INTRODUCTION
Nocturia, a common lower urinary tract symptom defined as waking to void during the main sleep period [1], is a major health problem in middle-aged and older adults, affecting approximately 65% of adults aged >50 years [2]. Nocturia disrupts sleep, increases daytime fatigue, impairs quality of life, and increases the risks of falls, fractures, cardiovascular diseases, and all-cause mortality. Multiple factors contribute, including reduced bladder capacity, disturbed sleep, and nocturnal polyuria (NP), with NP, defined as overproduction of urine at night, accounting for 50%–80% of cases [1, 3].
Excessive dietary salt intake contributes to NP [4], and salt reduction alleviates nocturnal urine production. Further, age-related decline in nitric oxide (NO) production contributes to salt-induced NP. In our clinical study, patients with reduced systemic NO exhibited a positive correlation between dietary salt intake and diurnal polyuria index (DPi), supporting an interaction between decreased NO and salt-induced NP [5]. In animal experiments, pharmacological inhibition of NO production excessively activated the intrarenal renin-angiotensin system (RAS), reducing sodium excretion during the active phase and shifting compensatory excretion to the inactive phase, increasing nocturnal urine output [6]. We recently demonstrated that moderate-intensity exercise, which enhances endogenous NO production, ameliorates salt-induced NP, supporting the critical role of NO in renal sodium and water excretion circadian regulation [6]. Therefore, reduced NO production, high salt intake, and intrarenal RAS activation are key pathophysiological factors in salt-induced NP. However, the mechanism underlying intrarenal RAS activation remains unknown.
Renal sympathetic nerves are crucial in modulating intrarenal RAS activity and sodium reabsorption [7]. Enhanced renal sympathetic activity activates the intrarenal RAS and increases tubular sodium reabsorption, promoting sodium retention and hypertension; thus, renal sympathetic nerve overactivity is a major hypertension mechanism. The cerebral sympathetic nervous system, particularly the paraventricular nucleus (PVN) of the hypothalamus, a central autonomic integration center, regulates renal sympathetic nerve activity in response to high salt intake and reduced NO availability [8]. In this context, increased c-Fos expression in the PVN, a widely used molecular marker of acute neuronal activation, reflects enhanced neuronal activation associated with increased sympathetic outflow. Renal denervation (RD) disrupts renal sympathetic nerves, is linked to resistant hypertension, and reduces blood pressure and sympathetic activity [9]. Therefore, we hypothesized that renal sympathetic nerves and upstream cerebral sympathetic nervous systems mediate intrarenal RAS activation caused by reduced NO production and high salt intake. Consequently, we assessed cerebral sympathetic nervous system activity in an NP mouse model treated with an NO synthase inhibitor plus high salt and evaluated the effects of RD on intrarenal RAS activity and micturition behavior.
MATERIALS AND METHODS
Experiments involving male C57BL/6J mice (SLC Japan, Japan) were performed in compliance with the relevant laws and institutional guidelines (including the National Institutes of Health [NIH] Guide for the Care and Use of Laboratory Animals) and were approved by the Animal Ethics Committee of the University of Osaka (approval No. J008231-002). Mice were maintained in a temperature-controlled room on a 12/12-hour light/dark cycle (active period: 8:00 PM–8:00 AM) with ad libitum food and water access. Systolic blood pressure (average of three measurements) was measured at weeks 0, 2, and 8 using the tail-cuff method (BP-98A; Softron Corp., Japan). Automated voided stain on paper (aVSOP) measurements were performed at weeks 0 and 8. At week 8, mice were sacrificed by isoflurane inhalation, and kidney and brain tissues were collected.
Feed and Drinking Water Administration
Normal salt diet (NSD) and 1% high-salt diet (HSD) were purchased from Oriental Yeast (Japan). Nω-nitro-L-arginine methyl ester (L-NAME) (N5751-10G, Sigma-Aldrich) and hydralazine hydrochloride (Hyd) (H1753, Sigma-Aldrich) were dissolved in drinking water at 0.5 g/L and 0.2 g/L, respectively, and provided to mice ad libitum. Hydralazine lowers blood pressure without directly suppressing sympathetic activity, and hence is an appropriate control to distinguish blood pressure-dependent effects from sympathetic nervous system-dependent effects on DPi.
