RDNx and T-RDNx versus sham controls, highlighting a crucial role for renal afferent nerves in cystogenesis. In contrast, blood pressure was improved with T-RDNx but not A-RDNx. Importantly, treatments produced similar results in both males and females. Direct renal afferent nerve recordings revealed that ARNA was twofold greater in PCK rats versus noncystic controls and was directly correlated with cystic severity. To our knowledge, we are the first to demonstrate that PCK rats have greater ARNA than noncystic, age-matched controls. The findings of this study support a novel and crucial role for renal afferent innervation in cystogenesis in the PCK rat.NEW & NOTEWORTHY This is the first study to dissect the contributions of renal sympathetic and afferent innervation in the PCK rat, a preclinical model of autosomal recessive polycystic kidney disease. We demonstrated that resting afferent renal nerve activity is greater in the PCK rat than noncystic controls and that basal afferent renal nerve activity is directly correlated with the extent of renal cystogenesis.

Renal Denervation Improves Renal Afferent Nerve Activity After High Sodium Intake

Renal Denervation Improves Renal Afferent Nerve Activity After High Sodium Intake

Abstract MP48: Crispr/cas9 Trpv1 Deletion In Rats Does Not Impair Mechano- And Chemo-sensory Renal Afferent Nerve Responses

Elevated renal afferent nerve activity (ARNA) or dysfunctional renal reflexes contributes to hypertension and chronic kidney disease. The transient receptor potential vanilloid type-1 (TRPV1) channel is expressed in renal sensory nerves, and intrarenal administration of the TRPV1 agonist capsaicin increases ARNA. Nonselective denervation of renal sensory nerves using high-concentration capsaicin reduces arterial blood pressure (ABP) in experimental models of hypertension. However, the role of TRPV1 channels in ARNA responses to chemo- and mechano-sensitive stimuli has not been directly tested. To test this hypothesis, we generated a novel TRPV1 rat knockout model (TRPV1 -/- ) using CRISPR/CAS9 to delete exon 3 . ARNA multifiber recordings were performed in male and female TRPV1 -/- and wild-type littermates (250-400g) after decerebration or Inactin anesthesia (data combined). Wild-type and TRPV1 -/- rats had no significant differences in baseline mean ABP (126±4 mmHg vs 138±5 mmHg, respectively; n=8-10) or heart rate (451±25 bpm vs 432±24 bpm, respectively; n=8-10). Baseline ARNA was not different between wild-type and TRPV1 -/- rats (16±3 Hz vs 28±6 Hz, respectively; n=8-10). Intrarenal artery infusion of the TRPV1 agonist capsaicin (0.1-10μM, 50μL per 15s) significantly increased ipsilateral ARNA in wild-type but not TRPV1 -/- rats (Δ discharge with 10μM: 65±3 Hz vs 6±1 Hz, respectively; n=5-7). As a second chemosensitive stimulus, intrarenal artery infusion of bradykinin (0.1-10μM, 50μL per 15s) produced similar increases in ipsilateral ARNA between wild-type and TRPV1 -/- rats (Δ discharge with 10μM: 52±6 Hz vs 73±18 Hz, respectively; n=5-6). Finally, elevated renal pelvic pressures (0-20mmHg; 30s) significantly increased ipsilateral ARNA in both wild-type and TRPV1 -/- rats; however, the ARNA response was significantly greater in TRPV1 -/- versus wild-type rats (Δ discharge with 20mmHg: 47±14 Hz versus 18±6 Hz, respectively; n=5-8). In conclusion, mechanosensitive and chemosensitive ARNA responses remain intact in TRPV1 -/- rats. The mechanisms responsible for renal sensory nerve activation remain unidentified and the impact of TRPV1 deletion in rat models of hypertension and kidney disease remains to be tested.

Sympathetic modulation by antihypertensive drugs.

Sympathetic modulation by antihypertensive drugs.

Responsiveness of afferent renal nerve units in renovascular hypertension in rats

Previous data suggest that renal afferent nerve activity is increased in hypertension exerting sympathoexcitatory effects. Hence, we wanted to test the hypothesis that in renovascular hypertension, the activity of dorsal root ganglion (DRG) neurons with afferent projections from the kidneys is augmented depending on the degree of intrarenal inflammation. For comparison, a nonhypertensive model of mesangioproliferative nephritis was investigated. Renovascular hypertension (2-kidney, 1-clip [2K1C]) was induced by unilateral clipping of the left renal artery and mesangioproliferative glomerulonephritis (anti-Thy1.1) by IV injection of a 1.75-mg/kg BW OX-7 antibody. Neuronal labeling (dicarbocyanine dye [DiI]) in all rats allowed identification of renal afferent dorsal root ganglion (DRG) neurons. A current clamp was used to characterize neurons as tonic (sustained action potential [AP] firing) or phasic (1–4 AP) upon stimulation by current injection. All kidneys were investigated using standard morphological techniques. DRG neurons exhibited less often tonic response if in vivo axonal input from clipped kidneys was received (30.4% vs. 61.2% control, p < 0.05). However, if the nerves to the left clipped kidneys were cut 7 days prior to investigation, the number of tonic renal neurons completely recovered to well above control levels. Interestingly, electrophysiological properties of neurons that had in vivo axons from the right non-clipped kidneys were not distinguishable from controls. Renal DRG neurons from nephritic rats also showed less often tonic activity upon current injection (43.4% vs. 64.8% control, p < 0.05). Putative sympathoexcitatory and impaired sympathoinhibitory renal afferent nerve fibers probably contribute to increased sympathetic activity in 2K1C hypertension.

