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

Abstract

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.

INTRODUCTION

Arterial blood pressure (ABP), renal function, and sympathetic nerve activity (SNA) oscillate within a 24-h circadian period (1–4). In rodents, ABP and heart rate are higher during nighttime (active period) versus daytime (inactive period) (3, 5, 6). Renal function similarly oscillates within a 24-h circadian period as glomerular filtration rate, renal plasma flow, renin secretion, and the excretion of water and electrolytes are higher during circadian active periods (7–9). Direct recordings of renal, splanchnic, or lumbar SNA in unanesthetized rodents demonstrate a clear circadian rhythmicity with higher levels at nighttime versus daytime (3–6, 10). In humans, microneurography recordings of muscle SNA also show a circadian rhythmicity with higher SNA activity during daytime versus nighttime periods (11–13). Transsynaptic viral tracing studies using pseudorabies virus demonstrate that sympathetically innervated organs such as the kidneys are polysynaptically connected to multiple autonomic centers and to the central circadian clock or the suprachiasmatic nucleus (SCN) (14, 15). Therefore, an anatomical framework exists by which central circadian circuits influence SNA to control the circadian oscillations in renal function and ABP.

The kidneys are innervated by renal nerves, which are comprised of both sympathetic efferent and afferent (sensory) fibers. Renal afferent fibers sense changes in the chemical composition of the renal parenchyma or changes in renal pressures and relay this information to the central nervous system. Thus, activation of renal afferent nerves by endogenous renal chemokines (e.g., bradykinin, substance P, or adenosine) (16–19) or changes in intrarenal pressures (20–22) produces sympathetic reflex responses in efferent SNA and ABP (16, 17, 23, 24). Such reno-renal reflexes help maintain homeostasis (25). Although the absolute level of SNA, renal function, and ABP vary with the circadian cycle, little information exists on whether reflex changes in SNA and ABP evoked by visceral inputs arising from the kidney are also influenced by the circadian cycle. Although we recently demonstrated that afferent renal nerve activity (ARNA) responses to chemosensory and mechanosensory renal stimuli were not different between nighttime and daytime (22), the extent to which SNA and hemodynamic responses to renal sensory stimuli are different between nighttime and daytime remained to be tested. Since SNA and ABP are higher during nighttime versus daytime in rodent models, we hypothesized that the circadian cycle would produce greater SNA reflex responses during nighttime active period. To test this hypothesis, we performed in vivo multifiber nerve recordings of renal, splanchnic, and lumbar SNA and simultaneously measured ABP and heart rate during intrarenal arterial infusion of bradykinin or capsaicin and during elevations in renal pelvic pressure.

METHODS

Animals

All experimental procedures conform to the National Institutes of Health’s Guide for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh Institutional Animal Care and Use Committee. Male and female Sprague–Dawley rats (250–400 g, Charles River Laboratories) were pair-housed in a temperature-controlled room (22 ± 1°C) with a 12-h:12-h dark/light cycle (lights on at 0700–1900) and given ad libitum access to deionized water and 0.1% NaCl chow diet (Research Diets, D17020). Experiments performed during the light cycle (daytime) began at 1000 and data collection started 2.5–3 h later. For experiments performed during the dark cycle (nighttime), rats were transported to the laboratory for experimentation at 1700, placed in a darkened enclosure at 1900, anesthetized, and their eyelids secured shut under darkness. To minimize light exposure, room lights were kept to a minimum and surgical lamps were directionally blocked to only illuminate working spaces. Animal instrumentation began at 2100, and data collection started 2.5–3 h later. Animals studied during daytime periods were reported in a prior study (24) and are presented here as the daytime control group (n = 6; 8 males, 8 females) to limit unwarranted animal numbers.