Renal Denervation
Mice were anesthetized by intraperitoneal injection of a medetomidine Hyd (0.75 mg/kg), midazolam (4 mg/kg), and tartaric acid butorphanol (5 mg/kg) mixture at 0.1 mL per 10 g of body weight. A midline dorsal incision exposed the kidneys and arteries. The renal nerves were carefully dissected and removed microscopically. Following manual RD, 10% phenol in ethanol was administered to the renal arteries. Sham mice underwent the same procedure without nerve removal or phenol application.
Urine Volume and Time Measurements
Urine volume was measured using the aVSOP method [10, 11]. Mice were housed in cages (110 mm×160 mm×75 mm) for 4 days. A laminated, colorimetric filter paper was moved under a water-repellent grid at 10 cm/hr. Urine spots were analyzed with ImageJ (v1.53e, NIH), and DPi—the ratio of urine volume during the inactive period to total daily urine volume—was calculated.
Norepinephrine Content Measurement
Norepinephrine (NE) content was measured from 100 mg of right and left renal tissues homogenized in 500 μL of 0.2 M perchloric acid, or from urine, followed by centrifugation. Supernatants were filtered, and NE was extracted using a phenylboronic acid spin column conditioned with acetic acid and phosphate buffer. Samples were spiked with the internal standard 3,4-dihydroxybenzylamine, and eluted catecholamines were analyzed by high-performance liquid chromatography equipped with an Eicom ECD-800 electrochemical detector using a COSMOSIL 3PBr column at 50°C. NE content was quantified by internal-standard calibration curves based on peak-height ratios [12].
Immunohistochemical Quantification of c-Fos in the PVN
c-Fos–positive cells in the PVN were quantified in coronal sections through brain tissue immunohistochemistry (primary antibody: rabbit anti-c-Fos [ab190289, Abcam, 1:1,000]; secondary: anti-rabbit IgG horseradish peroxidase [HRP] [1:5,000]). Serial 30-μm sections spanning the entire PVN were collected. Images were captured using a bright-field microscope. An observer blinded to the experimental groups performed manual counts. Only cells with distinct nuclear staining clearly above background were considered positive. Typically, 5 representative sections per animal were analyzed bilaterally, and the mean number of c-Fos–positive cells per section was calculated.
Immunoblotting
Proteins were extracted from whole kidneys using a lysis buffer and prepared in sodium dodecyl sulfate (SDS) sample buffer (Cosmo Bio, Japan). Protein concentrations were determined using the Lowry method. Samples (20 μg) were subjected to 8%/10% SDS-PAGE (polyacrylamide gel electrophoresis) and immunoblotting. Membranes were blocked with Blocking One (Nacalai Tesque, Japan), incubated overnight with the primary antibodies anti-angiotensinogen (Abcam, AB213705; 1:1,000) and anti-β-actin (A1978; 1:1,000; Sigma-Aldrich, USA), and then with the HRP-conjugated anti-rabbit IgG secondary antibody (7074, 1:5,000; Cell Signaling Technology, USA). Signals were detected using Chemi-Lumi One (Nacalai Tesque, Japan) and visualized with ChemiDoc XRS Plus (Bio-Rad Hercules, USA).
Statistical Analysis
Group comparisons were performed using Student t-test, paired t-test, 2-way repeated measures analysis of variance, and Tukey test, as applicable. Holm correction was used for multiple comparison tests. All data are presented as means±standard errors. Statistical significance was set at P<0.05. All analyses were performed using JMP ver. 17.1.0 (SAS Institute, USA).
