Loss of circadian gene Bmal1 in the collecting duct lowers blood pressure in male, but not female, mice

Abstract

Kidney function follows a 24-h rhythm subject to regulation by circadian genes including the transcription factor Bmal1. A high-salt diet induces a phase shift in Bmal1 expression in the renal inner medulla that is dependent on endothelin type B (ET_B) receptors. Furthermore, ET_B receptor-mediated natriuresis is sex dependent. Therefore, experiments tested the hypothesis that collecting duct Bmal1 regulates blood pressure in a sex-dependent manner. We generated a mouse model that lacks Bmal1 expression in the collecting duct, where ET_B receptor abundance is highest. Male, but not female, collecting duct Bmal1 knockout (CDBmal1KO) mice had significantly lower 24-h mean arterial pressure (MAP) than flox controls (105 ± 2 vs. 112 ± 3 mmHg for male mice and 106 ± 1 vs. 108 ± 1 mmHg for female mice, by telemetry). After 6 days on a high-salt (4% NaCl) diet, MAP remained significantly lower in male CDBmal1KO mice than in male flox control mice (107 ± 2 vs. 113 ± 1 mmHg), with no significant differences between genotypes in female mice (108 ± 2 vs. 109 ± 1 mmHg). ET_B receptor blockade for another 6 days increased MAP similarly in both male and female CDBmal1KO and flox control mice. However, MAP remained lower in male CDBmal1KO mice than in male flox control mice (124 ± 2 vs. 130 ± 2 mmHg). No significant differences were observed between female CDBmal1KO and flox mice during ET_B blockade (130 ± 2 vs. 127 ± 2 mmHg). There were no significant genotype differences in amplitude or phase of MAP in either sex. These data suggest that collecting duct Bmal1 has no role in circadian MAP but plays an important role in overall blood pressure in male, but not female, mice.

INTRODUCTION

Mammalian biological processes follow oscillating cycles over a 24-h period. The circadian clock is recognized as the central clock and peripheral clocks intertwined with one another to function in coordination to maintain physiological processes appropriate for the time of day. The central clock in mammals is located in the suprachiasmatic nucleus (SCN) of the brain, while peripheral clocks prevail in every other part of the body. In general, circadian biologists understand that the central clock in the SCN is entrained by light and, thereafter, entrains peripheral clocks in a tissue-specific manner (13, 31, 33). At its most basic molecular level, the circadian clock system consists of four main circadian genes that form transcription-translation feedback loops. In the positive arm of the feedback loop, the circadian proteins Bmal1 and Clock heterodimerize and bind to E-box elements of the circadian genes Per and Cry to initiate transcription. The negative arm of the loop consists of Per and Cry proteins translocating back into the nucleus and inhibiting transcription of Bmal1 and Clock. Although light is the dominant environmental cue for entrainment of the central clock, timing of food and type of food influence the clock mechanisms. Mukherji et al. (22) showed that limiting food intake to the resting phase in mice caused disruption of peripheral clock gene expression as well as disruption of hormone secretion. Our laboratory recently reported that high salt (HS) intake impacts the rhythms of several clock genes distinctly within each region of the kidney (29).

Under normal light-dark conditions, blood pressure (BP) oscillates over a 24-h period, with a 10–20% lower nighttime than daytime average. It has been shown that individuals are at higher risk of developing cardiovascular disease if they exhibit a nondipping BP phenotype, where BP reduction at night is <10% (12). Interestingly, genetic deletion of circadian genes in rodents causes distinct BP phenotypes. For instance, Bmal1 knockout mice are arrhythmic and do not exhibit an active rise in BP (5). Per1 knockout mice are hypotensive but maintain a rhythmic BP phenotype (26). Deletion of Cry leads to salt-sensitive hypertension, yet a rhythmic day-night variation is maintained (7). In addition, several groups have shown that water and electrolyte homeostasis is also under tight control of the circadian system (9, 15, 32). Given the importance of fluid-electrolyte control in BP regulation and the prevalence of salt-sensitive hypertension, it is important that we determine the relation between clock genes and salt intake.

