RECENT ADVANCES IN UNDERSTANDING THE CIRCADIAN CLOCK IN RENAL PHYSIOLOGY

Abstract

Accumulating evidence suggests a critical role for the molecular circadian clock in the regulation of renal function. Here, we consider the most recent advances in our understanding of the relationship between the circadian clock and renal physiology.

Introduction

Our bodies are challenged on a daily basis with variables that can potentially change our internal milieu. There are systemic cues that prepare our bodies for these daily events in order to maintain temporal homeostasis. The process by which the body anticipates varying conditions each day is performed by fluctuating mechanisms, which comprise the circadian clock [1]. The circadian field has gained recent attention due to three distinguished scientists (Jeffrey Hall, Michael Rosbasch, and Michael Young) being awarded the Nobel Prize in Physiology or Medicine in 2017 for their isolation and characterization of the circadian gene Period (Per) in fruit flies [2].

Briefly, the molecular circadian clock is comprised of a series of feedback loops that are primarily controlled by four key proteins (CLOCK, BMAL1, PER, and CRY). This molecular pathway is present in nearly every cell type and organ system in the body. The master clock, located in the suprachiasmatic nucleus (SCN), uses light input to give time cues to peripheral clocks in other cells of the body. Peripheral tissues also possess the ability to sustain their own circadian rhythms to meet individual demands. For a more thorough review on this process, see Partch et al [3]. Not only are circadian genes widespread in the body, they are also found in nearly every organism, ranging from bread mold to humans, and their function allows for adaptation to our planet with a 24-hour light and dark cycle. Importantly, these circadian genes are highly conserved between mice and humans [4]. It is well established that disruption of sleep and circadian pathways in humans are associated with increased risk for a number of pathologies including cancer and cardiovascular diseases [5,6]. Therefore, considering circadian clock function is imperative to our overall understanding of human physiology and pathophysiology.

Circadian regulation of renal function is particularly important with regards to daily variations in our nutrient and fluid intake. A major function of our kidneys is to maintain homeostasis via the fine-tuned regulation of electrolyte balance and fluid volume. A contributor to the control of fluid balance is the increased release of vasopressin by the brain during the active period compared to the inactive period [7]. Additionally, the expression of vasopressin receptors in the kidneys is controlled by clock genes [8]. Both of these mechanisms work together in order to maintain stable extracellular volume control throughout the day. Contribution of the circadian clock to renal physiology is an active area of investigation. The purpose of this review is to provide a succinct summary of the most recent literature pertaining to the circadian clock in the kidney and speculate on future directions for these important findings. For a more comprehensive summary of this topic, please see [9,10].

Circadian Rhythms Research in the Kidney

Rodents are a useful model when studying circadian rhythms in the kidney due to the similarities in renal function and the daily fluctuations that occur in these functions when compared to humans [10,11]. An important note to consider is that mice and rats are nocturnal, or active during the night, as opposed to humans who are diurnal, or normally active in the day. Consequently, renal functions that exhibit circadian rhythms, such as electrolyte excretion, renal blood flow, filtration, and oxygenation of renal tissue, peak in the night-time when the mice and rats are active instead of the daytime as seen in humans [12]. Although the kidneys are not the only organs that express clock genes, they are second only to the liver in terms of the number of genes that exhibit circadian cycles [13] suggesting that the circadian clock is particularly important to the health and proper function of the kidneys. Indeed, components of the circadian clock have been linked to each tubule segment via in vitro cell culture studies or in vivo animal experiments focused on transporters associated with specific cell types (Figure 1).

Figure 1. Summary of Recent Evidence Linking the Circadian Clock to Renal Function.

Diagram of a nephron indicating the section of the tubules of recent focus as determined by the transporters that were examined in in vitro or in vivo studies in the last four years. For a more comprehensive review of this topic, see [9,10].

