Impact of sleep on chronobiology of micturition among healthy older adults

Abstract

Nocturia (waking to void) is prevalent among older adults. Disruption of the well-described circadian rhythm in urine production with higher nighttime urine output is its most common cause. In young adults, their circadian rhythm is modulated by the 24-h secretory pattern of hormones that regulate salt and water excretion, including antidiuretic hormone (ADH), renin, angiotensin, aldosterone, and atrial natriuretic peptide (ANP). The pattern of hormone secretion is less clear in older adults. We investigated the effect of sleep on the 24-h secretion of these hormones in healthy older adults. Thirteen participants aged ≥65 yr old underwent two 24-h protocols at a clinical research center 6 wk apart. The first used a habitual wake-sleep protocol, and the second used a constant routine protocol that removed the influence of sleep, posture, and diet. To assess hormonal rhythms, plasma was collected at 8:00 am, 12:00 pm, 4:00 pm, and every 30 min from 7:00 pm to 7:00 am. A mixed-effects regression model was used to compare subject-specific and mean trajectories of hormone secretion under the two conditions. ADH, aldosterone, and ANP showed a diurnal rhythm that peaked during sleep in the wake-sleep protocol. These nighttime elevations were significantly attenuated within subjects during the constant routine. We conclude that sleep has a masking effect on circadian rhythm amplitude of ADH, aldosterone, and ANP: the amplitude of each is increased in the presence of sleep and reduced in the absence of sleep. Disrupted sleep could potentially alter nighttime urine output in healthy older adults via this mechanism.

NEW & NOTEWORTHY Nocturia (waking to void) is the most common cause of sleep interruption among older adults, and increased nighttime urine production is its primary etiology. We showed that in healthy older adults sleep affects the 24-h secretory rhythm of hormones that regulate salt-water balance, which potentially alters nighttime urine output. Further studies are needed to elucidate the impact of chronic insomnia on the secretory rhythms of these hormones.

INTRODUCTION

Nocturia, defined by the International Continence Society as waking at night to void (1), is prevalent in older adults. Ninety-three percent of those over the age of 70 yr report waking up at least once a night to void, and 60% report waking at least twice (2). Nocturia is underreported and difficult to treat (3). Nocturnal polyuria, or increased urine production during sleep, is a major contributor to nocturia in the elderly (4, 5), and inadequate understanding of its pathophysiology is a major impediment to improving therapy (6).

Normally, renal excretion follows a well-defined circadian rhythm; <20% of 24-h urine volume is excreted at night (7). Nocturnal polyuria is defined as nocturnal output that exceeds 33% of total daily urine production. Loss (and eventual reversal) of the natural circadian pattern of urine output, without change in 24-h urine volume, is seen with aging (8–10). Nocturnal mobilization of peripheral edema, sleep apnea, or fluid overload conditions, such as poorly controlled diabetes and heart failure, contribute to nocturnal polyuria in the elderly, but its etiology in the absence of these conditions is not clear (3, 4, 9, 11, 12).

Most biological processes including the sleep-wake cycle, body temperature regulation, metabolic homeostasis, and pattern of urinary excretion are closely regulated by 24-h variations in secretion of the hormones that modulate these processes. For some hormones (e.g., melatonin and cortisol), the 24-h fluctuations are strongly regulated by endogenous biological timing system controlled by the suprachiasmatic nucleus of the hypothalamus, whereas for others, sleep, food intake, and general behavior may affect their secretions. For example, human growth hormone secretion demonstrates a sleep-dependent rhythm: the hormone levels peak within 90 min of sleep onset and the surge disappears in the absence of sleep (13). Hormones regulating the circadian pattern of urinary excretion can be divided into two antagonizing systems: vasoconstrictor/Na⁺-retaining systems, such as antidiuretic hormone (ADH) and the renin-angiotensin-aldosterone system (RAAS), and vasodilator/natriuretic systems, such as atrial natriuretic peptide (ANP). Secretion of these hormones shows an endogenous rhythm among young adults with a nighttime time peak. Attenuation of the nocturnal surge is seen with acute sleep deprivation, leading to natriuresis and diuresis (14). Knowledge about the diurnal rhythm of these hormones in healthy older adults and the effect of sleep is limited.