Endothelin (ET)-1 is a peptide produced by various types of cells and participates in BP regulation via binding to its ET type A (ET_A) and type B (ET_B) receptors (20). Work from the Kohan and colleagues and our laboratory has demonstrated a specific role for ET_B receptors in the collecting duct (CD) in facilitating efficient natriuresis with HS intake (16, 20). Previous studies from our laboratory have demonstrated a potential involvement of ET-1 in the circadian system (18, 28, 29). Speed et al. (29) showed a 5.5-h phase delay in Bmal1 expression in the inner medulla (IM) of wild-type rats in response to a HS diet. However, the HS diet-induced Bmal1 phase shift was abolished in rats lacking functional ET_B receptors. In addition, Johnston et al. (18) showed that loss of functional ET_B receptors impairs Na⁺ excretion in a time of day-dependent manner. We hypothesized that the circadian gene Bmal1 in the CD regulates BP and Na⁺ excretion through the ET_B receptors. We generated a novel mouse model of a CD-specific deletion of Bmal1 to determine the role of CD Bmal1 in BP control.

METHODS

Animals.

All animal experiments were conducted with the approval of the University of Alabama at Birmingham Institutional Animal Care and Use Committee in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Aquaporin-2 (AQP2)-Cre recombinase (AQP2-Cre) mice were obtained from our local colony originally developed by Dr. Donald Kohan (1). Floxed Bmal1 mice were generously supplied by Dr. Martin Young (39). Male floxed Bmal1 mice were bred with female AQP2-Cre mice to obtain principal cell-specific knockout of the Bmal1 gene. Mice homozygous for floxed Bmal1 and heterozygous for AQP2-Cre were referred to as CDBmal1KO, whereas mice homozygous for floxed Bmal1 were used as controls. Both male and female mice (12–18 wk of age) were used in this study. Animals were kept in a room with a 12:12-h light-dark condition (lights on from 7 AM to 7 PM) throughout the study.

Validation of CDBmal1KO mice.

PCR was performed using the forward primer 5′-AGAATATGTCAGTACAGATTGCCTCC-3′ and reverse primer 5′-ACTTTTCACATCAGCTAACAAGGCAC-3′ (reference nos. 137796082 and 138103230, Integrated DNA Technologies) to determine if recombination occurred. Products were run on a 1.5% agarose gel in 0.5× Tris, acetic acid, and EDTA buffer. Primers were designed against the floxed exon 8 region of the Bmal1 gene that yields a 2,151-bp product in nonrecombined DNA and a 494-bp product in recombined DNA. For histology, kidneys were removed, immediately fixed in 10% formalin for 24 h, and embedded in paraffin for immunohistochemical analysis, as previously described (19), with anti-Bmal1 primary antibody (1:3,000 dilution, catalog no. 14020, Cell Signaling Technology, Danvers, MA) (35) and polymer-conjugated secondary antibody (Biocare Medical, Concord, CA).

BP, pulse pressure, and heart rate measurements by telemetry.

BP, pulse pressure, and heart rate (HR) were measured by telemetry (model TA11-PAC10, Data Science, St. Paul, MN). Telemetry devices were implanted into the right carotid artery of mice, as previously described (3). Mice were allowed to recover from surgery for ≥7–10 days before any experiment began. The sampling rate was 180 s every 20 min. A custom nonlinear regression equation [y = mesor + amplitude × cos(2 × π × x/period + arcophase)] was used for cosinor analysis on each individual mouse (2). Since the animal room was kept on a regular 24-h light-dark cycle, a period of 24 h was assumed. Mean estimated statistic of rhythm (mesor), amplitude, and acrophase were reported from the regression for each individual mouse.

Dietary salt intervention and metabolic cage experiments.

Mice were maintained in metabolic cages designed for urine collection and had free access to food and water. To prevent contamination of urine by regular pellet chow, a gel diet (Micro-stabilized Rodent Liquid Diet, TestDiet, Richmond, IN) containing 6.3% agar was used throughout the study. Mice were given 3 days to acclimate to the metabolic cage and gel diet before any dietary intervention. Twelve-hour food and water intake, as well as urine output, was monitored daily at 7 AM and 7 PM, corresponding to lights on and lights off, respectively. Mice were maintained on a normal-salt (NS; 0.4% NaCl) diet for 6 days followed by 6 days on a HS (4% NaCl) diet. To investigate the ET-1/ET_B pathway, mice were fed the ET_B receptor antagonist A-192621 (30 mg·kg⁻¹·day⁻¹) mixed in the HS food for the subsequent 6 days. This dose of A-192621 produces a near-maximal inhibition of ET_B receptor activity (34, 36).

