Circadian Control of Sodium and Blood Pressure Regulation

Abstract

The attention for the control of dietary risk factors involved in the development of hypertension, includes a large effort on dietary salt restrictions. Ample studies show the beneficial role of limiting dietary sodium as a lifestyle modification in the prevention and management of essential hypertension. Not until the past decade or so have studies more specifically investigated diurnal variations in renal electrolyte excretion, which led us to the hypothesis that timing of salt intake may impact cardiovascular health and blood pressure regulation. Cell autonomous molecular clocks as the name implies, function independently to maintain optimum functional rhythmicity in the face of environmental stressors such that cellular homeostasis is maintained at all times. Our understanding of mechanisms influencing diurnal patterns of sodium excretion and blood pressure has expanded with the discovery of the circadian clock genes. In this review, we discuss what is known about circadian regulation of renal sodium handling machinery and its influence on blood pressure regulation, with timing of sodium intake as a potential modulator of the kidney clock.

Blood pressure follows a diurnal rhythm, with a peak during the active period and a nadir during inactive period in both humans and animals. This day–night difference, in both diurnal and nocturnal species, creates what is identified as a “dip” in blood pressure at night. Numerous studies have associated nondipping and reversed dipping patterns of blood pressure with increased cardiovascular incidents, chronic kidney disease, and higher mortality rates.¹ Essential/primary hypertension, as the name implies, indicates that a definitive pathological culprit is not clearly identifiable. However, many pathophysiological mechanisms can contribute to the development of essential hypertension.^2–4 It is fair to say that most, if not all of these mechanisms, involve imbalance of extracellular sodium homeostasis either directly or indirectly.^4,5 Under normal physiological conditions of sodium intake, sodium excretion reaches a maximum when most food is consumed, that being during the day in humans and at night in rodents. Under steady-state conditions, renal excretion of sodium and water is equal to intake. This leads to stable blood pressure levels in response to fluctuations in sodium and water intakes. Dysfunction in a wide array of sodium regulatory mechanisms that include but are not limited to the kidney leads to sodium retention, and consequently chronic elevation of arterial blood pressure.

Circadian rhythms are endogenously generated 24-hour cycles that are deeply imbedded in all living organisms including plants, bacteria, and mammals.^6,7 Physiological functions in the body are timed to match physiological requirements at different times of day. This is achieved through coordination between central and peripheral clocks. The central clock which is located in the suprachiasmatic nucleus (SCN), responds to time cues, such as light–dark, rest–activity, and time of feeding and orchestrates rhythmicity of function throughout the body. The peripheral clock conducts its function through a complexity of interacting loops of transcription factors that oscillate at different times of day. Clock/Bmal1 represent the positive transcription arm of the loop, while Period/Cryptochrome heterodimer is the repressor arm, that feeds back to suppress Clock/Bmal1 transcription.⁸ There is strong evidence that the loss of rhythmicity in blood pressure is associated with circadian misalignment and clock gene dysfunction.¹

In this review, we will look into circadian regulation of renal sodium handling along the nephron. We will discuss published evidence of clock-mediated blood pressure regulation, with consideration of the impact of timing of sodium intake on temporal patterns of sodium excretion and blood pressure rhythms. Given the strong relationship between impaired renal sodium excretion and the development of hypertension, we will discuss the possible impact of circadian dysfunction on the diurnal regulation of blood pressure and the pathophysiology of hypertension.

Molecular Basis Of Circadian Rhythms

The SCN in the hypothalamus is the home for the central clock that acts as the conductor by coordinating the rhythmicity of different organs.⁷ This is achieved through a transcriptional–translation feedback loop (Figure 1). The central clock responds to external cues, including light signals that reach the SCN through the retinal–hypothalamic tract, which leads to a series of downstream signals yielding the generation CLOCK and Bmal1 gene transcripts. Circadian locomotor output cycles Kaput (CLOCK) and brain and muscle Arnt-like protein-1 (Bmal1) genes are transcription factors that form a heterodimer and represent the positive arm of the loop. They bind to the E-box response elements in the promoter region of cryptochrome (Cry) and period (Per) genes stimulating their transcription. Per and Cry transcripts form the suppressor arm of the loop, they undergo casein kinase (CK1δ/ε)-mediated phosphorylation which facilitates their nuclear entrance and feedback to inhibit Clock/Bmal1 activity.^6–8 Dephosphorylation of Per1 through the action of Protein phosphatase-1 (PP-1) releases the inhibitory dimer from its nuclear binding site and is recycled back to the cytoplasm.⁸ Clock:Bmal1 heterodimer stimulates the transcription of the orphan nuclear receptors ROR and Rev-ERBα, that feeds back to regulate Clock and Bmal1 transcription.⁹ Each arm of the loop peaks during certain times of day, with Bmal1 peaking at the beginning of the inactive period (nighttime in humans and daytime in nocturnal animals), with a corresponding trough in Per1 expression.

Figure 1.

The transcription translation feedback loop: Clock:Bmal1 heterodimer binds to the E-box element of the promotor regions of Per and Cry genes, driving their transcription. They also drive the transcription of many clock-controlled genes (CCG) in different organs, promoting rhythmicity of physiological functions. Cry and Per transcripts heterodimerize and through phosphorylation of Per by the action of CK1δ/ε, they enter the nucleus to inhibit Clock:Bmal1 binding to the E-box element. Clock:Bmal1 binding promotes the transcription of REV-ERBα binds to the response element (RE) of Bmal1 promotor and inhibits its transcription.

Peripheral clocks are imbedded within the genome of all peripheral tissues with variable rates of expression. While the liver has the highest percentage of oscillating genes in the body, only 3% of protein-encoding transcripts oscillate in the hypothalamus.¹⁰ The central clock in the SCN influences peripheral tissues metabolic functions through neural and hormonal factors. However, rapidly growing convincing evidence showed that cells have autonomous molecular clocks that function independently of SCN influence.