Sodium Handling

Per1

The kidneys exhibit circadian rhythms in function including blood pressure control [14], glomerular filtration rate [15], and tubular handling [15,16]. The majority of recent circadian research focuses on sodium balance and its effects on blood pressure. The diurnal rhythm of tubular handling, particularly sodium handling, occurs independent of posture and food/water intake [17]. Per genes 1 and 2 have been described as the best candidates for conducting circadian oscillations partly due to their short half-life [18]. The clock gene Per1 in the kidney is an early aldosterone target gene that transcriptionally regulates targets such as the expression of epithelial sodium channel (ENaC), sodium-glucose linked transporter-1, sodium-hydrogen exchanger-3, and endothelin-1 (ET-1) which are essential in controlling sodium reabsorption [19–22]. When Per1 knockout mice are challenged with a relatively mild treatment of high salt (HS) and deoxycorticosterone pivalate (DOCP), which is a long-acting analog of aldosterone, the night/day difference of sodium excretion is attenuated and the blood pressure dip during the inactive period is lost [23,24].Blood pressure normally dips 10– 20% during the inactive period. Individuals who do not exhibit this drop in blood pressure are termed ‘non-dippers’. Along with this non-dipping phenotype, Per1 knockout mice following HS/DOCP treatment also exhibit dysfunction in the expression of sodium handling genes causing altered sodium excretion [24].Furthermore, a distal nephron-specific Per1 knockout mouse was created using cadherin Cre recombinase, which resulted in improper regulation of sodium transporter genes [24].These studies suggest that Per1-dependent transcriptional regulation of Na handling genes is important for the maintenance of 24-hour blood pressure and that this effect is dependent on the actions in the distal nephrons. This model can be used to determine the contribution of non-dipping to changes in kidney function and development of injury.

Bmal1.

Bmal1 stimulates transcription of numerous genes that have been linked to maintaining proper kidney function [25,26]. This relationship has also been shown in kidney-specific knockout mice where Bmal1 was knocked out specifically in renin-producing cells (Renin1dCre-positive). These mice exhibited decreased urine osmolality and increased urine volume compared to controls [26]. Global Bmal1 knockout mice lost their circadian rhythm in blood pressure such that their blood pressure did not rise during the active period. This response was associated with a reduction in plasma catecholamines [27]. Spontaneously hypertensive rats (SHR) are another animal model that is often used to study the role of Bmal1 with blood pressure regulation [28]. In this study, single nucleotide polymorphism (SNP) databases were analyzed to discover that SNPs in the Bmal1 gene are associated with hypertension in humans [28]. In a recent study, SHR treated with a combination of a calcium channel blocker and an angiotensin receptor blocker to reduce blood pressure also exhibited a reduction in Bmal1 mRNA levels in the kidneys [29]. Inducible, kidney-specific knockout mice have been studied to determine the role of Bmal1 specifically in renal nephrons using Bmal1lox/lox/Pax8-rtTA/LC1 mice [30]. The metabolome in the plasma of the whole-nephron-specific Bmal1 knockout mice was altered compared to littermate controls [30]. There was an increase in plasma urea and creatinine levels in the knockouts which was attributed to the decreased renal expression levels of OAT3 [30]. Blood pressure measurements indicated that inducible, nephron-specific Bmal1 knockout mice exhibit significantly lower systolic blood pressure compared to control. The circadian rhythm of blood pressure in the knockout mice was comparable to controls [30]. Bmal1 in the adult male mouse kidney thus appears to play a role in blood pressure regulation and renal function; however, its role in mediating the circadian rhythm of blood pressure may be extra-renal. Exploring the mechanisms by which Bmal1 affects blood pressure may give insight on how SNPs in Bmal1 could lead to pathophysiology in humans.

Timing Cues; Intrinsic and Extrinsic

Endothelin.