Urinary measurements.

Urinary Na⁺ and K⁺ concentrations were determined by atomic absorption spectrometry (Analyst 200, Perkin-Elmer, Waltham, MA). Urinary ET-1 concentrations were determined by ELISA (QuantiGlo ET-1 Kit, R&D Systems, Minneapolis, MN). Reagents for measuring urinary aldosterone were generously provided by Dr. Elise Gomez-Sanchez and Dr. Celso Gomez-Sanchez (University of Mississippi Medical Center) (14). Briefly, a 96-well plate was coated with mouse γ-globulin overnight and washed three times before the addition of goat anti-mouse IgG. The plate was incubated for 30 min and then washed three times. Thereafter, 50 μL of Aldo A2E11 Integra antibody (1:75,000 dilution) were added to each well, along with 50 μL of standards or samples and 50 μL of aldosterone-3-CMO-biotin. The plate was incubated for 2 h and then washed, and avidin-horseradish peroxidase was added. The chromogenic substrate tetramethylbenzidine was added, and the plate was incubated for 30 min in a dark room before it was read. Urinary nitrite and nitrate (NO_x) concentration was analyzed using the ENO-30 HPLC system (Eicom, Kyoto, Japan), as previously described (11).

Statistical analysis.

Values are means ± SE. Student’s t test was used to compare differences in the mesor, amplitude, and acrophase of BP and HR from individual cosinor analysis as well as urinary excretion of ET-1, aldosterone, and NO_x. Two-way ANOVA with Sidak’s multiple comparison was used to compare food intake, water intake, urine production, and urinary Na⁺ and K⁺ excretion. P < 0.05 was considered significant.

RESULTS

Verification of CD principal cell Bmal1 knockout.

DNA from the renal cortex, outer medulla (OM), IM, tail, and liver was extracted from flox and CDBmal1KO mice. We detected recombined bands in the IM and OM (494 bp) of CDBmal1KO, but not flox, mice (Fig. 1A). There appeared to be a trace amount of recombined DNA in the cortex, but none was detected in tissue from the tail or liver. Since recent studies have shown that AQP2-Cre can be detected in certain parts of the mouse brain (21, 25), we also dissected mouse brain and extracted DNA from the frontal cortex, brain stem, hypothalamus, and cerebellum. In similar previous reports, we detected recombined alleles in the brain cortex and hypothalamus (Fig. 1B). To confirm loss of Bmal1 from the CD, we performed immunohistochemistry using kidney sections from flox and CDBmal1KO mice (Fig. 1C). In flox mice, Bmal1 colocalized with hematoxylin counterstaining in the nuclei; however, Bmal1 was absent in a large number of nuclei within the CD of the renal OM of CDBmal1KO mice. Using light microscopy, we randomly chose 16 visual fields and calculated the percentage of cells without Bmal1 staining (34 ± 10%).

Fig. 1.

Validation of collecting duct Bmal1 knockout (CDBmal1KO). A and B: PCR analysis for conditional Bmal1 knockout from kidney and other tissues. Samples from male and female mice yielded identical results (male mouse shown). CTX, cortex; OM, outer medulla; IM, inner medulla; NC, negative control. C: representative immunohistochemistry staining for Bmal1 in the kidney outer medulla. Absence of Bmal1 expression was clearly observed in kidneys of CDBmal1KO mice (blue, red arrow) in contrast with tissue from flox mice (brown).

Telemetry analysis in CDBmal1KO mice.