In the beginning of the last century, studies showed that urine output has a day–night difference regardless of time of water or food intakes,^11,12 which was then described as “night water retention.” ¹¹ Subsequent studies found that the majority of kidney functions display diurnal rhythms, including glomerular filtration rate, urine osmolarity, urine electrolyte excretion,⁶ endothelin-1 (ET-1) excretion,¹³ and autonomic nervous system activity.¹⁴

In this review, we present evidence in support of the intrinsic mechanisms regulating time of sodium excretion along the nephron and the influence of environmental time cues, as timing of food and possibly salt intake on rhythms of sodium excretion and the subsequent effect on blood pressure.

Sodium Transport Along The Nephron And Kidney Clock

Sodium channels are abundantly expressed along the nephron, with the ascending limb of the loop of Henle being the only part of the nephron devoid of sodium channels.¹⁵ The multistop journey of sodium along the nephron is tightly regulated by hormonal, neuronal, and paracrine factors, reflecting the importance of sodium balance on body fluid homeostasis and as will be discussed in this review, the diurnal regulation of blood pressure (Figure 2). More than 99% of the filtered sodium is reabsorbed along the nephron. The complex machinery of transporters along the renal tubule is responsible for microadjustment of the remaining 1% to achieve extracellular fluid balance and cellular homeostasis.¹⁵

Figure 2.

Distribution of the major oscillating sodium channels and transporters along the rodent nephron with approximation of the circadian rhythm of Na⁺/H⁺ exchanger 3 (NHE3),²¹ Na⁺/K⁺/2Cl⁻ cotransporter (NKCC),³² Na⁺/Cl⁻ cotransporter (NCC),⁴⁰ and epithelial Na⁺ channel (ENaC)^13,32 in the proximal tubule (PT), thick ascending limb (TAL), distal convoluted tubule (DCT), and collecting duct (CD), respectively.

The proximal tubule reabsorbs approximately two-thirds of filtered sodium. This is driven by the Na⁺/K⁺ ATPase on the basolateral border of tubular cells.¹⁶ Sodium reabsorption in this part of the nephron is achieved largely through the function of sodium-hydrogen exchanger 3 (NHE3) and an array of symporters, that include, but not limited to sodium-glucose cotransporter 1 and 2 (SGLT1/2)¹⁷ at the apical border of the cell membrane. NHE3 is also expressed in the thick ascending loop of Henle and together with Na⁺/K⁺/2Cl⁻ cotransporter (NKCC2), 25% of sodium is reabsorbed in this part of the nephron.⁷ Sodium reabsorption along the distal convoluted tubule (DCT) is tightly regulated and critical for extracellular fluid homeostasis. It is also the target of most pharmacological agents in the treatment of hypertension that DCT1 is often referred to as the thiazide sensitive segment and DCT2 and collecting duct epithelial sodium channel (ENaC) is described as the amiloride sensitive sodium channel. The collecting duct is the main target of the mineralocorticoid, aldosterone, which is critical for sodium reabsorption in the distal part of the nephron.¹⁸ The DCT reabsorbs 6%–10% of the total filtered sodium and only 1% of sodium now reaches the collecting duct. Precise matching of sodium intake to excretion occurs primarily through physiological adjustments in the collecting duct yet can have a large impact on blood pressure control.¹⁵

SODIUM-HYDROGEN EXCHANGER 3

NHE3 is critical for sodium reabsorption and acid–base balance along the nephron¹⁹ especially in the proximal tubule.^19–21In situ hybridization studies and laser capture microdissection techniques showed evidence of circadian oscillation of NHE3 in the thick and thin ascending limbs of the nephron, with an early active period peak.²² However, proximal tubular cells were not assessed in this study. Saifur Rohman et al. provided the first report of circadian rhythmicity in NHE3 gene expression in rodent kidneys, which is directly regulated by Clock:Bmal1 heterodimer.²¹Cry1/2 null mice showed loss of circadian oscillation of NHE3, Bmal1, and Per2 mRNA in the kidney, and transfecting opossum kidney cells with mPer2 and mCry2 downregulated NHE3 expression as measured by luciferase activity. They further utilized opossum kidney cells and luciferase reporter plasmids with NHE3 5′ flanking regions of variable lengths to determine the promotor region essential for gene transcription, and in this fragment, the consensus sequence for Clock:Bmal1 E-box element was identified. Mutations in NHE3 promotor led to failure of Clock and Bmal1 transactivation, while opossum kidney cells expressing intact NHE3 promotor responded by 5-fold increase in transcriptional activity upon treatment with both Clock and Bmal1. This study provides evidence that circadian rhythms in sodium homeostasis are driven by the autonomous kidney clock. The study also showed evidence for NHE3 and Per2 colocalization in the brush border of the proximal convoluted tubules. Blood pressure was not measured in the Cry1/2 null mice, however, we know from other studies that these mice are normotensive with preserved blood pressure rhythm under normal salt conditions,²³ suggesting that circadian regulation of NHE3 is not the key determinant of blood pressure phenotype. This is supported by studies showing that knockout of NHE3 from the proximal tubule had no effect on systolic blood pressure.²⁴ It is also of note that the application of computational methods predicted Bmal1 and Clock modulation of NHE3 circadian rhythmicity, and a loss of this circadian variation, but not sodium reabsorption, in the absence of either clock gene.²⁵ Similarly, Per1 was detected at the promotor region of NHE3.²⁶ Per1 cannot enter the nucleus without phosphorylation by the casein kinase isoform δ/ε (CK1δ/ε).^8,27 Injection of a CK1δ/ε inhibitor into 129/sv wild-type (WT) mice resulted in suppression of NHE3 mRNA expression in mouse cortex collected at midnight. This observation was further confirmed by suppression of NHE3 expression and transcriptional activity in pharmacologically induced blockade of Per1 nuclear entry and siRNA-induced knockdown in HK-2 cells.²⁶

Taken together, these studies show that NHE3 is a direct target for circadian clock genes (Table 1). Clock, Bmal1, and Per1 appear to serve as positive direct regulators, while Cry1, Cry2, and Per2 are likely suppressors of channel expression. Further characterization of NHE3 expression and activities in different animal models of clock gene dysfunction, will further establish the role of the critically important proximal tubule channel in modulating sodium reabsorption in response to changes in timing of sodium intake.