ADH, the major hormone responsible for renal water handling, demonstrates a well-documented diurnal variation in young people (15). This rhythm is closely linked to the sleep-wake cycle and is only weakly associated with the endogenous circadian rhythm (16). The data are less consistent in older adults. One study found a diurnal variation in levels of ADH (17), but others did not (18–20). Asplund et al. (18) reported blunting of the nocturnal phase of ADH secretion in the elderly. Some studies have indicated that daytime plasma ADH concentration is not affected by aging (21–23), but others (24, 25) reported increased basal plasma ADH levels in healthy older adults. These conflicting results may be explained by the heterogeneity of the study populations and methods and study design used. Some studies included subjects with lower urinary tract symptoms and nocturia (17, 19, 20), but others excluded them (18, 26). Experimental methods and timing of specimen collection varied from study to study; blood specimens were collected 4−12 h apart (17, 19, 20, 26), and participants were awakened to draw the overnight samples (18). Sleep was not assessed in these studies, nor was sleep apnea ruled out.

The RAAS regulates renal Na⁺ handling. Plasma renin activity (PRA) shows weak circadian rhythmicity, but there is also evidence that PRA is driven by the sleep-wake cycle. There is a strong concordance between nocturnal oscillations in PRA and sleep cycles, with increasing PRA observed during nonrapid eye movement sleep. The frequency and amplitude of PRA oscillations depend on the regularity and length of nonrapid eye movement sleep cycles, and spontaneous or provoked awakenings blunt the rise in PRA and aldosterone (27–30). In healthy young adults, sleep deprivation has a dramatic impact on urine output, with a >50% increase in nocturnal urine volume (14). Sleep deprivation significantly suppresses nocturnal PRA levels, leading to increased Na⁺, K⁺, and water excretion (14, 31). Although these data suggest an impact on sleep, such studies have been conducted in younger individuals, and the impact of the aging kidney and chronic insomnia process on hormone secretion or electrolyte handling is not known.

The aim of the present study was to better characterize, in healthy older adults: 1) the circadian rhythms of hormones that regulate salt and water excretion, including ADH, renin, angiotensin II (ANG II), aldosterone, and pro-ANP; and 2) the impact of sleep on these rhythms. We postulated that these hormones have a weak endogenous circadian rhythmicity and that sleep plays a role in regulating nocturnal urinary output by increasing secretion of the aforementioned hormones.

MATERIALS AND METHODS

Subjects

Thirteen healthy community-dwelling older adults (8 women and 5 men, age: 65–80 yr) without hypertension, heart disease, peripheral edema, sleep complaints, or nocturia participated in this study. Participants were recruited using the University of Pittsburgh research registry Pitt + Me. The Institutional Review Board of the University of Pittsburgh approved all protocols, and written informed consent was obtained from each participant before enrollment in the study.

Clinical Assessment

All participants were screened for sleep apnea using ApneaLink Plus (ResMed, San Diego, CA), a Federal Drug Administration-approved type III home sleep apnea test device. Participants with ≥15 apnea/hypopnea events per hour were excluded because of the association of moderate sleep apnea with nocturia (32, 33). We also excluded those with a history of bladder cancer, spinal cord lesion, multiple sclerosis, pelvic radiation, interstitial cystitis, urethral obstruction, urinary retention (PVR > 100 mL after noninstrumented void), unstable medical issues including signs of congestive heart failure by exam or NT-pro β-natriuretic peptide (NT-proBNP) > 30 pmol/L (34), chronic kidney disease at stages III−V (estimated glomerular filtration rate < 60), use of >14 alcohol drinks per week or >3 caffeinated drinks (∼300 mg) per day, or significant mental or cognitive impairment (Montreal Cognitive Assessment score < 26). Also excluded were older adults with current use of specific medications that may affect sleep or renal electrolyte excretion: hypnotics, antipsychotics, steroids, β-blockers, angiotensin-converting enzyme inhibitors, angiotensin receptor blockers, or diuretics.