General Procedures

Rats were anesthetized with isoflurane (2% to 3% in 100% O₂), and the depth of anesthesia was assessed by the absence of paw withdrawal responses to foot pinch. Brachial artery and femoral vein catheters (PE-50 tubing) were implanted to monitor ABP (BPM-832 Dual Pressure Monitor, CWE, Inc.) and infuse saline (0.25 mL/h iv), respectively. Animals were artificially ventilated via tracheotomy to maintain end-expiratory CO₂ between 3.5% and 4.5% and O₂ between 35% and 45% (Gemini Respiratory Gas Analyzer, CWE, Inc.). Body temperature was continuously monitored and maintained at 37 ± 0.5°C using a rectal thermometer and circulating water pad. Through a ventral midline incision, a heat-stretched catheter (0.037 OD × 0.023 ID micro-renthane, Braintree Scientific) was placed at the entrance of the right renal pelvis (contralateral to the renal SNA recording) via the ureter and exteriorized at the level of the bladder. The presence of urine flow without blood indicated proper catheter placement. The animals were then placed in a stereotaxic frame. The right kidney was approached through a retroperitoneal incision, and the adrenal artery that branches off the right renal artery was cannulated with heat-stretched micro-renthane tubing (17, 22). After all surgical procedures were completed, isoflurane anesthesia was replaced by Inactin (120 mg/kg, 0.2 mL/min iv; Sigma T133). Experiments began at least 45 min later. Number of animals (including sex) per group (n) is reported in the figure or table legends.

Experimental Protocol. Experiment 1: SNA and Hemodynamic Responses to Chemo- and Mechanosensitive Stimuli

Animals were prepared as described in General Procedures and simultaneous recording of ABP and lumbar, renal, and splanchnic SNA as previously described (26–28). The lumbar sympathetic nerve was isolated and placed on bipolar stainless-steel electrodes and insulated with KWIK-SIL (WPI, Sarasota, FL). The left renal and splanchnic nerves were isolated through a left retroperitoneal incision by gentle retraction of the kidney (contralateral to the adrenal and renal pelvic catheters). Nerves were placed on separate sets of bipolar electrodes and insulated with KWIK-SIL. Nerve signals were amplified (×10,000) and filtered (0.3–1.0 kHz) using a differential AC amplifier (A-M Systems; Sequim, WA), monitored on a Tektronix TDS 2004 digital oscilloscope, and then digitized at 2 kHz using a Micro1401 and Spike2 software (Cambridge Electronic Design, Inc.). SNA from all three nerves showed clear cardiac-related bursts and sympathoinhibition to acute injection of phenylephrine (4 μg/kg iv; Sigma P6126, data not shown). Lumbar, renal, and splanchnic SNA were rectified and integrated (1-s time constant). The nerve signal was calculated by subtracting the signal obtained after ganglionic blockade (30 mg/kg iv; hexamethonium; Sigma H0879). All nerves had a signal-to-noise ratio greater than 2:1. Baseline values were normalized and set at 100%.

Stimuli were tested in a randomized order using chemokine concentrations and renal pelvic pressure steps (22, 24). Intrarenal artery infusion of bradykinin (0.1–10.0 μΜ; Sigma, B3259), capsaicin (0.1–10.0 μΜ; Sigma, M2028), or saline vehicle was infused (0.05 mL over 15 s) via an adrenal artery catheter branching from the right renal artery in a randomized order (17, 24). Solutions were flushed through the catheter with 0.12 mL saline at 0.4 mL/min. A 3-min recovery period between bradykinin doses and a 6-min recovery period between capsaicin doses were used as previously described (22). SNA was averaged in 1-s bins. A rolling 5 s average was calculated, and responses that achieved two SDs above a 30-s baseline segment were analyzed using the area under the curve. Responses were not tested in a small subset of animals lacking a renal-branching adrenal artery. Increased renal pelvic pressure was performed by connecting the ureteral-pelvic catheter to a pressure transducer and column of water by y-connector. Pelvic pressure was elevated (0, 2, 5, 10, and 20 mmHg) for 30 s followed by a 2-min recovery period. At the end of the pressure steps, the y-connector was disconnected. Data were averaged in 1-s bins. A 3-s rolling average was calculated, and responses were assessed by area-under-the-curve analysis.

Experiment 2: ARNA Responses to Intrarenal Bradykinin Infusion

We previously reported that intrarenal artery infusion ofcapsaicin or elevated renal pelvic pressure similarly increases ARNA between nighttime and daytime (22). However, ARNA responses to intrarenal infusion of bradykinin were not previously tested. Thus, a second cohort of rats was anesthetized, ventilated, and instrumented as described in General Procedures for monitoring ABP. Through a retroperitoneal incision, the right adrenal artery was cannulated with heat-stretched micro-renthane tubing. Then, the right renal nerve was placed on bipolar stainless-steel electrodes, insulated with KWIK-SIL, and sectioned proximally to the recording electrode to isolate ARNA. Intrarenal artery infusion of bradykinin (0.1–10.0 µM) or saline vehicle was infused (0.05 mL over 15 s) via the adrenal catheter. Solutions were flushed with 0.12 mL saline at 0.4 mL/min. A 3-min recovery period between bradykinin doses. Data were averaged in 1-s bins and a 3-s rolling average was calculated. The ARNA signal was calculated by subtracting the noise obtained after sectioning the nerve distal to the recording electrode. Values were normalized to baseline values set at 100% or as nerve discharge (Hz) using a window discriminator (22).