Table 1.

Circadian clock regulation of sodium channels and transporters along the nephron

Channel/transporter	Regulator	Function	References
NHE3	Clock, Bmal1, Cry1/2	NHE3 circadian oscillation	21,25
	Per1	Stimulates (↑) transcription	26
SGLT1/2	Per1	Stimulates (↑) transcription	26
NKCC2	Per1	↓ mRNA expression under HS/DOCP	35,36
		Blood pressure regulation under HS diet
NCC	Per1	Decreases (↓) NCC mRNA expression	36,37
ENaC	Per1	Upregulation of αENaC expression	35,36,48–51

Abbreviations: ENaC, epithelial sodium channel; NCC, sodium chloride cotransporter; NHE3, sodium-hydrogen exchanger 3; NKCC2, Na⁺/K⁺/2Cl⁻ cotransporter; SGLT1/2, sodium-glucose cotransporter 1 and 2.

Sodium-Glucose Cotransporter

Glucose is exclusively reabsorbed in the proximal tubule coupled with sodium reabsorption and is achieved via the apical SGLT1 and SGLT2. Coupled reabsorption of sodium and glucose through this channel is driven by the basolateral Na⁺/K⁺ ATPase activity and intracellular glucose accumulation drives GLUT2-mediated glucose pump back to the plasma.²⁸

Evidence of circadian regulation of SGLT1 comes from studies of the gut. SGLT1 was identified as a circadian target in the small intestine, with loss of temporal pattern of SGLT1 expression in Clock mutant mice.²⁹ Interfering with Bmal1 in vitro resulted in upregulation of SGLT1 expression in differentiated Caco-2 cells through a PAX-4 mediated transcriptional suppression.³⁰ However, in the kidney, evidence of circadian control of SGLT1/2 expression and activity is limited (Table 1). Solocinski et al. identified Per1 as a positive regulator of SGLT1 in a mechanism that is similar to NHE3.²⁶ Studies on the role of SGLT1/2 in circadian regulation of renal sodium and glucose homeostasis are needed, particularly with the recently presented evidence showing association between loss of SGLT1 and higher systolic blood pressure in Atika mice.³¹ In addition to glucose, sodium reabsorption is also coupled with amino acids, lactate, and P_i with no available data on circadian regulation.

Sodium-Potassium-Chloride Cotransporter

While the loop of Henle accounts for roughly 25% of tubular NaCl reabsorption,⁷ little is known about a role for clock genes in this part of the nephron. Apart from the circadian expression of NKCC2 in the kidney,³² studies discussed here only show evidence of indirect circadian regulation of the Na⁺/K⁺ channel. Krid et al. studied circadian oscillation of the estrogen-related receptor β (ERRβ) in the thick ascending limb.³³ ERRβ showed a temporal pattern of expression with a light phase peak (zeitgeber time 4, ZT4), coinciding with NKCC2 peak of expression (Figure 2).^32,34 In fact, mice treated with the pan-ERR inhibitor, diethylstilbestrol, suffered sodium wasting and inability to concentrate urine manifested in marked increase in urine volume and significantly reduced osmolarity. Krid et al. suggested that this phenotype is an ERRβ-mediated NKCC2 dysregulation based on several observations. ERR inhibition increased Na⁺/K⁺ excretion ratio in the urine and decreased NKCC2 mRNA expression and activity. These changes were observed despite no significant effect on sodium chloride cotransporter (NCC) expression. Further, they reported that a mouse thick ascending limb (MKTAL) cell line treated with selective ERRβ agonist showed a 2-fold increase in the relative expression of NKCC2.³³ However, whether pharmacological manipulation of ERRβ was associated with changes in core clock gene profiles was not assessed. Male mice lacking the circadian transcription factor Per1 showed a nondipping hypertension phenotype under combined high salt diet and short-term mineralocorticoid treatment³⁵ (Table 1). While WT mice suppressed the expression of renin in response to treatment, Per1KO mice showed no renin suppression as well as no change in NKCC2 channel expression in the renal cortex relative to normal salt conditions.^35,36 However, the temporal pattern of expression of NKCC2 was not investigated in this study, and whether loss of Per1 will lead to a complete loss or phase shift in NKCC2 expression and activity under high salt diet conditions is yet to be determined.

Sodium Chloride Cotransporter

The NCC is the main apical sodium channel in the first part of the DCT (Figure 2) with reduced expression in the late part of the DCT¹⁵ and is characterized by its sensitivity to thiazide diuretics and response to changes in plasma potassium concentration.³⁷ NCC functions through a multistep signal cascade that involves phosphorylation for channel activity.³⁷ However, mice lacking NCC showed no changes in sodium excretion or blood pressure under normal conditions,^37,38 possibly through downstream ENaC-mediated compensation.³⁹ Many studies have shown circadian regulation of NCC through multiple mechanistic pathways, reflecting a role for NCC in time-of-day sodium regulation. A newly published study by Bailey and colleagues showed that glucocorticoid receptor activation increased NCC activity via phosphorylation that was associated with increased expression of Bmal1, Per1, Per2, and Cry1 in the kidneys of C57BL6 mice.³⁷ However, mineralocorticoids increased NCC abundance without affected phosphorylation. Previous studies have shown a peak of the phosphorylation level of NCC at the beginning of the active period, but limited oscillation of the inactive form of the channel protein⁴⁰ and attenuation of the diurnal rhythmicity of pNCC and Per1 with chronic GR blockade.³⁷