Mean arterial pressure (MAP) from the last 2 days of the NS diet, HS diet, or HS diet + ET_B antagonist treatment was used to perform cosinor analysis for each individual mouse. Mesor, amplitude, and acrophase were averaged in each group to compare genotype differences (Table 1). Under NS and HS diet conditions, 24-h MAP was significantly lower in male CDBmal1KO mice than in male flox control mice (Fig. 2, A and B). MAP increased in both male CDBmal1KO and flox control HS diet-fed mice in response to ET_B receptor blockade (Fig. 2C) and remained at a lower level in CDBmal1KO mice compared with flox control mice (Fig. 2C). No significant genotype differences in MAP of female mice were observed for any of the treatment groups (Fig. 2, G–I). Cosinor analysis was performed for each individual mouse, and figures were generated using GraphPad Prism (Fig. 2, D–F and J–L). No significant differences in amplitude or acrophase were observed between flox and CDBmal1KO in either male or female mice. In addition, we performed the same analysis to determine if there was any modification in systolic BP (SBP), diastolic BP (DBP), pulse pressure, and HR (Table 1). We did not observe significant differences in mesor, amplitude, and acrophase of SBP between flox control and CDBmal1KO mice in either sex. However, mesor of DBP was significantly lower in male CDBmal1KO than flox mice under NS, HS, and ET_B antagonist treatment (Table 1). There was no significant difference in pulse pressure between flox and CDBmal1KO mice in either sex. Under the NS condition, HR was slightly lower in male CDBmal1KO mice than in male flox mice; however, there was no significant difference under the HS condition or with ET_B antagonist treatment.

Table 1.

Cosinor analysis of telemetry data from male and female mice

	Flox			CDBmal1KO
	Mesor	Amplitude	Acrophase	Mesor	Amplitude	Acrophase
Systolic blood pressure
Male mice
NS diet	130 ± 3	9.8 ± 0.7	17.1 ± 1.2	124 ± 2	9.5 ± 0.6	18.2 ± 1.0
HS diet	132 ± 2	13.6 ± 0.8	14.9 ± 2.2	129 ± 2	12.7 ± 0.8	18.1 ± 1.2
HS diet + A-192621	152 ± 2	15.9 ± 0.7	17.6 ± 1.2	150 ± 3	14.5 ± 1.4	17.0 ± 1.0
Female mice
NS diet	126 ± 1	9.4 ± 0.4	18.1 ± 2.1	122 ± 1	9.2 ± 1.1	17.2 ± 2.1
HS diet	127 ± 2	11.1 ± 0.9	17.4 ± 1.3	126 ± 2	11.9 ± 1.3	18.0 ± 2.5
HS diet + A-192621	150 ± 4	11.6 ± 1.9	13.2 ± 1.1	153 ± 2	14.2 ± 1.7	12.3 ± 2.7
Diastolic blood pressure
Male mice
NS diet	94 ± 2	8.1 ± 0.5	17.1 ± 1.2	85 ± 2†	7.9 ± 0.6	17.6 ± 1.1
HS diet	95 ± 1	11.0 ± 0.7	16.8 ± 1.2	86 ± 2†	10.8 ± 0.7	17.0 ± 1.1
HS diet + A-192621	109 ± 2	12.6 ± 0.5	18.5 ± 1.1	100 ± 3*	11.0 ± 0.8	15.0 ± 1.4
Female mice
NS diet	90 ± 1	7.8 ± 0.6	18.2 ± 1.3	89 ± 1	7.0 ± 1.1	15.3 ± 2.3
HS diet	90 ± 2	9.1 ± 0.8	17.4 ± 2.1	90 ± 2	9.2 ± 0.9	16.3 ± 1.3
HS diet + A-192621	107 ± 2	8.6 ± 1.3	12.2 ± 1.4	108 ± 3	9.9 ± 1.1	11.8 ± 3.2
Mean arterial pressure
Male mice
NS diet	112 ± 2	8.9 ± 0.6	17.9 ± 1.7	105 ± 2*	8.6 ± 0.6	16.9 ± 1.1
HS diet	113 ± 1	12.3 ± 0.8	16.8 ± 1.8	107 ± 2*	11.5 ± 0.7	17.0 ± 1.1
HS diet + A-192621	130 ± 2	14.1 ± 0.5	17.9 ± 2.0	124 ± 2‡	12.4 ± 1	16.3 ± 1.0
Female mice
NS diet	108 ± 1	8.5 ± 0.5	17.1 ± 2.1	106 ± 1	8.2 ± 0.9	16.1 ± 1.3
HS diet	109 ± 1	9.9 ± 0.8	15.3 ± 1.9	108 ± 2	10.4 ± 1.0	18.4 ± 2.1
HS diet + A-192621	127 ± 2	9.8 ± 1.4	14.3 ± 1.6	130 ± 2	11.6 ± 1.4	14.3 ± 1.6
Pulse pressure
Male mice
NS diet	36 ± 2	1.8 ± 0.3	16.0 ± 1.7	39 ± 2	1.8 ± 0.2	17.1 ± 1.5
HS diet	37 ± 2	2.7 ± 0.2	15.6 ± 1.7	42 ± 3	2.1 ± 0.2	13.1 ± 2.1
HS diet + A-192621	43 ± 2	3.4 ± 0.6	17.3 ± 1.2	49 ± 4	3.7 ± 0.8	15.1 ± 1.9
Female mice
NS diet	36 ± 2	1.7 ± 0.2	22.2 ± 2.2	34 ± 1	2.2 ± 0.2	17.0 ± 4.2
HS diet	36 ± 2	2.1 ± 0.4	17.9 ± 1.5	36 ± 2	2.8 ± 0.5	14.5 ± 1.9
HS diet + A-192621	43 ± 4	3.1 ± 1.0	12.2 ± 2.1	45 ± 3	4.3 ± 0.6	11.4 ± 2.6
Heart rate
Male mice
NS diet	607 ± 8	47.2 ± 4.7	15.2 ± 0.8	597 ± 4	49.8 ± 4.5	13.5 ± 2.1
HS diet	579 ± 6	65.2 ± 4.9	16.6 ± 1.1	562 ± 3*	62.7 ± 4.0	12.2 ± 2.1
HS diet + A-192621	570 ± 4	63.5 ± 5.4	12.1 ± 2.7	564 ± 5	64 ± 5.8	14.4 ± 1.7
Female mice
NS diet	631 ± 17	38.7 ± 3.8	10.2 ± 2.1	637 ± 4	35.7 ± 2.9	9.2 ± 2.3
HS diet	596 ± 9	47.9 ± 9.0	6.8 ± 3.6	609 ± 6	41.2 ± 2.6	5.6 ± 3.8
HS diet + A-192621	592 ± 12	39.1 ± 4.5	7.2 ± 3.5	592 ± 4	34 ± 6.6	3.1 ± 1.5