Experiment 3: ARNA Responses to Intrarenal End PO Intravenous Bradykinin and Capsaicin

A third group of male rats was anesthetized, ventilated, and prepared for recording of ABP and ARNA as described in General Procedures. Although the right kidney is frequently innervated by multiple nerve branches, the nerve isolated for ARNA recordings was the only nerve transected. After a 60-min stabilization period, bradykinin (0.1, 1.0, and 10 µM), capsaicin (0.1, 1.0, and 10 µM), or saline was infused (0.05 mL over 15 s) via adrenal artery (intrarenal) or femoral venous (intravenous) catheter. Solutions were flushed with 0.12 mL over 15 s. Solutions were tested in a randomized order and separated by 3 min. Data were analyzed as described in experiment 2.

Statistical Analysis

All data were analyzed using GraphPad Prism v.9.0 software. Daytime data for efferent SNA responses were previously published (24) and have been included for statistical comparisons between nighttime datasets. Data were tested for normality using Shapiro–Wilk test and equal variance using Bartlett’s test. Data that did not pass normality were log-transformed. A repeated-measures one-way ANOVA with a Dunnett’s post hoc test or a mixed-effect two-way ANOVA followed by a Student’s t test was performed for statistical testing. All data were presented as means ± SE. P values < 0.05 were statistically significant.

RESULTS

Baseline Sympathetic Nerve Activity and Hemodynamics

Table 1 summarizes baseline mean ABP, heart rate, and SNA measured as a voltage in Inactin-anesthetized rats. Mean ABP and heart rate were significantly higher during nighttime versus daytime (Table 1). Renal SNA was also significantly higher during nighttime versus daytime. In contrast, splanchnic SNA and lumbar SNA were not significantly different between groups.

Table 1.

Baseline mean ABP, heart rate, renal SNA, splanchnic SNA, and lumbar SNA during daytime and nighttime

	n	Mean ABP, mmHg	Heart Rate, beats/min	SNA, μV
				Renal	Splanchnic	Lumbar
Daytime
Total	16	97 ± 3	343 ± 7	1.60 ± 0.54	1.48 ± 0.24	0.48 ± 0.09
Male	8	89 ± 4#	342 ± 10	2.75 ± 0.94#	1.98 ± 0.38#	0.67 ± 0.14#
Female	8	105 ± 3	343 ± 10	0.46 ± 0.07	0.98 ± 0.18	0.30 ± 0.06
Nighttime
Total	22	104 ± 3*	372 ± 10*	2.32 ± 0.46*	1.20 ± 0.14	0.66 ± 0.09
Male	12	100 ± 3	376 ± 16	2.70 ± 0.69	1.41 ± 0.22	0.53 ± 0.10
Female	10	108 ± 4	367 ± 10	1.87 ± 0.59	0.97 ± 0.13	0.81 ± 0.14

Values are means ± SE; n, number of rats. ABP, arterial blood pressure; SNA, sympathetic nerve activity. Baseline values were analyzed between groups using a Student’s t test (*P < 0.05). Differences between males and females were analyzed using a two-way ANOVA with Bonferroni post hoc test (#P < 0.05).

Table 1 further shows baseline hemodynamic and SNA values between male and female animals. Daytime mean ABP was significantly lower in males versus females, whereas renal, splanchnic, and lumbar SNA were significantly higher in males versus females. Despite these baseline diurnal differences, daytime ARNA or efferent SNA responses to renal capsaicin infusion or elevated renal pelvic pressure is not influenced by sex (22). Moreover, we did not find sex differences in SNA or hemodynamic reflex responses in this study. Therefore, male and female data are combined but individual data points, for male and female animals, are represented within each data set.