A report of upregulated NCC relative expression in a Ksp-cadherin Cre-model of distal nephron-specific Per1KO mouse³⁶ supported the hypothesis of clock-mediated regulation of sodium transport in the DCT (Table 1). NCC is important in blood pressure regulation and its activation follows a diurnal rhythm.^41,42 Inactive phase upregulation of pNCC through chronic corticosterone infusion leads to rise in daytime blood pressure and development of nondipping phenotype in C57BL6 mice.⁴² The blood pressure phenotype was associated with impaired circadian rhythm of mRNA expression of Bmal1, Per1, and Sgk1. Glucocorticoid-induced kinase 1 (SGK1) functions as a regulator of a number of tubular sodium transporters.⁴³ Few studies have attempted to identify direct and indirect clock-mediated mechanisms in the regulation of channel expression and activity. However, there has been 1 report showing that Per1 regulates NCC through the WNK pathways⁴⁴ but whether corticosterone-induced upregulation of Sgk1 and glucocorticoid-induced leucine zipper (GILZ), an oscillating transcriptional regulator responding to glucocorticoids, are potential pathways for clock-induced NCC regulation is to be determined. This is particularly important, since little is known about molecular mechanisms integrating NCC rhythmicity and the kidney clock transcription–translation feedback loop.

Epithelial Sodium Channel

ENaC has a hetero-multimeric structure of α-, β-, and γ-subunits and the functionality of the channel depends on αENaC proteolytic cleavage^45,46 (Figure 2). ENaC is localized to the DCT2, connecting tubule and the collecting duct.⁴⁷ This amiloride sensitive channel is critical for final control of sodium homeostasis, responding to changes in tubular sodium delivery, ET-1, aldosterone, nitric oxide (NO) as well as growing evidence of clock gene regulation to tightly regulate sodium excretion to match the intake and to achieve fluid–electrolyte balance.^15,46 Studies by our group has shown rhythmic oscillations of αENaC in rat inner medulla, with an active period peak and more than 50% reduction in the amplitude in response to high salt diet.¹³ Testing this observation through benzamil blockade at different times of day, showed no difference in natriuresis in response to drug administration at ZT0 (corresponding to the beginning of the inactive period, light phase in rodent) vs. ZT12 (active period or dark phase).⁴⁶ This reflects a disconnect between mRNA expression and function and calls for further studies on channel conductivity at different times of day at baseline vs. high salt conditions.

Given the critical role of ENaC in the regulation of blood pressure, studies have focused on investigating circadian regulation of ENaC under different dietary salt conditions (Table 1). Data provided from in vitro studies supported the hypothesis that Per1 positively regulates αENaC expression and directly promotes channel activity. Mouse inner medullary collecting duct (IMCD-3) cells were treated with siRNA directed against Per1, showed 80% reduction in Per1 mRNA expression and marked reduction in αENaC expression that was not responsive to aldosterone.⁴⁸ Using both luciferase activity and protein abundance assays, cortical collecting duct cells (mpkCCD_c14) transfected with dominant negative Per1 vector or Per1-specific siRNA, respectively, showed reduction in αENaC promotor activity and expression.⁴⁹ Similarly, targeting Per1 in Xenopus 2F3 distal nephron cells with siRNA, showed a reduction in channel density and open probability.⁵⁰

The first reported global Per1KO mouse, on a 129/sv background,⁵¹ showed a lower blood pressure phenotype⁵² and studies by Gumz et al. reported downregulation of αENaC expression in the cortex of the KO mice.⁴⁹ However, the C57BL/6J Per1KO mouse model showed no blood pressure phenotype and similar ENaC expression profile compared with WT controls under normal dietary conditions.³⁵ Likewise, the loss of Per1 from the distal part of the mouse nephron was associated with upregulation of alpha αENaC expression in the cortex.³⁶ An interesting observation of the C57BL/6J Per1KO mouse model was that HS/DOCP treatment stimulated the expression of αENaC that was correlated with the serine/threonine kinase SGK1 expression in this group, promoting salt sensitivity and nondipping hypertension. Whereas, the higher SGK1 in the WT mice, was not associated with changes in αENaC expression, suggesting that missing Per1 is contributing to the inappropriately higher αENaC.³⁶

Collectively, these studies highlight a disconnect between the in vitro and in vivo models as well as strain-specific phenotypes. This calls for the utilization of other models of circadian and blood pressure dysfunction, both in humans and rodents, to provide a better understanding of circadian regulation of renal salt handling and blood pressure homeostasis.

Hormonal Regulation

Renin–angiotensin–aldosterone system

When activated, the renin–angiotensin–aldosterone system generally promotes sodium reabsorption, extracellular fluid volume expansion and increases in blood pressure. This multistep signaling cascade allows for tight control of extracellular fluid and blood pressure in response to changes in dietary salt intake, especially in when salt conservation is needed. Although limited, there have been some studies addressing the role of the renin–angiotensin–aldosterone system in control of circadian sodium excretion.

The intrarenal renin–angiotensin system (RAS) has emerged in recent years as an important contributor to blood pressure control, particularly in disease states. However, much has yet to be learned regarding circadian mechanisms. Measuring urinary angiotensinogen (AGT) reflects intrarenal renin activity and while AGT excretion was low regardless of time of day in healthy individuals, patients with chronic kidney disease had higher urinary daytime AGT, positively correlating to higher blood pressure and protein excretion at the same time frame.^53,54 An animal model of chronic progressive glomerulonephritis, showed a positive correlation between loss of urinary AGT rhythm and renal damage. Wistar rats injected with anti-thymocyte serum developed chronic glomerulonephritis, and were assessed for the protein expression of AGT, Angiotensin II (Ang II), and Angiotensin II type 1 receptor (AT1R) compared with the control group. Anti-thymocyte serum rats showed higher daytime AGT, Ang II, and AT1R protein expression compared with controls. Daytime activation of intrarenal RAS was positively correlated with blood pressure and proteinuria, suggesting circadian rhythmicity of intrarenal RAS influences renal function and blood pressure rhythms.⁵⁵ Limited studies have investigated the role of the kidney clock on intrarenal RAS-mediated regulation of fluid and electrolyte homeostatsis. A mouse model with a conditional deletion of Bmal1 gene in renin-producing cells driven by Ren1^dCre was developed by Firsov group. These mice showed increased glomerular filtration rate, increased urine excretion and changes in nighttime urinary sodium excretion compared with the control group. mRNA expression of αENaC, NCC, and Sgk1 showed an attenuated circadian pattern in the Bmal1^lox/lox/Ren1^dCre mice compared with the control group.⁵⁶