Values are means  ±  SE. Units for mesor and amplitude: mmHg for systolic, diastolic, and mean arterial pressure and beats/min for heart rate; units for acrophase: zeitgeber time. NS, normal salt; HS, high salt.

^*P < 0.05 vs. flox mice, same sex, same treatment;

^†P < 0.01 vs. flox mice, same sex, same treatment;

^‡P = 0.05 vs. flox mice, same sex, same treatment.

Fig. 2.

Mean arterial pressure (MAP) of male (A–F) and female (G–L) mice. MAP from the last 2 days during normal-salt diet (A, D, G, and J), high-salt diet (B, E, H, and K), or high-salt diet + the endothelin type B (ET_B) receptor antagonist treatment (C, F, I, and L) treatment is presented as 24-h averages. Nonlinear cosinor analysis was used to determine the average MAP (mesor; see Table 1) of male (n = 8–9) and female (n = 6) mice. Student’s t test was used to compare CDBmal1KO vs. flox mice: *P < 0.05.

Urinary Na⁺ and K⁺ excretion.

In both male and female mice, the expected increase in Na⁺ excretion was observed during the active period compared with the inactive period (Fig. 3). However, there was no significant genotype difference in Na⁺ excretion in either sex on any diet. K⁺ excretion was not significantly changed by diet in any genotype or sex (Fig. 4).

Fig. 3.

Urinary Na⁺ excretion (U_NaV) during the last day of normal-salt diet (A and D), high-salt diet (B and E), or high-salt diet + the endothelin type B (ET_B) receptor antagonist A-192621 (C and F) treatment. Data are presented as active and inactive phases during different treatments. Two-way ANOVA was used to compare time and genotype.

Fig. 4.

Urinary K⁺ excretion (U_KV) during the last day of normal-salt diet (A and D), high-salt diet (B and E), or high-salt diet + the endothelin type B (ET_B) receptor antagonist A-192621 (C and F) treatment. Data are presented for active and inactive phases. Two-way ANOVA was used to compare time and genotype.