Endothelin-1 (Edn1) is a PER1 target gene [31] which exhibits a circadian expression at the level of mRNA [32]. Gene expression of ETa and ETb receptors in the kidneys changes depending on the time of day. The expression of these genes is not altered in Per1 heterozygote mice, however, endothelin-1 levels during the active period are higher in inner medullary collecting duct cells isolated from Per1 heterozygote mice compared to wild type mice on the 129/sv background strain [31]. HS stimulates renal ET-1 production and activation of the ETb receptor [33]. Additionally, HS can alter the circadian rhythm of blood pressure. Therefore, recent studies looked at the role of ET-1 receptors in response to HS. In a comprehensive study, Johnston et al. demonstrated that rats lacking ETb receptor function have an impaired natriuretic response to an acute salt load which depends on the time of day [34]. This response suggests that there is an interaction between endothelin and the circadian clock to regulate the renal excretion of salt. In an additional study, Speed et al. found that Bmal1 displayed a phase shift of expression in control rats after HS, however, this shift was not present in rats lacking ETb receptors [35] suggesting that ET-1, through ETb, is necessary for normal circadian clock gene expression rhythms. The sodium channels that were affected in this study were not determined; however, cultured inner medullary collecting duct cells treated with ET-1 exhibited a drop in alpha ENaC protein expression [35]. Taken together, these data suggest that ET-1 is both a target of and perhaps a timing cue for the circadian clock.

Vasopressin.

There is a rhythm in vasopressin release which is highest during sleep in order to retain water due to the lack of water intake at this time [36]. A recent study by Hara et al. found that the rat inner medulla, but not the cortex, has a diurnal rhythm in osmotic pressure, where the peak is reached during the active phase and the nadir during the inactive phase [37]. This rhythm was also displayed with sodium, chloride, and urea inner medullary concentrations. These patterns were attributed to matching rhythms in vasopressin receptors V1aR and V2R, aquaporin 2, and the urea transporter UT-A2 occurring in the inner medulla. To beautifully demonstrate the circadian rhythms of clock proteins and localization in the renal medulla, a PER2-Luciferase-2 knock-in mouse was used for macroscopic bioluminescence imaging [37]. The renal medulla regulates urine osmolality by several mechanisms included in this study, which strengthens the importance of the circadian clock on this process. Vasopressin is a critical circulating hormone that is rhythmically released in order to synchronize peripheral clocks.

Corticosteroids.

Cortisol has long been considered a primary synchronizing cue for the peripheral clocks [38]. There is mounting evidence of the contribution of the adrenal glands to the circadian clock in the kidney. A seminal study in 1977 demonstrated that plasma cortisol plays a key role in the circadian rhythm of renal function [39]. Hormones released by the adrenal glands, such as cortisol and aldosterone, have a circadian rhythm [39,40]. The removal of the adrenal glands, eliminating any production of these hormones, lowers the amplitude of mRNA from renal core clock genes and clock-output genes [41]. Alternatively, chronic administration of corticosterone to healthy mice, maintaining plasma levels midway between the physiological peak and trough, resulted in an increase in Bmal1 and Per1 during the inactive phase. This effect was associated with increased phosphorylation of the thiazide-sensitive sodium-chloride cotransporter (NCC) and a ‘non-dipping’ blood pressure phenotype [42]. Night-time dosing of hydrochlorothiazide treatment in these mice reversed the non-dipping phenotype back to normal, which was attributed to the long half-life of thiazides. Furthermore, adrenalectomy resulted in a blunted rhythm of phosphorylated NCC expression in the kidney [42]. Future studies of this type with other natriuretic or diuretic agents may shed light on additional mechanisms that likely contribute to the normal blood pressure rhythm. Several hormones are key for communication throughout the body with regard to circadian cues. Understanding the role of adrenal hormones may be a crucial link between the central clock in the SCN and peripheral clocks, especially with kidney function.

Renal Nerves.