Ang II has a prominent effect on NHE3 activity to promote sodium reabsorption in the proximal tubule.⁵⁷ Global loss of NHE3 attenuated the effect of Ang II on the elevated blood pressure phenotype.¹⁷ Loss of NHE3 in the kidney or proximal tubule-specific KO models showed similar attenuation of the hypertensive response to Ang II.^17,58 A common finding in pharmacological and genetic models of RAS-induced hypertension is circadian dysfunction, manifested in the development of nondipping hypertension in many of these models.^59,60 While plasma AGT, Ang II precursor, does not oscillate,⁶¹ studies have reported diurnal rhythms of plasma Ang II with an early morning peak.⁶² The hypertensive Ren-2 transgenic TGR (mREN2)27 rat overexpresses renin due to an additional copy of the renin gene.⁶³ This model not only overexpresses the full range of renin–angiotensin–aldosterone system components and displays fulminant hypertension, but also shows a loss of circadian rhythm of blood pressure,⁶⁴ indicating the possibility that abnormally high renin levels can result in circadian disruption. Fukuda et al. found that blocking Ang II AT₁ receptor with olmesartan restored diurnal blood pressure rhythmicity.^65,66 They attributed this effect to suppression of daytime renal tubular sodium reabsorption.⁶⁵

The DCT, mainly its late segment, as well as the cortical collecting duct are sensitive to the mineralocorticoid, aldosterone,¹⁵ which through influencing clock genes in this part of the nephron, promotes sodium reabsorption. Similar to Ang II, plasma, and urinary aldosterone follow diurnal rhythms with an early morning peak in healthy humans and at the corresponding active phase in rodents.^44,67 The interaction between the transcription–translation feedback loop of the circadian clock and aldosterone has recently been the center of attention in terms of aldosterone-dependent hypertension (Figure 3). For example, Per1 was found to be upregulated by aldosterone, both in vivo and in vitro.^44,48,68 Aldosterone injection into male Sprague-Dawley rats induced more than 3-fold increase in Per1 mRNA and since aldosterone activates αENaC, treating mIMCD-3, OMCD, and mpkCCD cell lines with aldosterone resulted in significant increase in αENaC expression. Aldosterone-induced stimulatory effect on αENaC was proceeded with Per1 induction and was suppressed when cells are treated with Per-specific siRNA.⁴⁸ Aldosterone regulation of Per1 occurs at the transcriptional level, and so does Per1 through stimulating αENaC transcription. These results likely explain the sodium-wasting and hypotensive phenotype of the Per1-deficient mice. Clock-null mice showed disrupted circadian rhythms of plasma aldosterone, with no effect on 24-hour mean values.⁴³

Figure 3.

Proposed interaction between clock genes, aldosterone, and endothelin-1 (ET-1) in the regulation of diurnal rhythms of sodium excretion that involve the function of ENaC. Abbreviations: 20-HETE, 20-Hydroxyeicosatetraenoic acid; ENaC, epithelial sodium channel; ET-1, endothelin-1; UNaV, urinary sodium excretion.

Circadian profiling of the whole kidney transcriptome along with real-time PCR showed a significant reduction in the number of oscillating genes in Clock^−/− mice, as well as disruption of many transcript-encoding genes irrespective of circadian rhythmicity.⁴³ This includes transcripts encoding for cytochrome p450 enzymes, leading to impaired 20-Hydroxyeicosatetraenoic acid (20-HETE) synthesis and excretion, as well as an observed acrophase shift in kidney microsomes of the KO animals. 20-HETE, in addition to its vasopressor action, exhibits a bimodal role in sodium balance. It inhibits luminal sodium channels as well as the Na⁺/K⁺ ATPase activity in the thick ascending limb, promoting sodium excretion.^43,69 This is in addition to promoting the production of aldosterone through induction of angiotensin converting enzyme.⁷⁰ Aldosterone is tightly linked to clock genes, and many genetic models of clock gene dysfunction exhibit disrupted aldosterone balance. Cry-null mice, with complete loss of Cry1 and 2, have high circulating plasma aldosterone and low renin activity.²³ Studies have also shown accelerated renal damage and proteinuria as a secondary effect to hyperaldosteronism.⁷¹

Aldosterone is a key modulator of the renal clock and its downstream targets in a time-specific manner, and studies are needed to explore aldosterone-driven circadian dysfunction in different models of hypertension and its relation to timing of salt intake as a strong candidate for chronotherapy of patients with hypertension.

Paracrine Regulation

Endothelin-1

ET-1 is 23-amino acid polypeptide that functions in an autocrine and paracrine manner through binding to 2 G-protein-coupled receptors, ET_A and ET_B receptors. While considered the most potent vasoconstricting factor through its binding to the ET_A receptor, ET-1/ET_B binding promotes sodium excretion through NO-mediated ENaC inhibition.^72,73 ET-1 is expressed in all vascular endothelial cells and promotes vasoconstriction in response to a wide variety of stimuli, including hypoxia, thrombin, pressure, and mechanical stretch.⁷⁴ Studies then followed to reveal that ET-1 is not exclusive to endothelial cells, but is expressed and secreted from epithelial cells such as renal tubular cells and contributes to sodium homeostasis and blood pressure regulation.⁷⁵ Intrinsic renal production of ET-1 mainly by the collecting duct is the major source of urinary ET⁷⁶ and studies have shown that its expression and excretion follow a diurnal rhythm.¹³ There is growing evidence of clock-mediated regulation of the endothelin axis (Figure 3). Dysfunction of ET-1 system is deleterious to diurnal sodium handling and is associated with chronic kidney disease.^73,77 ET-1 gene promotor binds many transcription factors, including the hormone response element⁷⁸ and E-box element,⁷⁹ and contribute to circadian regulation of ET-1 transcription.