Food intake, water intake, and urine volume.

Table 2 shows food intake, water intake, and urine volume from the last 24-h period during NS, HS, or HS + ET_B antagonist treatment. As expected, all three measures were higher during the active period than the inactive period with NS, HS, or HS + ET_B antagonist; however, there was no significant difference between genotypes in either sex on any diet/treatment.

Table 2.

Food intake, water intake, and urine production in flox and CDBmal1 KO mice

	Flox		CDBmal1KO
	Active period	Inactive period	Active period	Inactive period
Food intake, g/12 h
Male mice
NS diet	6.3 ± 0.1	4.2 ± 0.2	5.4 ± 0.4	4.8 ± 0.8
HS diet	5.4 ± 0.3	2.5 ± 0.3§	5.9 ± 0.3	2.9 ± 0.1§
HS diet + A-192621	5.5 ± 0.2	2.3 ± 0.1§	5.5 ± 0.2	2.7 ± 0.1§
Female mice
NS diet	5.9 ± 0.1	2.1 ± 0.3§	6.2 ± 0.3	2.0 ± 0.4§
HS diet	5.4 ± 0.3	2.1 ± 0.4‡	5.8 ± 0.2	2.0 ± 0.4§
HS diet + A-192621	5.3 ± 0.1	2.4 ± 0.5§	5.5 ± 0.3	2.6 ± 0.2‡
Water intake, mL/12 h
Male mice
NS diet	1.0 ± 0.3	0.6 ± 0.1	0.9 ± 0.1	0.6 ± 0.1
HS diet	8.1 ± 0.1	2.1 ± 0.2§	8.0 ± 0.3	2.6 ± 0.1§
HS diet + A-192621	11.2 ± 0.5	3.1 ± 0.3§	10.6 ± 0.4	3.5 ± 0.2§
Female mice
NS diet	1.0 ± 0.1	0.6 ± 0.2	1.2 ± 0.2	0.8 ± 0.1
HS diet	8.1 ± 0.4	2.7 ± 0.3§	7.5 ± 0.9	2.8 ± 0.3‡
HS diet + A-192621	9.7 ± 0.3	2.9 ± 0.1§	10.2 ± 0.7	3.9 ± 0.6§
Urine volume, mL/12 h
Male mice
NS diet	1.3 ± 0.5	0.4 ± 0.1	1.5 ± 0.5	0.5 ± 0.1
HS diet	6.1 ± 0.7	1.3 ± 0.4§	5.9 ± 0.4	1.8 ± 0.1‡
HS diet + A-192621	7.3 ± 1.5	2.5 ± 0.2†	7.5 ± 0.5	2.9 ± 0.2†
Female mice
NS diet	1.2 ± 0.3	0.3 ± 0.1*	1.0 ± 0.2	0.2 ± 0.1
HS diet	5.7 ± 0.5	1.6 ± 0.3*	3.8 ± 1.3	1.6 ± 0.2
HS diet + A-192621	6.6 ± 0.5	2.2 ± 0.3‡	6.7 ± 0.6	2.7 ± 0.5‡

Values are means  ±  SE; n = 3−4. NS, normal salt; HS, high salt.

^*P < 0.05 vs. the respective active period, same sex, same treatment;

^†P < 0.01 vs. the respective active period, same sex, same treatment;

^‡P < 0.001 vs. the respective active period, same sex, same treatment;

^§P < 0.0001 vs. the respective active period, same sex, same treatment.

Urinary ET-1 and aldosterone excretion.

Using urine samples collected from the 12-h active period of the last day (urine volume from the inactive phase was not adequate for the ELISA protocol) during NS, HS, or HS + ET_B antagonist treatment, we determined urinary excretion of ET-1 and aldosterone. We did not observe any significant genotype difference in either male or female mice fed the NS or HS diet (Fig. 5, A, B, D, and E). ET-1 excretion following ET_B antagonist treatment was significantly higher in female, but not male, CDBmal1KO mice than flox control mice (Fig. 5, C and F). As expected, urinary aldosterone excretion was much lower in all mice on the HS diet. However, no significant differences in urinary aldosterone excretion were observed between genotype or sex (Fig. 6). Also, no significant differences in urinary excretion of NO_x during the active period of NS treatment were observed between genotype or sex (700 ± 178 and 574 ± 106 nmol/12 h for male flox and CDBmal1KO mice, respectively, and 668 ± 85 and 474 ± 79 nmol/12 h for female flox and CDBmal1KO mice, respectively).