Study Protocol

The dietary intake of eligible participants was assessed over three separate phone calls, 5–7 days apart, for 24-h dietary recall (35). Dietary recall data were limited to intake on weekdays because of the tendency of most persons to alter eating patterns during the weekend. All dietary recalls were conducted without prior notification to avoid changes in diet on the reporting day. Their intake during their normal routine was used to formulate their diet plans during the 24-h study protocols at our clinical research center (CRC). Participants were admitted to the CRC for two 24-h study protocols 6 wk apart: 1) a habitual wake-sleep protocol and 2) a constant routine protocol. Participants were instructed to fast for at least 8–10 h before arriving at the CRC (7:00 am) for each protocol to eliminate potential confounding due to oral intake variability.

During the habitual wake-sleep protocol, participants arrived by 0700 at the CRC. Participants were allowed to pursue usual activities and hobbies like listening to music, watching television, etc. Participants were not time isolated (i.e., clocks, real-time radio, television, and computer/internet were allowed). Meals and sleep were at regular habitual times for individual participants. They were allowed physical activity including walking around the CRC floor but were not allowed to leave the CRC unit. Illumination was maintained at 150 lux during daytime, and lights were turned off during sleep. All three meals were served based on individualized fluid/diet intake in their normal routine. Participants were not allowed to nap. Polysomnography was completed overnight to determine sleep.

During the constant routine protocol, participants arrived by 0700 at the CRC. Starting at 8:00 am and ending 24 h later, participants stayed in continuous wakeful bedrest in a semirecumbent position with the head tilted up by 45° (36). Participants were under temporal isolation, removed from all time cues with no windows, clocks, telephone, or real-time radio, television, or computer. Constant light of <15 lux was maintained during the 24-h duration. Participants were allowed to use a bedside commode. A trained team of technicians looked after the participants, socially interacting with them and monitoring them in person. Meals were replaced by hourly nutritional supplement drinks calibrated to the participant’s 24-h caloric intake in their normal routine.

At 0730 on the day of admission, venous access was established through a heparinized cannula in a cubital vein. Starting at 0800, blood samples were taken every 4 h during the day (8:00 am to 7:00 pm) and every 30 min at night (7:00 pm to 7:00 am the next day). Participants were ambulatory during the daytime for the habitual wake-sleep protocol but were asked to remain seated for at least 10 min before blood sampling. A volume of 10 mL isotonic saline and 0.5 mL heparin (100 U/mL) was injected subsequent to blood sampling to prevent catheter clotting. Care was taken not to wake the participants during blood sampling during the nights when sleep was allowed. Blood sampling during sleep was completed using a catheter passed through a small port next to the bed to the adjoining room.

Blood pressure and heart rate were measured at 2 h intervals from 8:00 am to 8:00 pm and every hour from 8:00 pm to 8:00 am the next morning using a noninvasive ambulatory blood pressure monitor (OnTrak 90227, SpaceLabs Healthcare, Snoqualmie, WA). Participants also underwent overnight polysomnography the night sleep was permitted.

Plasma samples were assayed for the following: ADH, renin, ANG II, aldosterone, and pro-ANP. Circadian rhythms were assessed with phase and amplitude of plasma melatonin rhythms.

Blood was drawn in heparinized syringes and divided into two tubes. For the assessment of ANG II and ADH, aprotinin (kallikrein inhibitor, 500 U/mL) was added to one of the tubes. All samples were immediately centrifuged at 3,000 g for 20 min at 4°C; extracted plasma was aliquoted into individual freezing tubes with a volume of 0.55 mL and stored at −80°C. During the habitual wake-sleep protocol, most participants were asleep between 10:00 pm and 6:00 am the next day. Samples collected during these times were labeled sleep time for analysis.

Biochemistry Determinations

Melatonin was measured by radioimmunoassay (Alpco, Salem, NH). The intra-assay coefficient of variance (CV) for duplicate samples was 4.61%, and the interassay CV was 10.14%. The detection limit was 0.2 pg/mL. Renin was measured by ELISA (Alpco). The intra-assay CV for duplicate samples was 4.1%, and the interassay CV was 12.22%. The detection limit was 0.8 pg/mL. ANG II was measured by ELISA (Phoenix Pharmaceuticals, Burlingame, CA). The intra-assay CV for duplicate samples was 6.53%, and the interassay CV was 8.18%. The detection limit was 5.9 pg/mL. Aldosterone was measured by ELISA (Alpco). The intra-assay CV for duplicate samples was 6.51%, and the interassay CV was 10.47%. The detection limit was 10 pg/mL. ADH was measured by ELISA (Alpco). The intra-assay CV for duplicate samples was 3.31%, and the interassay CV was 3.76%. The detection limit was 2.84 pg/mL. Pro-ANP was measured by ELISA (Alpco). The intra-assay CV for duplicate samples was 5.03%, and the interassay CV was 10.27%. The detection limit was 0.05 nmol/mL.