SNA and Hemodynamic Responses to Intrarenal Bradykinin Infusion Are Exaggerated during Nighttime

To determine the extent to which circadian time influences SNA and hemodynamic reflex responses to renal chemosensory stimuli, we measured contralateral renal SNA, splanchnic SNA, lumbar SNA, and ABP during intrarenal bradykinin infusion. Figure 1 illustrates SNA and ABP responses to 10 μΜ bradykinin infusion during daytime and nighttime. Intrarenal arterial infusion of bradykinin promptly increased renal SNA, splanchnic SNA, and ABP in both groups. Lumbar SNA exhibited a biphasic response profile during nighttime that increased in parallel with renal and splanchnic SNA but decreased after ABP increased. Therefore, the analysis of lumbar SNA was restricted to the response profile during the bradykinin infusion and increase in ABP. Figure 2 shows summary data. Intrarenal bradykinin infusion produced concentration-dependent changes in SNA during both daytime and nighttime. Infusion of bradykinin (1.0–10.0 μΜ) significantly increased renal SNA (daytime: P ≤ 0.05; nighttime: P ≤ 0.05; ANOVA) and splanchnic SNA (daytime: P ≤ 0.05; nighttime: P ≤ 0.05; ANOVA). Bradykinin only increased lumbar SNA using 10 μM bradykinin (daytime: P ≤ 0.05, nighttime. P ≤ 0.05; ANOVA). Interestingly, intrarenal bradykinin infusion produced significantly larger responses in renal SNA (10.0 μΜ; P < 0.05) and splanchnic SNA (10.0 μΜ; P < 0.05) during nighttime versus daytime. Lumbar SNA was not significantly different between groups (Fig. 2).

Figure 1.

Examples of rectified/integrated ($\int$) renal SNA, splanchnic SNA, and lumbar SNA, raw SNA, heart rate, ABP, mean ABP (white) to intrarenal arterial infusion of 10.0 μΜ bradykinin (black line; 0.05 mL; 0.2 mL/min) during daytime and nighttime. Raw SNA tracings (0.5 s) highlight baseline and peak bradykinin responses. ABP, arterial blood pressure; SNA, sympathetic nerve activity.

Figure 2.

Means ± SE and individual data points of renal SNA, splanchnic SNA, lumbar SNA, heart rate, and mean ABP responses to intra-arterial renal infusion of bradykinin (0.1–10.0 μΜ) during daytime [white bar; n = 8 males (●), n = 5–7 females (□)] and nighttime [gray bar; n = 11 males (●), n = 10 females (□)]. Mixed-effects two-way ANOVA followed by a Student’s t test was performed for significance between groups (*P < 0.05). A repeated-measures one-way ANOVA was performed followed by Dunnett’s post hoc test for significance within groups compared with saline infusion (#P < 0.05). ABP, arterial blood pressure; SNA, sympathetic nerve activity.

In addition, intrarenal infusion of bradykinin significantly increased ABP at 10.0 μΜ bradykinin (Fig. 2; daytime: P ≤ 0.05, nighttime: P ≤ 0.05; ANOVA). A significant increase in heart rate was only observed during nighttime using 10.0 μΜ bradykinin (P ≤ 0.05; ANOVA). A direct comparison of nighttime versus daytime periods revealed significantly larger pressor responses during nighttime versus daytime. No significant differences in heart rate were observed between groups.

SNA and Hemodynamic Responses to Intrarenal Capsaicin Infusion during Nighttime and Daytime

To further assess the extent to which circadian time influences SNA and hemodynamic responses to renal chemoreflex activation, the TRPV1 agonist capsaicin was infused into the renal artery. Figure 3 illustrates SNA and ABP responses to 10 μΜ capsaicin. Intrarenal arterial infusion of capsaicin produced prompt increases in renal SNA, splanchnic SNA, and ABP during daytime and nighttime. Interestingly, lumbar SNA was unchanged during daytime but increased during nighttime. The lumbar SNA also exhibited a biphasic response at nighttime. Again, the analysis of lumbar SNA was restricted to the capsaicin infusion and increase in ABP. Figure 4 shows summary data for SNA and hemodynamic responses to capsaicin. Intrarenal arterial capsaicin infusion produced concentration-dependent increases renal SNA (daytime: P ≤ 0.05; nighttime: P ≤ 0.05; ANOVA), splanchnic SNA (daytime: P ≤ 0.05; nighttime: P ≤ 0.05; ANOVA). The magnitude of renal SNA and splanchnic SNA responses was significantly larger between daytime and nighttime at 10 μΜ capsaicin. In contrast, lumbar SNA significantly increased during nighttime in response to 1.0 μΜ capsaicin (P ≤ 0.05; ANOVA). However, lumbar SNA responses were not significantly different between daytime and nighttime.