RESULTS
Cerebral and Systemic Sympathetic Nerve Activity in NP Model Mice
First, we evaluated cerebral and renal sympathetic nerve activities in NP model mice randomized into NSD, 1% HSD, L-NAME, and L-NAME+HSD groups. We measured urine volume every 4 hours over 3.5 consecutive days before and after the intervention (Fig. 1A) and assessed temporal changes in the DPi. The DPi significantly increased in the L-NAME+HSD group (from 0.229±0.006 to 0.348±0.010, P=0.007) but not in the NSD (0.217±0.019 to 0.216±0.001, P=0.959), HSD (0.208±0.009 to 0.227±0.008, P=0.077), and L-NAME (0.238±0.006 to 0.234±0.022, P=0.583) groups (Fig. 1B). After an 8-week intervention, renal AGT/β-actin (ACTN) levels in the L-NAME+ HSD group significantly exceeded those in the NSD, HSD, and L-NAME groups (1.437±0.174 vs. 0.438±0.084 vs. 0.639± 0.115 vs. 0.795±0.111 arbitrary units, P=0.001, P=0.013, and P=0.004, respectively) (Fig. 1C). Next, we investigated the relationship between NP and the cerebral sympathetic nervous system activity. The number of c-Fos–positive cells in the PVN of the L-NAME+HSD group was significantly higher than those of the NSD, HSD, and L-NAME groups (27.50±3.11 vs. 3.50±0.99 vs. 9.67±1.12 vs. 8.00±1.59 cells/section, respectively; P<0.001 for all comparisons) (Fig. 1D and E). To evaluate systemic sympathetic nervous system activity, we examined the 24-hour urinary NE excretion, which was significantly higher in the L-NAME+HSD group than in the NSD group (0.369±0.053 μg/day vs. 0.109±0.022 μg/day, P=0.028). No significant differences were observed between the other groups (Fig. 1F). Systolic blood pressure during the active period was not changed in the NSD (from 106.5±2.9 to 104.1±3.2 mmHg, P=0.294) and L-NAME groups (from 106.6±3.2 to 117.4±1.7 mmHg, P=0.053) but increased progressively in the HSD (from 109.4±1.6 to 117.6±2.0 mmHg, P=0.037) and L-NAME+ HSD (from 104.3±6.1 to 118.0±3.1 mmHg, P=0.034) groups (Fig. 1G). Therefore, NO suppression and high salt intake synergistically activate the cerebral and systemic sympathetic nervous systems and intrarenal RAS.
RD Effects on Intrarenal RAS Activity and DPi in NP Model Mice
To clarify the effects of increased cerebral sympathetic nervous system activity on intrarenal RAS activity and micturition behavior, we evaluated intrarenal RAS activity and DPi changes induced by RD in mice randomized into Sham and RD groups. In RD, sympathetic nerves were resected along with the surrounding tissue around the renal artery and vein (Fig. 2A). After a 2-week postsurgical recovery period, both groups were treated with 1% HSD and L-NAME for 8 weeks. To confirm successful renal sympathetic nerve blockade, kidney NE concentration was evaluated, which was significantly lower in the RD group than in the Sham group (12.27±2.40 vs. 124.86± 14.24 ng/g tissue, P=0.002) (Fig. 2B). Renal AGT/ACTN level in the RD group was also significantly lower than in the Sham group (1.070±0.091 vs. 1.595±0.136 arbitrary units, P=0.009) (Fig. 2C). Thus, RD may reduce the renal sympathetic nervous system and intrarenal RAS activity.
Next, we examined temporal changes in micturition behavior. We analyzed the urine volume every 4 hours for 3.5 consecutive days before and after the intervention (Fig. 2D). The DPi increased significantly in the Sham group (from 0.212±0.009 to 0.338±0.021, P<0.001) but not in the RD group (from 0.198±0.011 to 0.182±0.018, P=0.223) (Fig. 2E). Moreover, we examined the systemic sympathetic nervous activity via 24-hour urinary NE excretion, which was significantly lower in the RD group than in the Sham group (0.131±0.024 μg/day vs. 0.301± 0.030 μg/day, P=0.001) (Fig. 2F). Systolic blood pressure during the active period did not change significantly in the RD group (from 96.7±6.7 to 102.2±5.7 mmHg, P=0.344), whereas it increased progressively in the Sham group (93.7±4.3 to 112.2±4.2 mmHg, P=0.004) (Fig. 2G). Therefore, RD may suppress intrarenal RAS activity and reduce DPi in the NP mouse model; the activation of the cerebral sympathetic nervous system and renal sympathetic nerves may contribute to intrarenal RAS activation and DPi elevation.