High salt stimulates renal ET-1 to activate the ET_B receptor to promote sodium excretion⁸⁰ such that ET_B-mediated natriuresis is critical for blood pressure control.⁸¹ Loss of ET_B function leads to the development of hypertension under high salt conditions.^81,82 Mice lacking ET-1 in their collecting duct develop salt sensitive hypertension,⁸³ reflecting the critical role of the renal endothelin system in body fluid and blood pressure regulation.

Circadian regulation of ET-1 has gained special interest after the identification of preproendothelin and period homolog as early aldosterone-responsive genes.⁶⁸ Work from the Gumz laboratory has provided evidence for a role of the circadian clock gene Per1 in the regulation of ET-1 function and blood pressure homeostasis.^44,52,84 Transfecting mpk cortical collecting duct cells (mpkCCD) with Per1–8 siRNA was associated with ET-1 mRNA induction and increased ET-1 concentration in the culture media.⁵² In their model of 129/sv Per1KO mouse, ET-1 mRNA expression was higher in the cortex and medulla of kidneys collected at noon and at midnight compared with WT controls.⁵²Per1 heterozygous mice, showed elevated ET-1 levels in the renal inner medulla, with no effect on the mRNA expression of ET_B or ET_A receptors, that were expressed in a diurnal, but opposite rhythms in both WT and Per1 het mice.⁷⁹ These studies show that Per1 is a direct negative regulator of ET-1, but not its receptors and supports the hypothesis that ET-1 is involved in circadian regulation of renal rhythms and blood pressure regulation.

ET-1 is essential in excreting high salt loads and high salt diet induced a phase shift in Bmal1 mRNA expression in rat’s inner medulla,¹³ which proposed a role for the core clock transcription factor Bmal1 in the diurnal regulation of the ET-1 system and circadian rhythms of sodium excretion. This was recently confirmed with the observed loss of diurnal rhythms of sodium excretion in global Bmal1KO rat.⁸⁵ In cultured mouse IMCD cells, ET-1 attenuated Bmal1 mRNA expression.¹³ Johnston et al. reported delayed natriuretic response in ET_B-deficient rats in response to an acute salt load given either at the beginning of the inactive or active periods, supporting the role of ET-1/ET_B interaction in the diurnal regulation of sodium excretion.⁷⁷ ET_B-deficient rats on high salt diet, showed suppression of Cry1 and Per2 expression in their kidneys with no effect on Bmal1 in contrast to transgenic controls that showed high salt-induced phase shift of Bmal1 as previously mentioned.¹³ This suggests that high salt-induced circadian disruption of Bmal1 requires an intact endothelin axis, with potential compensatory mechanisms promoting Cry and Per genes in the rat kidney.

Clinical studies showed evidence of ET system overactivity and involvement in diurnal regulation of blood pressure in chronic kidney disease patients.^86,87 Patients with chronic kidney disease had higher midnight plasma ET-1 compared with healthy controls, with loss of day–night difference in circulating ET-1 levels, in addition to loss of nighttime dip in systolic and diastolic blood pressure.^86,88 Subjecting 27 chronic kidney disease normotensive patients to ET_A receptor antagonist, sitaxentan, as part of a reno-protective protocol for 6 weeks, promoted a reduction in nighttime blood pressure with more than 8% dip in systolic blood pressure compared with baseline (week 0).⁸⁶ This study further builds on the evidence of ET system involvement in diurnal regulation of blood pressure.

Our knowledge of clock-mediated regulation of the endothelin system is growing, however, studies elucidating mechanisms that govern clock-mediated responses of the endothelin system and its subsequent effect on cardiovascular and renal health are still lacking.

Nitric oxide

Interaction between the endothelin system and NO signaling in the kidney is essential for sodium homeostasis and blood pressure regulation. As mentioned earlier, ET_B-dependent natriuresis through binding to ET-1, is mediated via NO-induced suppression of distal nephron sodium reabsorption.⁸⁹ NO is produced secondary to l-arginine oxidation, catalyzed by nitric oxide synthase (NOS).⁹⁰ Cell membranes are permeable to NO, which freely diffuses to induce a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade.⁹⁰ In addition to the established role of NO and NOS in the vasculature,^91,92 NOS isoforms were also identified in different parts of kidney, with evidence showing NO as a natriuretic promoting factor.^93–95 NO signaling downregulates NKCC2 and ENaC activity, mainly through NOS3 activity, promoting sodium and chloride excretion.⁹⁶ On the other hand, in rats infused with aldosterone, NO was found to positively regulate NHE3 in the proximal tubule and NKCC in the thick ascending limb^90,97 suggesting a compensatory mechanism for aldosterone-induced sodium retention.

Although no mechanistic evidence exists, NOS activity in the kidney was found to follow a circadian rhythm that appears to be under the influence of light–dark conditions. BALB/C mice were subjected to 2 different light settings, 1 group was under a 14:10 (L:D), while the other group was housed under constant light conditions (LL). Cosinor analysis of NOS activity in kidney tissues collected every 4 hours followed a circadian rhythm under prolonged light conditions, while this rhythm was abolished under constant lighting. NOS activity in the aorta was opposite to that in the kidney. Plasma NO and brain NOS activity exhibited a diurnal rhythm under both lighting protocols. This study suggests that while rhythmicity of the NO system is independent of exogenous light cues in the brain and plasma, the kidney clock is influenced by disruption in environmental time cues.⁹⁸ There is ample evidence that disruption of NO signaling leads to hypertension and salt sensitivity, however, no evidence exists on direct interaction between the renal clock and tubular NOS-mediated natriuresis.