Statistical Analyses

Paired t tests were performed to assess overall differences in mean hormone secretion between awake and sleep time during the habitual wake-sleep protocol.

To assess the diurnal rhythmicity of hormone secretion, a mixed-effects regression model was fitted to the data for each hormone. The models included fixed intercepts, linear effects to capture “elapsed time” trends (i.e., time as a function from the start of the constant routine), and sine and cosine terms to capture possible 24-h rhythmic temporal trends (circadian patterns), condition effect, and two-way interactions between conditions and all time-dependent effects (linear, sine, and cosine) to capture differences in mean trajectories between the two conditions. The models included a subject random effect to account for correlation from the same subject between conditions (habitual wake-sleep vs. constant routine), and an autoregressive correlation structure to account for within-subject over-time correlations. From the fitted model, we then 1) formally tested for the 24-h rhythm of hormone secretion (awake vs. sleep) during the habitual wake-sleep protocol using the 2-degree of freedom likelihood ratio test of the sine and cosine coefficients for the habitual wake-sleep condition, 2) formally tested any within-subject differences in the mean trajectories between the two conditions using a 4-degree of freedom likelihood ratio test of differences in intercept, linear time, sine, or cosine terms between the two conditions; 3) displayed estimated mean trajectories with 95% confidence intervals for each condition; and 4) displayed estimated within-subject mean trajectories with 95% confidence intervals. These can be used to identify the time periods at which mean differences occur if the hypothesis of equal trajectories within conditions in 2 is rejected. Similar analyses were completed to assess differences in mean arterial pressure (MAP) and heart rate between the two protocols.

RESULTS

Thirteen participants completed both 24-h study visits. All participants were healthy, community-dwelling older adults. Comorbidities included hypothyroidism, degenerative joint disease, acid reflux, osteoporosis, asthma, and seasonal allergies. All had normal renal function at baseline with an estimated glomerular filtration rate of ≥60.

Plasma Hormone Profiles

Mean 24-h profiles of plasma melatonin, ADH, renin, ANG II, aldosterone, and pro-ANP are shown in Fig. 1, A–F. We found significantly higher mean hormone levels during sleep time versus awake time in the habitual wake-sleep protocol for melatonin (P = 0.005), ANG II (P = 0.05), and pro-ANP (P = 0.0002). Mixed-effect regression models showed significant 24-h rhythms for melatonin (P < 0.001), ADH (P < 0.001), aldosterone (P < 0.05), and pro-ANP (P < 0.01). Each of these rhythms showed peak values during the nighttime hours.

Figure 1.

Mean hormonal levels during the habitual wake-sleep (WS) and constant routine (CR) protocol for melatonin (A), antidiuretic hormone (ADH; B), renin (C), angiotensin II (ANG II; D), aldosterone (E), and pro-atrial natriuretic peptide (pro-ANP; F). During the habitual WS protocol, significantly high mean hormonal levels were found during sleep time compared with awake time for melatonin (t = 3.37, P = 0.006, 95% confidence interval: 22.68, 105.82), ANG II (t = −2.11, P = 0.05, 95% confidence interval: −0.13, 0.002), and pro-ANP (t = 5.13, P < 0.001, 95% confidence interval: 0.29, 0.72) using paired t tests. Avg, average. *Statistical significance, P < 0.05.