Ablation of TRPV1 Abolishes Salicylate-Induced Sympathetic Activity Suppression and Exacerbates Salicylate-Induced Renal Dysfunction in Diet-Induced Obesity.

Sodium salicylate (SA), a cyclooxygenase inhibitor, has been shown to increase insulin sensitivity and to suppress inflammation in obese patients and animal models. Transient receptor potential vanilloid 1 (TRPV1) is a nonselective cation channel expressed in afferent nerve fibers. Cyclooxygenase-derived prostaglandins are involved in the activation and sensitization of TRPV1. This study tested whether the metabolic and renal effects of SA were mediated by the TRPV1 channel. Wild-type (WT) and TRPV1−/− mice were fed a Western diet (WD) for 4 months and received SA infusion (120mg/kg/day) or vehicle for the last 4 weeks of WD feeding. SA treatment significantly increased blood pressure in WD-fed TRPV1−/− mice (p < 0.05) but not in WD-fed WT mice. Similarly, SA impaired renal blood flow in TRPV1−/− mice (p < 0.05) but not in WT mice. SA improved insulin and glucose tolerance in both WT and TRPV1−/− mice on WD (all p < 0.05). In addition, SA reduced renal p65 and urinary prostaglandin E2, prostaglandin F1α, and interleukin-6 in both WT and TRPV1−/− mice (all p < 0.05). SA decreased urine noradrenaline levels, increased afferent renal nerve activity, and improved baroreflex sensitivity in WT mice (all p < 0.05) but not in TRPV1−/− mice. Importantly, SA increased serum creatinine and urine kidney injury molecule-1 levels and decreased the glomerular filtration rate in obese WT mice (all p < 0.05), and these detrimental effects were significantly exacerbated in obese TRPV1−/− mice (all p < 0.05). Lastly, SA treatment increased urine albumin levels in TRPV1−/− mice (p < 0.05) but not in WT mice. Taken together, SA-elicited metabolic benefits and anti-inflammatory effects are independent of TRPV1, while SA-induced sympathetic suppression is dependent on TRPV1 channels. SA-induced renal dysfunction is dependent on intact TRPV1 channels. These findings suggest that SA needs to be cautiously used in patients with obesity or diabetes, as SA-induced renal dysfunction may be exacerbated due to impaired TRPV1 in obese and diabetic patients.

Renal Sympathetic Nerve Activity and Heart Rate Responses to Renal Pelvic Infusion of Bradykinin and Capsaicin in Rats Exposed to Intermittent Hypoxia

Renal oxidative stress and inflammation are evident in individuals with obstructive sleep apnea and in animals exposed to intermittent hypoxia (IH). It is known that kidney injury alters renal afferent nerve activity disrupting reno-renal reflex mechanisms. This study investigated reno-renal reflex changes in response to intra-renal pelvic infusion of bradykinin and capsaicin in rats exposed to IH. Wistar rats were exposed to 96 cycles of IH (6% O2, n=10) or to normoxia (sham, 21% O2, n=11) daily for 14 days. On day 15, rats were anesthetized (induction: urethane, α-chloralose and sodium pentobarbitone mixture i.p. (416, 27 and 33mg/kg), maintenance: urethane and α-chloralose mixture i.v. (62 and 4mg/kg)) to record cardiovascular parameters, renal sympathetic nerve activity (RSNA), and urine flow (UF). Intra-renal pelvic infusion of three doses of bradykinin (53µg/kg, 141µg/kg, 247µg/kg, for 5 min at 20µl/min in random order) was performed, and was repeated following the blockade of bradykinin receptors 1 (BK1R) and 2 (BK2R). Finally, an intra-renal pelvic infusion of capsaicin (0.133µg/kg) was performed. During infusions, changes in blood pressure, heart rate (HR) and contralateral RSNA were recorded. Urine samples were collected from the contralateral kidney. Data are expressed as mean±SD and were analyzed using t-test or ANOVA, where appropriate. IH rats were hypertensive (128±17 vs. 112±22mmHg) with elevated HR (440±37 vs. 403±42bpm), and increased basal UF (56±18 vs. 26±12µl/min/kg); all p<0.05. IH rats had increased resting RSNA, but this was not statistically significant (% of max: 19.6±6.4 vs. 14.8±7.2, p=0.105). All intra-renal pelvic doses of bradykinin augmented RSNA in IH and sham rats (all p<0.05), without changes in blood pressure during infusions. However, UF during bradykinin infusion was unchanged in IH and sham rats. Increases in RSNA and HR in response to 247µg/kg bradykinin were blunted in IH rats compared with shams (RSNA p=0.005; HR p=0.021), with diminished increase in RSNA most evident during the last three mins of infusion (% of baseline: 126±20 vs. 108±5%, p=0.016) and attenuated increase in HR during the last 30s of infusion (∆HR: +7±7 vs. +18±15bpm, p=0.05). This was also evident by a decrease in the area under RSNA curves (AUC) in IH rats compared with shams (1884±970 vs. 5437±3950%.s, p=0.046). AUC analysis revealed that increases in RSNA in response to bradykinin were attenuated during BK1R blockade (p=0.012), and greater suppression of bradykinin-induced increase in RSNA was observed following concurrent BK2R blockade (p=0.005). Intra-pelvic infusion of capsaicin induced a similar increase in HR, RSNA and UF in IH and sham rats. Protein expression of bradykinin and TRPV1 receptors in kidney tissue was equivalent between groups. However, in isolated renal pelvic wall, BK2R expression was 53% less in IH (n=4) compared with sham (n=4), but not statistically significant (p=0.120), which might be related to the small sample size. Our results indicate a blunted reflex increase in RSNA and HR in response to intra-pelvic bradykinin in IH rats, which could be due to down-regulation of the BK2R pathway.