The Effect of Blood Pressure Reduction by Hyd on DPi
RD suppressed intrarenal RAS activity and reduced DPi in the NP mouse model. Nevertheless, it suppressed the elevated blood pressure. The DPi reduction by RD may be mediated either through intrarenal RAS activity suppression or blood pressure elevation downregulation. To clarify the effect of blood pressure suppression on the DPi, we conducted experiments using Hyd, an antihypertensive drug, in mice randomized into the L-NAME+HSD+Hyd and L-NAME+HSD groups; both were treated with the respective interventions for 8 weeks, and temporal changes in blood pressure and micturition behavior were examined.
In the L-NAME+HSD+Hyd group, systolic blood pressure did not change significantly (from 101.9±4.3 to 87.6±5.3 mmHg, P=0.083), whereas in the L-NAME+HSD group, it increased progressively (104.3±6.1 to 118.0±3.1 mmHg, P= 0.034) (Fig. 3A). We analyzed the urine volume every 4 hours for 3.5 consecutive days before/after intervention (Fig. 3B). In both groups, the DPi significantly increased (L-NAME+ HSD+Hyd: from 0.189±0.012 to 0.327±0.017, P=0.001; L-NAME+ HSD: from 0.219±0.010 to 0.328±0.015, P=0.001), with no difference between the two groups (Fig. 3C). Thus, although Hyd reduced blood pressure, DPi was not suppressed. Therefore, DPi reduction with RD may be mediated by intrarenal RAS activity suppression, rather than lowering blood pressure.
DISCUSSION
Decreased NO production combined with excessive salt intake induced overactivity in the cerebral/renal sympathetic nervous systems. RD attenuated intrarenal RAS activation and reduced the DPi in an NP mouse model. Because mice are nocturnal, polyuria during inactive periods corresponds to NP in humans. Thus, intrarenal RAS activation appears mediated by enhanced cerebral and renal sympathetic activity.
Excessive salt intake may trigger NP, but current evidence is inconsistent. Our previous studies have suggested that decreased NO, together with excessive salt intake, may promote intrarenal RAS activation and contribute to NP pathophysiology [5, 6]. Nevertheless, the precise underlying mechanisms remain unclear.
Cerebral/renal sympathetic nerve activation contributes to intrarenal RAS activation in NP model mice. NO within the hypothalamus PVN is essential for regulating cerebral sympathetic outflow. Reduced neuronal NO synthase expression and the resulting decline in NO production within the PVN are associated with increased sympathetic excitation [13]. PVN activation in the NP model is likely due to L-NAME-induced suppression of NO synthesis. Notably, c-Fos–positive cells did not increase with L-NAME alone but when combined with an HSD, indicating that salt loading, besides reduced NO, drives PVN activation. The activated PVN stimulates the renal sympathetic pathway, regulating intrarenal RAS activation [14]. Renal sympathetic nerves reach the kidney via the periarterial nerve plexus surrounding the renal artery and innervate both vascular and tubular segments of the renal parenchyma. This input mediates renal vasoconstriction, increases renin secretion, and activates the intrarenal RAS, ultimately enhancing renal sodium reabsorption [15]. In the NP model, this sequence likely decreased sodium excretion during the active period, with a compensatory increase in sodium excretion and urine volume during the inactive period.