The fitted mixed-effects regression model demonstrated no significant within-subject difference in melatonin levels between the two conditions (habitual wake-sleep vs. constant routine, P = 0.80; Fig. 2A). Compared with the sleep time of habitual wake-sleep, the ADH level was significantly lower during the nighttime of the constant routine protocol (P < 0.01; Fig. 2B). There were no significant differences in renin levels between the two conditions (P = 0.94; Fig. 2C). ANG II levels were significantly higher (P < 0.01; Fig. 2D), and aldosterone levels were significantly lower (P = 0.01; Fig. 2E), during the nighttime of the constant routine protocol compared with the sleep time of habitual wake-sleep. Compared with the levels during sleep in the habitual wake-sleep protocol, pro-ANP was significantly lower during the nighttime of the constant routine protocol (P = 0.038; Fig. 2F).

Figure 2.

Mean trajectories with 95% confidence intervals for each condition fitted using the mixed-effects regression model for 1) habitual wake-sleep, 2) constant routine (CR) protocol, and 3) within-subject differences for melatonin (A), antidiuretic hormone (ADH; B), renin (C), angiotensin II (ANG II; D), aldosterone (E), and pro-atrial natriuretic peptide (pro-ANP; F). The dotted red line when outside the shaded light gray area demonstrates a significant within-subject difference between the two conditions.

Hemodynamics

For hemodynamic evaluation, MAP and heart rate were analyzed. The well-described nighttime dip in MAP was evident during the sleep time of the habitual wake-sleep protocol (Fig. 3A) between 10:00 pm and 5:00 am. During nighttime of the constant routine protocol, this dipping was attenuated. Diurnal variations were also evident in heart rate (Fig. 3B), with lower levels during the sleep time of the habitual wake-sleep protocol. During the constant routine protocol, no endogenous circadian variation was noted in MAP (Fig. 4Aii) or heart rate (Fig. 4Bii), with a significant within-subject dip in MAP (P < 0.01) and heart rate (P < 0.01) during sleep in the habitual wake-sleep protocol (Fig. 4, A and B, respectively).

Figure 3.

Diurnal variations in mean arterial pressure (MAP; A) and heart rate (B) during the habitual wake-sleep (WS) and constant routine (CR) protocol. During the habitual WS protocol, both MAP (t = −22.07, P < 0.001, 95% confidence interval: −15.68, −27.97) and heart rate (t = −10.34, P < 0.001, 95% confidence interval: −8.34, −13.09) were significantly lower during sleep time compared with awake time using paired t tests. These differences were not significant during the CR protocol. *Statistical significance, P < 0.05.

Figure 4.

Mean trajectories with 95% confidence intervals for mean arterial pressure (MAP; A) and heart rate (B) fitted using the mixed-effects regression model for 1) habitual wake-sleep, 2) constant routine (CR) protocol, and 3) within-subject differences. The dotted red line when outside the shaded light gray area demonstrates significant within-subject difference between the two conditions.

DISCUSSION

The main hormonal systems (ADH, RAAS, and ANP) that influence urine output by the kidney were the focus of this study. Our results suggest that sleep and posture alter the characteristics of the nighttime secretion of these hormones in healthy older adults.

Our study demonstrated a peak in ADH secretion during sleep time among healthy older adults, with attenuation of this peak during the nighttime of constant routine with posture control and constant wakefulness. To the best of our knowledge, our results are the first to indicate that diurnal variation in ADH secretion with a peak during sleep time is preserved in healthy older adults (15). In young adults, a sleep time ADH peak results in the production of lower volumes of concentrated urine overnight (12), which reduces the likelihood of nocturia and facilitates uninterrupted sleep. Acute sleep deprivation, however, attenuates the sleep-related ADH peak and causes diuresis and increased nighttime urine production (14). As previously mentioned, data among older adults are conflicting, and the effect of sleep was not previously assessed. To address the gaps, we carefully selected healthy older adults without sleep apnea or comorbidities affecting nighttime urine production or any use of medications that may affect renal function. For the habitual wake-sleep protocol, we avoided sleep interruption during overnight blood sampling (see Study Protocol). Therefore, we conclude that in addition to the known triggers (plasma osmolality, plasma volume, electrolytes, and even psychological disturbances) (37, 38), levels of ADH are also altered by sleep or lack of it in healthy older adults. This knowledge is clinically relevant because poor sleep and prolonged period of inactivity are common in frail elderly patients. Future studies are warranted to investigate the effect of chronic insomnia on ADH secretion or nocturnal polyuria among older adults.