Figure 3.

Example of rectified/integrated ($\int$) renal SNA, splanchnic SNA, lumbar SNA, raw SNA, heart rate, ABP, and mean ABP (white) during intrarenal arterial infusion with 10.0 μΜ capsaicin (black line; 0.05 mL; 0.2 mL/min). Raw SNA tracings (0.5 s) highlight baseline and peak capsaicin responses. ABP, arterial blood pressure; SNA, sympathetic nerve activity.

Figure 4.

Means ± SE and individual data points of renal SNA, splanchnic SNA, lumbar SNA, mean ABP, and heart rate responses to intra-arterial renal infusion of capsaicin (0.1–10.0 μΜ) during daytime [white bar; n = 8 males (●), n = 5–7 females (□)] and nighttime [gray bar; n = 11 males (●), n = 10 females (□)]. Mixed effects two-way ANOVA followed by a Student’s t test was performed for significance between groups (*P < 0.05). A repeated-measures one-way ANOVA was performed followed by Dunnett’s post hoc test for significance within groups compared with saline infusion (#P < 0.05). ABP, arterial blood pressure; SNA, sympathetic nerve activity.

In addition, intrarenal infusion of capsaicin significantly elevated mean ABP at 10.0 μΜ capsaicin (Fig. 4; daytime: P ≤ 0.05; nighttime: P ≤ 0.05; ANOVA) but not heart rate. There were no significant differences in mean ABP or heart rate responses between daytime and nighttime.

SNA and Hemodynamic Responses to Elevated Renal Pelvic Pressure Are Not Different between Daytime and Nighttime

We also tested the extent to which the circadian cycle influences SNA and hemodynamic responses to elevated renal pelvic pressure (0, 1, 2, 5, 10, and 20 mmHg; 30 s each). Figure 5 illustrates SNA and ABP responses to 20 mmHg elevations in renal pelvic pressure during daytime and nighttime. Elevated renal pelvic pressure produced sympathoexcitatory responses in renal SNA and splanchnic SNA during both time periods. Lumbar SNA, mean ABP, and heart rate did not change.

Figure 5.

Example of rectified/integrated ($\int$) renal SNA, splanchnic SNA, lumbar SNA, heart rate, ABP, and mean ABP (white) during 30-s elevation in renal pelvic pressure (20 mmHg). Raw SNA tracings (0.5 s) highlight baseline and peak responses. ABP, arterial blood pressure; SNA, sympathetic nerve activity.

Figure 6 shows summary data for SNA and hemodynamic responses to elevated renal pelvic pressure. During daytime, renal SNA and splanchnic SNA responses were significantly different than a 0-mmHg pressure step at 10–20 mmHg (P ≤ 0.05; ANOVA). During nighttime, a significant difference in renal SNA and splanchnic SNA was observed at 20 mmHg (P ≤ 0.05; ANOVA) versus 0 mmHg. Lumbar SNA was not significantly different during either time period. However, there were no significant differences between daytime and nighttime responses for renal or splanchnic SNA (Fig. 6). Mean ABP and heart rate were not significantly different compared with a 0-mmHg step during daytime or nighttime. (Fig. 6).

Figure 6.

Means ± SE and individual data points of renal SNA, splanchnic SNA, lumbar SNA, mean ABP, and heart rate responses during renal pelvic pressure elevation (0–20 mmHg) during daytime [white bar; n = 10 males (●), n = 8 females (□)] and nighttime [gray bar; n = 12 males (●), n = 8 females (□)]. Mixed-effects two-way ANOVA was performed for significance between groups. A repeated-measures one-way ANOVA was performed followed by Dunnett’s post hoc test for significance within groups compared with 0 mmHg (#P < 0.05). ABP, arterial blood pressure; SNA, sympathetic nerve activity.