Neural Regulation

Interaction between the autonomic nervous system and rhythms of sodium excretion emerged from evidence showing that the autonomic nervous system activity regulates diurnal rhythms of blood pressure.⁹⁹ The morning blood pressure surge is associated with increased circulating norepinephrine, the key neurotransmitter in sympathetic nervous system activation, suggesting a role of the sympathetic nervous system in the diurnal regulation of blood pressure.¹⁰⁰ Higher sympathetic nervous system activity has been associated with a more robust daytime blood pressure variability in healthy human subjects.¹⁰⁰ Becker et al. investigated mechanisms of Ang II-induced stimulation of renal sympathetic nervous system and found that one of these mechanisms involves brain-derived neurotrophic factor (BDNF) interaction with its receptor (TrkB).¹⁰¹ Sprague-Dawley rats were subjected to intracerebroventricular infusion with Ang II with or without TrkB receptor antagonist (ANA-12) for 12-day duration. Ang II alone induced an increase in renal sympathetic nerve activity along with elevated mean arterial blood pressure and increased heart rate. Coinfusion of ANA-12 with Ang II prevented Ang II-induced renal sympathetic nerve stimulation with a moderate response on mean arterial pressure and a normal heart rate compared with vehicle-infused rats.¹⁰¹ This study proposes BDNF/TrkB signaling pathway as a modulator of Ang II-induced pressor effect, However, it is not clear whether BDNF/TrkB signaling has an influence on circadian rhythmicity of blood pressure as data showed the 24-hour averages of mean arterial pressure.

While studies on experimental animals indicate that renal sympathetic denervation improves the blood pressure phenotype in hypertension models, the use of this approach in humans has yielded mixed, albeit promising, results.^102–104 Bilateral sympathetic denervation reduced blood pressure in ET_B-deficient rats, while having no effect on diurnal sodium excretion or blood pressure rhythmicity. In fact, the data showed renal denervation had no influence on salt sensitivity in ET_B-deficient rats,⁸² suggesting that diurnal rhythms of sodium excretion are independent of neural regulation. Studies exploring interaction between the components of the central and peripheral circadian clocks with the sympathetic nervous system and their role in modulating timing of sodium excretion are still lacking with limited number of studies considering the effect of time of day of sodium intake and excretion as potential targets for sympathetic-mediated modulation of blood pressure.

Timing Of Sodium Excretion And Blood Pressure

The first report of autonomous rhythms in kidney function with a maintained day–night differences in urine flow regardless of rhythms of food intake¹² was as early as 1951. However, it was not until recent years that attention was given to circadian regulation of renal functions and its role in electrolyte homeostasis and blood pressure regulation. Studies have suggested that mis-timing of behavioral cues such as rest–activity, time of feeding, and sleep cycles can lead to misalignment of clock-mediated rhythms in sodium excretion.⁴³ Palm et al. on their studies on dogs, observed that continuous infusion of sodium, at a rate of 10 µmol/kg/minute, for 2 days, resulted in transient disruption of sodium excretion rhythms, with restoration of the day–night difference on the second day of the protocol. However, no effect on mean arterial blood pressure was observed during this short-term protocol.¹⁰⁵ Given the impact of sodium intake on blood pressure regulation further studies are needed to discern the nature of the relationship between circadian control of sodium handling and its potential impact on blood pressure.

Impaired diurnal rhythms of sodium excretion; i.e., decreased daytime urinary sodium excretion, was associated with higher nighttime blood pressure in humans.¹⁰⁶ Recruited individuals represented a broad age range (43–60 years old) and 15% were hypertensive. Twenty-four-hour urine collection was performed as 2 separate collections, daytime and nighttime and were self-reported. Ambulatory blood pressure monitoring was carried out concurrently, with no dietary intervention. The study sample was divided into 4 quartiles based on the level of daytime urinary sodium excretion. Subjects with the lowest daytime excretion rates, were older than 50 years of age, showed a nighttime systolic blood pressure that was 6 mm Hg higher than the group with highest daytime urinary sodium excretion and a significantly reduced nocturnal dip. This study suggests that the negative effects of a reduced diurnal patterns of sodium excretion (aka, renal circadian misalignment) on blood pressure is age dependent.

In the spontaneously hypertensive rat, a high salt diet disrupted circadian rhythms of plasma sodium but not mean arterial pressure rhythms although urinary sodium excretion was not reported.¹⁰⁷ The hypertensive ET_B-deficient rats showed a delayed natriuretic response to salt loads at different times of day, that was independent of changes in blood pressure.⁷⁷ These studies suggest a disconnect between timing or amount of salt intake and circadian rhythms in blood pressure.

Mice lacking the core clock gene, Clock, were assessed for light (L) vs. dark (D) periods sodium excretion, under regular 12:12 light:dark cycle. Clock^(−/−) expressed urinary sodium (L/D) excretion ratios that were close to one, indicating loss of diurnal rhythm in sodium excretion compared with their WT control,⁴³ which brings attention to Clock as a driving factor for the temporal pattern of sodium excretion, with target genes yet to be identified. High salt diet in Cry-null mice led to the development of salt sensitive hypertension. These mice have persistently higher levels of type VI 3β-hydroxyl-steroid dehydrogenase (Hsd3b6) which plays a key role in aldosterone synthesis. This study shows that Hsd3b6, which is most active in the adrenal gland, is regulated by clock genes.¹⁰⁸ This study proposes Hsd3b6 as a clock-controlled modulator of diurnal sodium excretion and blood pressure, through mediating aldosterone actions.