Although we did not observe any significant variations in renin levels (awake vs. sleep), ANG II and aldosterone were observed to have 24-h rhythms affected by sleep and posture control. Aldosterone had a nighttime peak, which was attenuated during the constant routine protocol. Aldosterone plays a key role in homeostatic regulation of electrolyte balance and extracellular compartment volume. The RAAS, plasma K⁺, and adrenocorticotropic hormone are the major regulators for aldosterone secretion, and it is known to display episodic secretory patterns over a 24-h period with mean plasma level, plasma frequency, and plasma amplitude increasing during the night and early morning hours (39). Studies in young subjects (27, 28) demonstrated that day-night variations in aldosterone secretion are closely tied to the sleep-wake cycle but not posture (40). Our findings are consistent with the studies in young adults and demonstrate a loss of nighttime peak when sleep and posture were controlled. Functionally, decreased aldosterone concentration is considered a predisposing factor to renal salt wasting, natriuresis, and nocturia in the elderly (7, 41).

However, contrary to findings in young adults, our study demonstrated an increase in ANG II during the constant routine protocol. The attenuation of aldosterone peak during the constant routine protocol occurred despite a significant increase in ANG II levels. The classical view of the RAAS pathway begins with renin cleaving its substrate angiotensinogen to produce inactive peptide angiotensin I, which is then converted to ANG II by endothelial angiotensin-converting enzyme. ANG II mediates vasoconstriction as well as aldosterone release from the adrenal gland, resulting in Na⁺ retention and increased blood pressure. We found that the ANG II elevation during nighttime of the constant routine protocol was not paralleled by aldosterone. These findings may indicate an uncoupling of ANG II-mediated aldosterone release with posture control and loss of sleep in older adults. A similar disconnect was noted by Cugini et al. (26) between PRA and aldosterone among older adults. Compared with young adults, the age-related renin decline among healthy older adults was not paralleled by aldosterone. ANG II was not checked in the study by Cugini et al. (26). A potential explanation for this disconnect between the ANG II-mediated aldosterone release during the constant routine protocol would be a difference in K⁺ balance. Decreased K⁺ levels in the blood suppress aldosterone release. K⁺ deprivation is also associated with increased renin release (42), which could increase ANG II levels. Perhaps there is a circadian rhythm to K⁺ handling that is driving some of these changes. If K⁺ balance explains the finding, then one would expect that K⁺ excretion would be relatively increased at night in the constant routine group, and perhaps this would suppress aldosterone.

ANP is another major modulator of Na⁺ excretion with a clear circadian rhythm among young adults (43) but not in older adults (26). Pro-ANP is a prohormone that is secreted from the heart and cleaved to an NH₂-terminal fragment (N-ANP) and a COOH-terminal, biologically active peptide (ANP). ANP is known to be a highly unstable peptide with an extremely short half-life (2–5 min) (44). Pro-ANP is found in the circulation along with ANP and serves as a circulating source of ANP (45); hence, we chose to use pro-ANP for this study. We found that under basal conditions, pro-ANP levels displayed a flat profile during the awake period with a steady increase during sleep, peaking in the early morning hours. This sleep time rise was blunted with posture control and sleep loss. ANP is produced mainly in atrial and ventricular myocytes and secreted in response to volume loading and cardiac wall stretching. ANP is a renal vasodilator and specifically inhibits RAAS-mediated Na⁺ absorption in the proximal renal tubule (46), leading to natriuresis and diuresis. Biologically, ANP has an antagonistic role to the RAAS (47); an increase in ANP results in the suppression of renal renin secretion and aldosterone levels suggesting indirect inhibition of aldosterone secretion by ANP (48). The early morning rise in pro-ANP seen in our study explains the previous findings: as the sleep time surge in ADH and aldosterone potentially leads to Na⁺ and water retention, an early morning rise in ANP works antagonistically and potentially leads to salt-water homeostasis with natriuresis and diuresis. These overnight variations of renal excretion are lost with constant wakefulness and posture.

Consistent with the concept that melatonin is driven primarily by a circadian rhythm rather than sleep (14, 49), we found that the 24-h melatonin profile remained uninfluenced by posture control and sleep loss during the constant routine protocol.