ARNA Responses to Bradykinin Did Not Differ between Daytime and Nighttime

To test whether greater SNA and hemodynamic responses to intrarenal artery infusion of bradykinin can be attributed to a greater ARNA response, we assessed ARNA responses to bradykinin during daytime and nighttime. Figure 7A illustrates examples of ARNA responses to 10.0 μM bradykinin during daytime and nighttime periods. Acute infusion of bradykinin produced prompt increases in integrated ARNA and ARNA discharge. ARNA activity returned to baseline levels after the infusion was completed. Saline infusion did not alter ARNA activity (examples not shown).

Figure 7.

A: representative traces of raw renal afferent nerve activity (ARNA), integrated ARNA, ARNA discharge, ABP, and mean ABP (white line) during acute intrarenal arterial infusion with 10.0 μΜ bradykinin (black line; 0.05 mL; 0.2 mL/min) during daytime and nighttime. Representative raw ARNA nerve tracings (0.2 s) highlight baseline and peak bradykinin responses. Intrarenal artery infusion of bradykinin promptly increased ARNA discharge and integrated ARNA across all groups. B and C: means ± SE and individual data points of the integrated ARNA (B) and peak change (Δ) in discharge during intra-arterial renal infusion of bradykinin (C) during daytime (white bar); n = 6 males (●), n = 4 females (□) and nighttime [gray bar; n = 6 males (●), n = 4 females (□)]. Mixed-effects two-way ANOVA was performed for significance between groups. A repeated-measures one-way ANOVA was performed followed by Dunnett’s post hoc test for significance within groups compared with saline infusion (#P < 0.05). ABP, arterial blood pressure; SNA, sympathetic nerve activity.

Figure 7, B and C, shows summary data for ARNA responses during daytime and nighttime. Bradykinin produced a dose-dependent increase in integrated ARNA and ARNA discharge during daytime (P ≤ 0.05; ANOVA) and nighttime (P ≤ 0.05; ANOVA) (Fig. 7B). However, these ARNA responses did not differ between daytime and nighttime (Fig. 7C).

Intrarenal but Not Intravenous Bradykinin and Capsaicin Infusions Increased ARNA

To assess whether the intrarenal infusions selectively activate renal sensory nerves versus a generalized systemic effect in the circulation, a final set of experiments compared ARNA and ABP responses with intrarenal versus intravenous infusions of bradykinin versus capsaicin. Intrarenal infusion of bradykinin produced concentration-dependent increases in ARNA and mean ABP (Fig. 8). However, intravenous infusion of bradykinin did not alter either variable. Similarly, intrarenal infusion of capsaicin produced concentration-dependent increases in ARNA and ABP (Fig. 8); however, intravenous infusion of capsaicin did not alter either variable.

Figure 8.

A: example of ARNA, rectified/integrated ARNA, ARNA discharge, ABP, and mean ABP (gray line) during intrarenal or intravenous infusion of bradykinin (1 µM, 50 µL). Representative raw ARNA insets are 0.6 s. B: means ± SE and individual data points of rectified/integrated ARNA, ΔARNA discharge, and Δmean ABP in response to intrarenal vs. intravenous infusion of bradykinin or capsaicin (n = 4 males). Intrarenal infusion of bradykinin and capsaicin produced concentration dependent increases in ARNA, ARNA discharge, and mean ABP. However, intravenous infusion did not alter any variable. A two-way repeated-measures ANOVA followed by paired t tests with a layered Bonferroni correction. *P < 0.05 intrarenal vs. intravenous. #P < 0.05 vs. saline. ABP, arterial blood pressure; ARNA, renal afferent nerve activity; SNA, sympathetic nerve activity.

DISCUSSION

The present study examined the extent to which the circadian cycle influenced efferent SNA and hemodynamic reflex responses to renal chemosensory and mechanosensory stimuli. First, intrarenal arterial infusion of bradykinin or capsaicin produced exaggerated sympathoexcitatory responses at nighttime versus daytime. However, ARNA responses to bradykinin were not different between nighttime and daytime. In contrast, SNA and ABP responses to elevated renal pelvic pressure were not different between daytime and nighttime. Thus, renal chemokines elicit greater sympathoexcitatory responses at nighttime versus daytime and suggest that the circadian cycle modulates sympathetic responses to visceral afferent activation.