Fig. 5.

Urinary endothelin (ET)-1 excretion during the active phase of the last day of normal-salt diet (A and D), high-salt diet (B and E), or high-salt diet + the ET type B (ET_B) receptor antagonist A-192621 (C and F) treatment. Data are presented for the active phase. Student’s t test was used to compare CDBmal1KO vs. flox mice: *P < 0.05.

Fig. 6.

Urinary aldosterone excretion during the active phase of the last day of normal-salt diet (A and D), high-salt diet (B and E), or high-salt diet + the endothelin type B (ET_B) receptor antagonist A-192621 (C and F) treatment. Data are presented for the active phase. Student’s t test was used to compare CDBmal1KO vs. flox mice.

Since there is some recent evidence showing that Bmal1 may regulate PGE₂ in nonkidney cells (4, 17), we determined PGE₂ excretion rate in flox and CDBmal1KO mice. However, we did not observe a significant difference between genotypes on either the NS diet (3.6 ± 0.6 and 4.6 ± 1.6 ng/12 h for male flox and CDBmal1KO mice, respectively, and 5.1 ± 0.7 and 3.7 ± 0.6 ng/12 h for female flox and CDBmal1KO mice, respectively) or the HS diet (10.6 ± 2.4 and 11.5 ± 2.6 ng/12 h for male flox and CDBmal1KO mice, respectively, and 10.0 ± 2.7 and 8.1 ± 2.5 ng/12 h for female flox and CDBmal1KO mice, respectively.)

DISCUSSION

Circadian misalignment predisposes individuals to increased risk of developing cardiovascular and renal diseases, including an abnormal BP rhythm (8). Previous rodent studies from our laboratory have suggested a sex-dependent difference in the circadian system of BP rhythms, as well as renal function, with or without dietary intervention (18). The present study was designed to determine the role of the circadian gene Bmal1 in the renal CD on BP rhythms. We originally hypothesized abnormalities in the BP rhythm (amplitude or phase) of CDBmal1KO mice; however, we did not see any significant alterations in the circadian characteristics of BP aside from the reduced mesor in male mice. Thus, our findings demonstrate that Bmal1 in the CD lowers overall MAP in male mice but does not significantly affect female mice. The genotype difference in MAP was primarily driven by the lower DBP in male CDBmal1KO mice, whereas SBP only trended to be different. In addition, CD Bmal1 does not affect the BP amplitude or acrophase in either sex. Moreover, CD Bmal1 does not cause a disruption of the diurnal urinary excretion of Na⁺ or K⁺. These data reveal a sex-specific, circadian-independent regulation of BP via Bmal1 in the CD.

Over the past decade, substantial research has been focused on deciphering the connection between the circadian system and diurnal BP regulation. In 2007, using global Bmal1 knockout mice, FitzGerald and colleagues (5) showed that deletion of a major circadian gene abolished circadian BP variation. In addition, they used Tek-Cre (Tie2-Cre) and generated endothelial Bmal1 knockout mice that maintained a rhythm, although BP in the active phase was lower in these endothelium-specific knockout than flox control mice (37). Xie et al. (38) reported that selective deletion of Bmal1 in the smooth muscle diminished the amplitude of BP rhythms without affecting locomotor activity, suggesting a role for Bmal1 in BP regulation independent of the SCN. Firsov and colleagues (23, 32) generated two transgenic models in which Bmal1 was deleted in either renal tubular (Pax8 Cre) or renin-secreting (Ren1d Cre) cells. They found that deletion of Bmal1 in both models resulted in a modest decrease in BP. However, the circadian BP rhythm remained intact. Our results reported here also suggest that Bmal1 in the kidney CD is not maintaining the circadian rhythm of BP or the diurnal rhythm of kidney excretory function under typical light-dark conditions.