While global Bmal1KO mice showed complete loss of rhythmicity in blood pressure,¹⁰⁹ urine and sodium outputs, the first global Bmal1KO rat model showed a disconnect between blood pressure and renal excretion throughout the 24-hour day.⁸⁵ Our group characterized the blood pressure phenotype of the CRISPR-Cas9-generated global Bmal1KO rat and showed that despite having lower blood pressure values, both male and female rats maintained a diurnal rhythm of blood pressure and activity, in contrast to the mouse model. However, night–day differences in urine output and sodium excretion were markedly diminished.⁸⁵ This brings into attention the redundancy in mechanisms controlling temporal patterns of sodium excretion and blood pressure regulation. This study also showed an intact acute natriuretic response in Bmal1KO rats regardless of time of sodium intake,⁸⁵ proposing a role of compensatory clock and sodium handling mechanisms independent of the canonical molecular clock. Assessment of sodium transporters as well as their direct regulators, including ET-1, NO and aldosterone in this animal model is crucial for further understanding of the clock–pressure–natriuresis axis.

Loss of Per2 resulted in a mild reduction of diastolic blood pressure in mice, with no effect on blood pressure or diurnal rhythmicity in light–dark as well as constant darkness conditions.^60,110 These studies show the implications of clock genes in the overall control of blood pressure, yet studies directed toward deciphering the complexities of clock mechanisms that drive blood pressure rhythms are still lacking. Species-specific differences in blood pressure phenotype in different models of circadian dysfunction invite efforts to explore neurohormonal and metabolic factors modulating the central and peripheral clocks.

Studies utilizing models of tissue-specific clock gene deletions are aiming at exploring circadian mechanisms driven by the autonomous peripheral molecular clocks. Mice with deletion of the Bmal1 gene in the collecting duct principal cells, the site of highest ET_B receptor abundance in the renal tubules, were developed by our group using AQP2-Cre and floxed Bmal1 mice.¹¹¹ Male CD-Bmal1KO mice were hypotensive compared with their littermate controls, However, blood pressure rhythm was preserved in both groups, with no genotype difference in urinary sodium, aldosterone, or ET-1 excretion. The mild increase in blood pressure following 6 days of high salt diet was similar between Bmal1KO and their flox controls, suggesting that Bmal1 in this part of the nephron is not driving blood pressure rhythms and has no role in response to high salt diet. A similar observation was reported in mice lacking Bmal1 expression in the renal tubules, particularly the in thick ascending limb of the loop of Henle, DCT, and the collecting duct.¹¹² This animal model, KS-Bmal1 KO, was developed by crossing Bmal1 exon 8-floxed mice, with a kidney-specific cadherin Cre⁺ mice. Similar to the AQP2-driven Bmal1KO, these mice showed preserved circadian rhythms of blood pressure, with an 8-mm Hg reduction in their systolic blood pressure. It is important to note that genotype differences in blood pressure was only observed in male and not female mice in both studies,^111,112 suggesting that Bmal1 is important for regulating blood pressure setpoint in male but not female mice and invites research into potential sex-specific compensatory mechanisms. This opens the door for future studies of sex-specific mechanisms underlying circadian blood pressure regulation. In alignment with studies by our group and others, mice with nephron-specific inducible Bmal1 knockout (Bmal1lox/lox/Pax8-rtTA/LC1) showed mild reduction, only in systolic blood pressure with maintained rhythms of blood pressure as well as urine and sodium excretion.¹¹³ Mice with a loss of Bmal1 from renin-producing cells showed disruption in rhythms of sodium excretion, but not blood pressure, despite being lower in the Bmal1^lox/lox/Ren1^dCre mice.⁵⁶ Overall, these studies show that peripheral circadian clocks are involved in keeping blood pressure within physiological limits, but their role in promoting blood pressure rhythmicity over the 24-hour period and modulating response to changes in salt intake is yet to be determined.

In contrast to the studies investigating the effect of high salt diets on circadian rhythms, Pati et al. looked into the role of Period gene in regulating blood pressure rhythms under conditions of low salt diet.⁶⁰ Mice lacking all 3 Period isoforms, Per1, 2 and 3 (Per-TKO), showed aberrant blood pressure responses to low salt diet (0.01%–0.02% NaCl). Both WT and Per-KO mice were placed on low salt diet to stimulate endogenous Ang II response. While WT showed a reduction in blood pressure, Per-TKO mice response was reversed, exhibiting a significant increase their daytime mean arterial pressure, compared with WT controls. The daytime rise in blood pressure resulted in a loss of circadian rhythmicity and a nondipping pattern of blood pressure. This was associated with higher plasma renin and AT₁ receptor protein expression in Per-KO mice. Restoration of circadian rhythms was achieved with losartan, an AT₁ receptor antagonist.⁶⁰ This study suggests that the response to Ang II is regulated by Period genes and highlights the role of the clock in maintaining blood pressure rhythmicity under conditions of salt restriction.

Recent clinical studies provided evidence that timing of food and subsequently salt intake impacts the diurnal rhythms of sodium excretion. Proteomic analysis of human plasma samples subjected to 6 days of circadian misalignment induced by nighttime feeding, showed alterations in circadian rhythms in the expression of a wide array of signaling proteins.¹¹⁴ Rhythmic disruption included proteins associated with cardiovascular and chronic kidney disease. Despite the small sample size of 6 subjects in this study, the findings highlight the detrimental effects of salt and food intakes at the wrong time of day and the hidden risks associated with shift work.

Based on the available data to date, we conclude that loss of one or more components of the molecular circadian clock can disrupt the temporal patterns of sodium excretion, expression of sodium transporters as well as a range of relevant regulatory mediators. These findings suggest with considerable confidence that the intrarenal clock can function independently of environmental and time cues. Preliminary evidence strongly suggests that the time of day in which sodium is consumed could lead to irregularities in blood pressure rhythms, and therefore, increases the risk of cardiovascular and/or renal complications associated with nondipping blood pressures.

Funding

This work was supported by National Heart, Lung, and Blood Institute (P01HL136267) and American Heart Association (20PRE35200157).

DISCLOSURE

The authors declared no conflict of interest.