ABP and SNA exhibit circadian patterns. Nocturnal animals such as rodents exhibit higher ABP and renal SNA during nighttime or active period versus daytime (5, 6). In the present study, baseline renal SNA and mean ABP of Inactin-anesthetized rats were higher at nighttime versus daytime. Such differences are attributed to light-sensitive changes in central circadian activity levels, particularly within the SCN (29, 30). Environmental light cues encode time-of-day differences into SCN neurons, which changes neuronal excitability and reverses day-night responsiveness to neurotransmitters such as GABA (31, 32). Lesion within the SCN or deletion of circadian SCN genes prevents day-night oscillations in ABP, HR, locomotion, and hormone secretion (33–37).

The activation of renal afferent fibers by endogenous renal chemokines increases SNA and ABP (16, 17, 23, 24). Bradykinin is an endogenous vasoactive peptide and a renal sensory stimulus that evokes a sympathoexcitatory response (24, 38), a renal nerve-dependent increase in vascular resistance and a pressor response (17, 19). Herein, intrarenal arterial infusion of bradykinin produced dose-dependent increases in renal SNA, splanchnic SNA, and ABP. The sympathoexcitatory renal SNA and splanchnic SNA agree with reported reductions in regional blood flow and pressor responses to intrarenal bradykinin measured in conscious animals (17, 39). Moreover, the present findings confirm that intrarenal but not intravenous infusion of bradykinin increases ARNA and ABP (17, 19). Directly related to the hypothesis, sympathoexcitatory responses were greater at nighttime versus daytime periods. Interestingly, lumbar SNA was not significantly altered by the circadian cycle indicating a nonuniform impact of the circadian cycle on lumbar SNA responses to bradykinin. This curious finding suggests that renal bradykinin evokes differential SNA to regulate kidney function, blood volume, and/or resistance beds. These circadian-dependent effects cannot be attributed to differences in renal afferent nerve activation as ARNA responses to bradykinin were not significantly different between nighttime and daytime periods. These new findings are similar to our previous report showing a lack of circadian influence on ARNA responses to intrarenal arterial infusion of capsaicin or elevated renal pelvic pressure (22). Thus, exaggerated reflex responses to renal bradykinin administration at nighttime are attributed to a change in the central excitability of central autonomic networks.

The TRPV1 agonist capsaicin was used to test SNA and hemodynamic responses to a second chemosensitive stimulus. TRPV1 channels are expressed in renal sensory nerves (40–42), and renal administration of capsaicin increases ARNA and produces renal and splanchnic sympathoexcitation but intravenous infusion does not (22, 24, 43, 44). Herein, intrarenal capsaicin infusion increased renal SNA, splanchnic SNA, and ABP. Like the findings with bradykinin, renal SNA, and splanchnic SNA responses were sensitive to the circadian cycle and exhibited larger responses during nighttime versus daytime. A small increase in lumbar SNA was observed only at night. It should be noted that there are limitations to using capsaicin as a renal sensory test stimulus. First, capsaicin is not endogenously produced by the kidneys. Second, capsaicin desensitizes TRPV1 channels at higher concentrations and can functionally attenuate primary afferent nerve transmission (45–47). Regardless, these data further support our hypothesis that SNA responses to renal chemokines are exaggerated at nighttime in rodents.

Increased renal pelvic pressures of 10–20 mmHg elevated renal SNA and splanchnic SNA but did not change lumbar SNA. These responses did not differ between daytime and nighttime periods. It is noteworthy that these data contrast previous studies showing increased renal pelvic pressure produces renal sympathoinhibition (20). However, there are key methodological differences. The present study employed short (30 s) pressure steps of lesser magnitude ≤20 mmHg using Inactin anesthesia versus longer pressure steps (10–20 min) of >20 mmHg using pentobarbital sodium anesthesia. Prolonged pressure steps of >20 mmHg may impede renal capillary or urine flow, prevent clearance of renal metabolites, and therefore activate chemosensory fibers (21, 48). In the current study, the lack of a circadian effect on SNA and ABP during increased renal pelvic pressure may have two possible explanations. First, distinct autonomic pathways may mediate SNA responses to renal chemo- versus mechanosensitive stimuli. Thus, the SCN or central clock may selectively influence renal chemosensitive pathways. Unfortunately, limited information exists regarding the central pathways mediating chemo- or mechanosensitive responses. Second, the degree of visceral afferent input for a given change in efferent output may determine whether the circadian cycle modulates SNA. For example, a circadian effect was only observed using concentrations of chemokines that evoke robust ARNA (22) and SNA responses. Lower chemokine concentrations evoke small ARNA and little or no SNA responses. The latter lacked a circadian effect. Increased renal pelvic pressure produces a degree of ARNA (and SNA) similar to that of low concentration chemokine infusions. Therefore, a circadian influence on reflex SNA and hemodynamic responses arising from visceral afferents may depend on the degree of afferent activation. Future studies are needed to test these possibilities.