RD significantly reduced the DPi in the NP model, suggesting a potential therapeutic approach for NP. Clinically, RD treats resistant hypertension via catheter-based radiofrequency ablation of the renal sympathetic nerves, with trials showing significant blood pressure reductions and favorable safety profiles [9]. Because RD exerts antihypertensive effects, we examined whether the observed DPi reduction was simply secondary to blood pressure lowering. Although RD significantly reduced blood pressure in NP model mice, pharmacological blood pressure reduction with hydralazine did not decrease DPi, indicating that blood pressure reduction alone is insufficient to ameliorate NP. In contrast, RD simultaneously suppressed renal sympathetic activity, leading to attenuation of intrarenal RAS activation and reduction of DPi. Therefore, the DPi-lowering effect of RD is mediated primarily by suppression of intrarenal RAS activation, rather than by its antihypertensive action per se. The NP model used in the present study was characterized by NP development driven by sympathetic hyperactivity and subsequent intrarenal RAS activation. Accordingly, hydralazine, which does not directly act on these mechanisms, did not improve NP, whereas RD, which directly suppresses sympathetic activity and intrarenal RAS activation, was considered responsible for the observed improvement in NP. RD also lowered urinary NE excretion, a marker of systemic sympathetic activity, indicating effects beyond the kidney and modulation of systemic sympathetic overactivity. Thus, RD may disrupt the vicious cycle between heightened sympathetic drive and intrarenal RAS activation.
To further characterize the phenotype of our NP model and address concerns regarding potential sodium-centered bias, we performed additional analyses regarding voiding behavior. Daytime urine volume was significantly reduced, and nighttime urine volume was increased, whereas total daily urine volume remained unchanged, indicating a redistribution of urine production rather than global polyuria. Voided volume per micturition showed no significant change during the active period, with only a modest increasing trend during the inactive period, and voiding frequency was not significantly altered during either period (Supplementary Fig. 1). These findings indicate that non–sodium-dependent alterations in voiding behavior were limited and that the present NP model should be regarded as a sodium-dependent, sympathetic/RAS-driven NP phenotype rather than a comprehensive model encompassing all clinical forms of NP.
Although RD effectively suppressed sympathetic activity and improved NP in our model, it is an invasive procedure and is not intended as first-line therapy for NP. From a clinical perspective, avoiding excessive salt intake represents a far more practical and feasible intervention and should be prioritized in patients with salt-induced NP. However, in cases where patients continue to experience NP despite adequate salt restriction, persistent sympathetic overactivity and intrarenal RAS activation may underlie the refractory phenotype, as demonstrated in the present study. Therefore, RD may represent a potential therapeutic option for selected patients refractory to dietary salt restriction. Further clinical studies are required to identify selection criteria for this subgroup.
This study had some limitations. First, although we elucidated how reduced NO production and high salt intake activate intrarenal RAS, clinical NP arises from complex physiological and behavioral factors, including aging, sleep disorders, and cardiovascular comorbidities. Our findings may therefore explain only a part of the pathophysiology. Second, although PVN activation led to intrarenal RAS activation and induced NP, it may also influence vasopressin secretion and renal water handling. However, the present study did not include direct assessment of vasopressin levels, and the contribution of vasopressin-mediated water reabsorption to NP in our model remains speculative. Future studies measuring vasopressin levels will be required to clarify the role of PVN-vasopressin pathways in the pathophysiology of this NP model. Finally, the efficacy of RD in NP of other etiologies remains unclear. The mechanistic conclusions of this study are restricted to salt-related NP driven by sympathetic hyperactivity and intrarenal RAS activation and should not be extrapolated to NP phenotypes primarily mediated by vasopressin dysregulation, bladder dysfunction, or sleep-related mechanisms. Further studies will be required to elucidate the pathophysiology of these NP phenotypes.
Overall, reduced NO production and excessive salt intake activate cerebral and renal sympathetic nervous systems, triggering intrarenal RAS activation and NP development. RD improves NP independent of blood pressure reduction by suppressing sympathetic activity. Cerebral and renal sympathetic nerves are pivotal in NP pathophysiology; RD may be a potential therapeutic strategy to improve NP.
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