This is an original study on nocturia and the role of peripheral circadian dysfunction of the bladder. Nocturia is defined by the International Continence Society (ICS) as “the number of times urine is passed during the main sleep period. Having woken to pass urine for the first time, each urination must be followed by sleep or the intention to sleep”. Commonly accepted causes of nocturia include global polyuria, nocturnal polyuria, reduced bladder capacity, sleep disorders, or a combination of these factors. While specific treatments are available for each of these causes, nocturia often persists. Recent advances in circadian biology and sleep science have suggested that we need to study nocturia as a form of circadian dysfunction. Prior studies have shown active circadian gene cycling in tissues throughout the body. It has been reported that dysregulated circadian rhythms, via shift work, can differentially affect the peripheral organ function of both kidney (urine production) and bladder (voiding). From this work, investigators found that the circadian pattern of urine production adapted rather quickly to circadian disruption following shift work, however the circadian rhythm of bladder capacity remained recalcitrant to rapid shifts in day-night rhythms, leading to aggravation of nocturia in shift workers. Prior studies in mouse models have also demonstrated the potential role of peripheral circadian rhythm dysregulation in nocturia. Leveraging previously published research, we hypothesized that peripheral circadian gene dysregulation plays an important role in nocturia. Our aims were:
a)	To define the presence and rhythmicity of peripheral circadian bladder gene expression in humans.
b)	To analyze the role of circadian rhythmicity of bladder genes in nocturia.

Institutional review board approved informed consent was obtained from all subjects prior to enrollment. For analysis of peripheral circadian rhythms, the most feasible method for repeated sampling of the bladder is through the collection of urine and bladder wash specimens at various time points. We recruited five nocturia subjects with ≥ 2 voids/night and two controls with ≤1 void/night. Bladder wash and whole urine specimens were obtained at the time of procedures in the operating room. With the bladder wash we aimed to collect only cells from the bladder to determine differential expression compared to whole urine specimens which may include cells from the upper urinary tract. Whole urine samples (50-70 ml) were also collected at various points during a 24-hour day-night cycle. Cells were isolated from each specimen for bulk mRNA sequencing, followed by data analysis.

Our results demonstrate several interesting findings in control and nocturia subjects as follows:
a)	In our preliminary data we compared daytime bladder wash gene expression in subjects with and without nocturia and found 536 differentially expressed genes. We then compared night versus day gene expression in subjects with and without nocturia and found 795 differentially expressed genes during the day cycle, and interestingly this number increased to 1919 in urine samples collected at night.
b)	We found that the PIEZO1 channel and CLOCK genes have coinciding rhythmic expression in controls, whereas there was no rhythmicity in expression of these genes in nocturia (Figure 1). 
c)	We analyzed connexin 43 and connexin 30 in our data and found that in control subjects there was a rhythmicity in the expression of these genes, but in nocturia subjects gene rhythmicity was absent. 
d)	Apart from these genes, we also found that aquaporin 9 (AQP9), a water and glycerol channel previously shown to have circadian rhythm in other organ systems, was noted to have very high expression in nocturia.

Our study is the first to analyze the role of peripheral circadian rhythm associated genes in nocturia using human samples and is consistent with previously published literature as follows:
a)	Our findings are suggestive of two things: i) most of the cells isolated from urine samples are of bladder origin when we compared differentially expressed genes in bladder washes (536) and urine samples (795) during the daytime; and ii) during the night cycle, a significant increase in the number of differentially expressed genes was noted in subjects with and without nocturia, suggestive of circadian dysregulation.
b)	Our results are consistent with previous studies in a mouse model, which found that sensation of bladder fullness follows a circadian rhythm, which is created by functional circadian changes of PIEZO1 being controlled by CLOCK genes to be active during wakefulness and inactive during sleep [Reference 1]. This study suggested a circadian rhythmicity between CLOCK genes and PIEZO1, which was also noted in our study using human samples (Figure 1).
c)	Our results are consistent with a study in a mouse model which showed that connexin 43, a bladder gap junction protein, loses its circadian rhythmicity of expression in Cry-knockout mice [Reference 2]. The inconsistency of connexin 43 and connexin 30 gene expression in our nocturia subjects is suggestive of circadian dysregulation.
d)	Although no previous data is available to suggest a role of AQP9 in nocturia, its role has been implicated in neurological conditions such as Alzheimer’s and Huntington’s disease, which are known to have circadian dysfunction [Reference 3]. Our findings suggest there might be a link between circadian dysfunction and high AQP9 expression in nocturia.

Our results suggest that peripheral circadian rhythms of bladder genes may be implicated in nocturia. There are limitations to our study given the limited number of subjects, limited timepoints of sample collection, and potential heterogeneity in the pathogenesis of each subject’s nocturia. Considering these limitations, our findings are the first to report on the gene rhythmicity of bladder in humans and are consistent with previously published work in animal models of circadian dysfunction. Further research in humans and animal models is needed to determine the role of CLOCK in the control of peripheral gene expression of the bladder.

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Continence 7S1 (2023) 100811
DOI: 10.1016/j.cont.2023.100811