This study provides novel evidence that the circadian cycle exaggerates sympathoexcitatory responses to renal visceral afferent stimulation using chemokines. The mechanisms responsible for such effects are unknown but may include multiple explanations. First, basal SNA is determined by the balance of excitatory and inhibitory inputs onto sympathetic regulatory neurons (e.g., rostral ventrolateral medulla). Presumably, the higher baseline SNA at nighttime could be attributed to a decreased inhibitory input or disinhibition. In turn, this would raise resting SNA and leave visceral inputs less opposed resulting in a greater sympathoexcitation to reflex activation. A second possibility is that the circadian cycle produces day/night fluctuations in the excitability or intrinsic properties of sympathetic neurons (e.g., input resistance) such that a greater sympathoexcitatory response can be evoked from a given visceral input and not necessarily raise baseline SNA. Transneuronal viral tracing studies from the kidney and other sympathetically innervated organs using pseudorabies virus have reported consistent labeling of key autonomic centers such as the rostral ventrolateral medulla, raphe, A5 region, and hypothalamic paraventricular nucleus (15). At longer survival times, the SCN is labeled suggesting that the central clock is anatomically linked and thus may influence the autonomic nervous system. Future experiments are required to assess the cellular mechanisms contributing to the greater sympathoexcitatory responses at nighttime and test whether the circadian cycle modulates the magnitude of other visceral afferent reflexes.

The current findings have several important implications. First, renal function exhibits a clear circadian pattern that is characterized by higher plasma renin levels and a greater excretory capacity during daytime in humans or nighttime in rodents (7, 49). The extent to which renal efferent or afferent nerves contribute to circadian patterns of renal function should be tested. Second, the circadian cycle clearly influences the absolute level of SNA and ABP (5, 6) but the findings highlight the circadian cycle also modulates sympathetic and hemodynamic responses to activation of renal visceral afferents. Surprisingly, there are limited data regarding the extent to which the circadian cycle also modulates reflex hemodynamic responses to activation of other visceral afferents such as cardiac, muscle, baroreceptors, and/or carotid chemoreceptors. This has important implications for disease states associated with visceral afferent dysfunction. In this regard, cardiovascular disease is prevalent when the circadian cycle is disrupted. For example, myocardial infarctions, arrhythmias, and stroke exhibit clear time-of-day patterns, and sleep-wake disturbances are common in patients with end-stage renal disease (50–52). Nightshift workers exhibit increased risks of hypertension, stroke, and coronary heart disease (53–55). Finally, the majority of endpoints in rodent studies are performed during the inactive period (daytime). Our findings indicate that time of day may influence functional endpoints related to SNA, ABP, and renal function, and therefore such experimental factors should be considered when conclusions are extrapolated to human disease.

In summary, the circadian cycle has a clear impact on the sympathetic nervous system, cardiovascular function, and renal function. The present study identified a novel circadian effect by which SNA and hemodynamic responses to renal chemokines are greater at nighttime versus daytime periods in rodents. In addition, renal afferent activation produced regional differences in SNA responses to chemokines. Altogether, our findings highlight that the circadian cycle is an important variable in understanding sympathetic responses to visceral afferent activation.

GRANTS

The research was supported by National Institutes of Health Grants R01 HL145875 (to S.D.S.), R01 HL152680 (to S.D.S.), and postdoctoral F32 DK123994 (to L.J.D.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

L.J.D. and S.D.S. conceived and designed research; L.J.D. and S.D.S. performed experiments; L.J.D. analyzed data; L.J.D. and S.D.S. interpreted results of experiments; L.J.D. prepared figures; L.J.D. and S.D.S. drafted manuscript; L.J.D. and S.D.S. edited and revised manuscript; L.J.D. and S.D.S. approved final version of manuscript.