Although the mechanisms are incompletely understood, nervous system signaling is an important timing cue among the peripheral clocks, which has also been described to be true for the kidneys. Renal nerves stimulate renin secretion and sodium reabsorption directly as well as indirectly by vasoconstriction of afferent and efferent arterioles. A rat model of metabolic syndrome (SHR/NDmcr-cp rats on standard diet) with non-dipping hypertension exhibited a large reduction in blood pressure, along with normalization of the blood pressure circadian rhythm following renal denervation. This was partially attributed to an increase in natriuresis due to suppression of renin production and reduction in NCC overexpression [43]. Although clock genes were not measured, NCC has been associated with some of these genes previously [44] as well as with glucocorticoid signaling and blood pressure rhythms [42]. An additional study also demonstrated that renal denervation partially corrected disruptions in diurnal autonomic tone in ETB-deficient rats [45]. Renal denervation has been tested as a clinical treatment for hypertension with controversial results. Targeting the effects of renal denervation that are beneficial could be considered to provide better outcomes.

Novel Links to the Renal Circadian Clock

Microbiota.

The effect of the microbiota on physiological processes in the body is a field that is quickly growing. A recent report was able to determine the circadian rhythms found in gut bacteria with the entrainment of the clock in peripheral organs, including the kidney [46]. This group concluded that the microbiota modulates the phase of the host peripheral clocks by the release of short-chain fatty acids (SCFA) released from the bacteria, specifically the SCFA lactate [46]. This relationship is intriguing given the extremely diverse microbiota and how the population can change drastically depending on the diet of the host. Promotion of a more beneficial composition of the gut microbiota in mice achieved by a high fiber diet prevented a DOCA induced elevation in blood pressure, circadian pressures were not provided. A similar response occurred following treatment with acetate, which is another SCFA that is released by bacteria. The expression of multiple genes in the kidney were changed following a high fiber diet and acetate supplementation where 244 similar genes were affected in the same fashion, particularly Bmal1 which was highly down regulated in both high fiber and acetate treated mice. An additional parameter that was assessed in this study was renal fibrosis. Acetate supplementation prevented fibrosis formation following DOCA treatment; however, it is important to note that a high fiber diet did not attenuate fibrosis. These studies provide evidence that continued research should include full integrative physiology.

Fibrosis.

Other studies have more directly examined the relationship between the clock and renal fibrosis. Circadian genes regulate the expression of genes that are pro-fibrotic such as CTGF, fibronectin, and tumor necrosis factor-α in cell culture [47]. Mice with inducible KO of BMAL1 in renal tubular cells did not display fibrosis or inflammation in the kidney compared to controls [30]. Furthermore, uninephrectomized CLOCK Δ19 mice, which express a mutant of the CLOCK protein that is unable to dimerize with BMAL1, had attenuated renal inflammatory responses compared to wild type following HS/DOCP treatment which is mediated by mineralocorticoid receptor signaling [48]. Conversely, CLOCK-deficient mice when challenged with unilateral ureteral obstruction had greater fibrosis compared to wild type [49]. Increased oxidative stress and fibrotic tissue assessed by immunohistochemistry and Western were observed following ureter obstruction in Clock knockout mice compared to wild type which was mediated by transforming growth factor beta [49]. It is possible that different stimuli result in opposing responses within the same pathway. It should also be noted that the dominant negative-acting CLOCKΔ19 model is distinct from the global Clock knockout due to the nature of the mutant CLOCK protein but also the background strain of mouse. Additional studies need to be conducted to clarify the role of circadian genes in renal fibrosis.

Sex.

It is well established that men have higher blood pressure than age-matched, premenopausal women [50], stressing the need to include both sexes in studies involving renal and cardiovascular diseases. However, the influence of sex on kidney circadian rhythms is not well studied. In the aforementioned studies that examined the contribution of circadian factors of sodium excretion and blood pressure, only male animals were used. Johnston et al. was an exception since this group included female rats when assessing night:day sodium excretion and blood pressure in control and ETb deficient mice. Females were reported to have more efficient natriuresis compared to males [34]. Circadian clock gene and protein levels were not assessed in this study. Sex hormones play a role in renal sodium handling [51], supporting the need to include females in future studies to determine if the circadian clock contributes to the lower blood pressure often observed in premenopausal female rodent models.