Our findings highlight the importance of conducting experiments in both male and female mice. All previous studies using various Bmal1 KO mice were conducted exclusively in male mice. Our laboratory and many others have shown that BP regulation and renal electrolyte handling are largely sex dependent. We therefore performed experiments in both male and female mice. Indeed, we found distinct BP phenotypes between the sexes. In addition, there was no significant difference in urine volume, urinary Na⁺ excretion, or urinary K⁺ excretion. Urinary ET-1 is considered an indicator of renal ET-1 production. We found a modest increase in ET-1 excretion in female CDBmal1KO mice in response the ET_B antagonist; however, this was not accompanied by modifications in either BP or renal excretory function. In addition, we showed that the lower level of BP in male CDBmal1KO mice was not directly related to the level of aldosterone or nitrite/nitrate.

Gumz et al. (15) showed that the circadian gene Per1 is a target of aldosterone and directly regulates the expression and abundance of the epithelial Na⁺ channel as well as ET-1. More recently, they discovered a sex-specific nondipping BP phenotype in response to mineralocorticoid hypertension in global Per1 knockout mice, where female mice were protected from nondipping hypertension (9, 10), which is consistent with the clinical phenomenon that premenopausal women have low incidence of developing nondipping hypertension (27). However, this sex-dependent BP phenotype was observed in global Per1 knockout mice. Whether the kidney contributes to the protective role in female mice remains unknown.

Many chronic diseases are accompanied by a compromised circadian rhythm of BP. For instance, patients with chronic kidney disease often present with nondipping nighttime BP. It has been suggested that, in addition to 24-h ambulatory BP level, nighttime BP should be a separate indicator for treatment and prognosis (24). Su et al. (30) showed a decrease in the circadian BP phenotype in a mouse model of diabetes, which is consistent with the clinical presentation in humans. In clinical trials to investigate nocturnal BP dipping and arterial stiffness in patients with chronic kidney disease and healthy controls, Dhaun et al. (6) found that elevated plasma ET-1 levels were associated with the nondipping BP in patients with chronic kidney disease. In addition, 6 wk of treatment with sitaxentan (an ET_A receptor antagonist) significantly improved the nocturnal dipping, suggesting involvement of the ET system in the diurnal variation of BP in the setting of renal diseases. Our laboratory has previously reported that rats lacking ET_B receptor function have salt-sensitive hypertension and an exaggerated BP rhythm, providing evidence for a connection between the ET system and circadian BP control. However, in our flox and CDBmal1KO mice, there was no significant difference in the diurnal BP variation following chronic ET_B receptor blockade, suggesting a species difference in circadian BP control. In addition, future studies are needed to determine if the ET system contributes to diurnal BP regulation under specific conditions such as acute kidney injury, chronic kidney disease, and diabetic nephropathy.

It is crucial that we understand how circadian BP rhythm is regulated. Overall, our study showed the sex-dependent regulation of BP by CD Bmal1, which aids our understanding of the connection between circadian misalignment and cardiovascular health. While Bmal1 is considered a core clock gene, the characteristic circadian pattern of BP remained intact, despite a lower overall BP throughout the day. Thus, the relation between rhythmicity of BP and overall BP appears to be distinct. One limitation of our model is that we do not have a thorough understanding of the possible involvement of Bmal1 knockout in the brain with the AQP2-Cre activity that we and others have detected. How this may impact circadian physiology and BP, if at all, remains to be investigated.

GRANTS

This work was funded by National Heart, Lung, and Blood Institute Grants P01-HL-136267 and P01-HL-069999 (to J. S. Pollock and D. M. Pollock), American Heart Association Strategically Focused Network Grant 15SFRN2390002 (to J. S. Pollock and D. M. Pollock), a University of Alabama Birmingham School of Medicine AMC21 Reload Multi-Investigator Grant (to J. S. Pollock and D. M. Pollock), National Heart, Lung, and Blood Institute Grant HL-127178 (to J. S. Speed), and National Institute of Diabetes and Digestive and Kidney Diseases Grants T32-DK-007545 (to M. K. Rhoads) and F31-DK-111067 (to J. S. Speed). This work was conducted as a part of the PhD thesis research of D. Zhang, which was supported by American Heart Association Predoctoral Fellowship 18PRE33990345.

DISCLOSURES

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

AUTHOR CONTRIBUTIONS

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