This study has several strengths. As previously noted, the participants were carefully screened to exclude any medical conditions or medication use that may impact renal salt and water excretion. In addition, to assess awake versus sleep time fluctuations, we were careful about maintaining sleep during the overnight blood sampling, which was lacking in previous studies of older adults. The findings thus reflect the physiological rhythm of these hormones in healthy older adults.

To differentiate the role of the endogenous circadian (near 24-h) component versus the contribution of exogenous rhythmic changes, such as food intake or sleep-wake cycle, to the observed rhythmicity in these hormonal variations, we conducted the experiments under constant environmental conditions. The constant routine protocol is a standard method for assessing circadian rhythms and the effect of periodic behaviors on physiological, cognitive, and psychological measures. It is indeed “artificial” compared with normal sleep-wake schedules. However, it remains the simplest method for assessing endogenous circadian phase and amplitude independent of the masking effects of sleep, posture, light-dark cycles, meals, and social interactions (50). During the constant rhythm protocol, rhythms of interest are measured at frequent intervals to obtain phase and amplitude information (51). This protocol enabled us to determine the effect of periodic behaviors (sleep, diet, and posture) on the release of renal regulatory hormones.

The study also has some limitations. First, unmeasured confounders could have affected the association between sleep loss and posture control, and hormonal levels. Second, this study included five men and eight women. We chose not to subdivide the data any further based on sex because such estimates would inherently have a greater instability due to being based on a very small number of observations and therefore we were unable to assess any sex-related differences in hormonal levels. Asplund et al. (52) have reported such differences with men having higher ADH levels than women. However, the study by Asplund et al. (52) was conducted in older adults with nocturia and nocturnal polyuria, and the occurrence of any sex-related differences in healthy older adults is not known. Third, the hormonal variations noted during the constant routine protocol indicate that loss of sleep affects the secretion of renal regulatory hormones; however, the impact of chronic insomnia on nighttime hormonal levels is not known. Our study lacks data on urine volume and urinary excretion of electrolytes during the study protocols. Further studies are needed to elucidate the impact of chronic insomnia on the 24-h rhythms of these hormones and their impact on urinary volume and electrolyte excretion. Finally, our participants were normotensive and demonstrated a nighttime dip in blood pressure during the habitual wake-sleep protocol that was attenuated during the constant routine protocol. Studies have shown that blood pressure has no endogenous circadian variation and that the 24-h blood pressure variations including nighttime dipping observed under normal circumstances are the result of environmental and behavioral factors such as sleep, activity, and food intake (53). Our study cannot determine whether the changes observed in renal regulatory hormones are more strongly related to blood pressure or to wakefulness per se. Further studies in older adults with hypertension and nondipping nighttime blood pressure are warranted to clarify this.

Conclusions

In healthy older adults, during basal conditions, ADH and aldosterone peak during sleep followed by a steady rise in ANP toward the early morning hours. The sleep time ADH and aldosterone peak likely contribute to Na⁺ and water reabsorption leading to decrease in nighttime urine production. The early morning rise in ANP may have an antagonistic effect on the RAAS and lead to natriuresis and diuresis to restore the water and electrolyte balance. All these hormonal rhythms as well as blood pressure dipping are attenuated with posture control and sleep loss. Contrary to findings in young adults, ANG II peaked during the sleep loss condition, likely contributing to the elevation in MAP.

Thus, hormonal regulatory mechanisms for salt-water balance are profoundly affected by sleep and posture in healthy older adults.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was supported by the National Institutes of Health Grants R21AG050892, R21AG060292, P30AG024827, R01GM113243, and R01GM140476.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

S.T., N.M.R., and D.J.B. conceived and designed research; S.T. performed experiments; S.T., B.D.C., G.Z., R.T.K., and S.P. analyzed data; S.T., N.M.R., R.T.K., S.P., A.R.S., and D.J.B. interpreted results of experiments; S.T., B.D.C., G.Z., R.T.K., and S.P. prepared figures; S.T. drafted manuscript; S.T., N.M.R., B.D.C., R.T.K., S.P., A.R.S., and D.J.B. edited and revised manuscript; S.T., N.M.R., and D.J.B. approved final version of manuscript.