Implications of the Kidney Clock for Human Health

Kidney Disease.

It is well reported in the clinical field that renal diseases are associated with the development of sleeping disorders, which are oftentimes treatable by a number of interventions [52]. The cause is difficult to determine, however, due to numerous comorbidities that are present alongside kidney disease that also likely contribute to altered sleep habits, such as obesity and Type 2 diabetes [53]. Furthermore, some studies associate the lower quality of sleep with worse chronic kidney disease outcomes [54,55]. Conversely, less is known regarding whether abnormal sleeping patterns lead to dysfunction of the kidney. A recent report concludes that a lack of sleep correlated with a more rapid decline in kidney function [56] which supports the theory that a disrupted circadian rhythm is damaging to the kidneys. Sleeping disorders have been linked to the development of diabetes and elevated blood pressure, which can also lead to kidney injury [6,57]. There is a growing field in basic research that is attempting to better understand the role of circadian rhythm in the kidney and its connection to renal pathophysiology.

Chronotherapy.

A potential strategy to improve the efficacy and reduce side effects of medication is chronotherapy, or the administration of medication at certain times of the day. This area is being actively studied in hypertensive patients. A MAPEC study examined this phenomenon among 2000 patients from a single health center, who were monitored after receiving medication either in the morning or evening. The patients who were administered blood pressure medication at night had lower nocturnal pressures and increased dipping compared to day which resulted in a lower risk of cardiovascular disease [58]. The Hygia Project is an ongoing assessment of a much larger population where clinical reports are analyzed from multiple health facilities [59,60]. Additionally, a recent study in rats used hypertensive therapy in the morning and evening and found that treatment before the inactive period was more beneficial for the rats than before the active [29]. However, this was only seen with short-term treatment of 1 week and no difference was seen with long-term treatment [29]. The American Diabetes Association has acknowledged these results and has even suggested administration of antihypertensive drugs in the evening [61]. The effect of chronotherapy on kidney health and function needs to be further examined.

Considerations for Research

The importance of rigor and reproducibility has been at the forefront of research after the publication by Begley and Ellis [62]. This article exposed the inability to reproduce results in a number of landmark studies leading to an emphasis on rigor and reproducibility. Several modalities have been raised to help improve this problem, including a new section to address these areas in NIH grant applications. An overlooked contributor to inconsistent results is the acknowledgement of circadian rhythms [63]. Few studies report the time of day in their methods let alone factor in the time of day when planning experiments. Consequently, data collected without considering the time of day may be difficult to reproduce especially if the collections/measurements in subsequent studies were taken at different times. Furthermore, measurements and collections are normally performed during the daytime, which for rodents is the inactive period when many of the renal physiological processes are in the nadir stage. These issues should be considered in all biomedical research studies.

Conclusion

A growing body of evidence suggests that the molecular circadian clocks located in our cells and tissues are critical to physiological function. The complex interaction between the brain, adrenal glands (hormones), and kidneys likely plays an important part in the regulation of circadian rhythms in renal function. Increasing our understanding of how the central and peripheral clocks work together to regulate physiological processes such as renal function and blood pressure will potentially lead to improved treatments for hypertension and kidney disease.

We wish to confirm that there are no known conflicts of interest associated with this manuscript, “Recent Advances in Understanding the Circadian Clock in Renal Physiology.” There has been no significant financial support for this work that could have influenced its outcome.

HIGHLIGHTS.

• Regulation of renal function is influenced by the circadian clock.

• Dysregulation of the molecular clock has been linked to worsened kidney disease and hypertension in rodent models.

• Changes in renal function depending on the time of day should be considered when planning and conducting experiments in rodent models.