Differences in renal BMAL1 contribution to Na⁺ homeostasis and blood pressure control in male and female mice

Abstract

The renal circadian clock has a major influence on the function of the kidney. Aryl hydrocarbon receptor nuclear translocator-like protein 1 [ARNTL; also known as brain and muscle ARNT-like 1 (BMAL1)] is a core clock protein and transcription factor that regulates the expression of nearly half of all genes. Using male and female kidney-specific cadherin BMAL1 knockout (KS-BMAL1 KO) mice, we examined the role of renal distal segment BMAL1 in blood pressure control and solute handling. We confirmed that this mouse model does not express BMAL1 in thick ascending limb, distal convoluted tubule, and collecting duct cells, which are the final locations for solute and fluid regulation. Male KS-BMAL1 KO mice displayed a substantially lower basal systolic blood pressure compared with littermate control mice, yet their circadian rhythm in pressure remained unchanged [male control mice: 127 ± 0.7 mmHg (n = 4) vs. male KS-BMAL KO mice: 119 ± 2.3 mmHg (n = 5), P < 0.05]. Female mice, however, did not display a genotype difference in basal systolic blood pressure [female control mice: 120 ± 1.6 mmHg (n = 5) vs. female KS-BMAL1 KO mice: 119 ± 1.5 mmHg (n = 7), P = 0.4]. In addition, male KS-BMAL1 KO mice had less Na⁺ retention compared with control mice in response to a K⁺-restricted diet (15% less following 5 days of treatment). However, there was no genotype difference in Na⁺ handling after a K⁺-restricted diet in female mice. Furthermore, there was evidence indicating a sex-specific response to K⁺ restriction where female mice reabsorbed less Na⁺ in response to this dietary challenge compared with male mice. We propose that BMAL1 in the distal nephron and collecting duct contributes to blood pressure regulation and Na⁺ handling in a sex-specific manner.

INTRODUCTION

Most physiological functions exhibit a circadian rhythm. These effects are mediated in part by a central clock that resides in the superchiasmatic nucleus of the brain, but it is well accepted that rhythms in physiological function are also influenced by peripheral circadian clocks (43). Both the central and peripheral clocks have the same core components that control the transcription of nearly half of all genes (46). Core proteins circadian locomotor output cycles kaput (CLOCK) and aryl hydrocarbon receptor nuclear translocator-like protein [ARNTL; also known as brain and muscle ARNT-like 1 (BMAL1)] heterodimerize and interact with E box promoter elements to drive transcription of target genes. This process leads to the translation of cryptochrome (CRY) and period (PER) proteins, which act to inhibit transcription of CLOCK and BMAL1, creating a feedback loop (11, 22). This transcriptional loop is active in cells throughout the entire body, affecting biological processes in an organ- and cell type-specific manner (46). Disruption of these circadian controlled processes is associated with many pathologies, including hypertension (25). The present study examined the role of the peripheral circadian clock, specifically the protein BMAL1, within cells of the kidney that are critical to solute homeostasis and blood pressure control.

Single-nucleotide polymorphisms in the BMAL1 gene have been associated with diabetes and elevated blood pressure in humans (21, 41). Use of BMAL1 knockout (KO) mouse models provides a way to gain a better understanding of the mechanism behind these human findings. Data from several mouse models have already demonstrated a link between BMAL1 and blood pressure, as summarized below and in recent review articles (45). Curtis et al. (5) demonstrated that male global BMAL1 KO mice have ~10 mmHg lower blood pressure than wild-type mice. Xie et al. (42) showed that smooth muscle cell-specific KO of BMAL1 leads to decreased blood pressure. Using renin cell-specific Cre recombinase to drive BMAL1 KO, Tokonami et al. (38) also demonstrated that KO mice exhibited a lower blood pressure phenotype compared with control mice. Nikolaeva et al. (20) showed that an inducible tubule-specific BMAL1 KO mouse model had lower blood pressure than controls. Although renal-focused BMAL1 KO mice have been reported to have different blood pressure phenotypes compared with control mice, the studies mentioned thus far looked exclusively at basal conditions in male mice. A recent study from Zhang et al. (44) not only demonstrated that male collecting duct-specific BMAL1 KO mice had a lower blood pressure phenotype compared with control mice but also that female mice did not display this difference. Additionally, this group administered a high-salt (HS) diet (4% NaCl) and HS diet with endothelin type B receptor antagonist to these mice, but there was no effect on genotype differences (44). To better understand the role of BMAL1 in the kidney, we used kidney-specific cadherin Cre recombinase to generate kidney-specific BMAL1 KO (KS-BMAL1 KO) mice and compared them with control mice. Both male and female mice were included in the assessment of basal blood pressure and the solute handling response to the administration of diets previously shown to influence solute homeostasis.

Our first objective was to determine the basal blood pressure phenotype of male and female KS-BMAL1 KO mice. Given the relationship between Na⁺ retention and blood pressure changes, the second goal of this study was to investigate the renal response to distinct diets that have been shown to promote Na⁺ retention. Genotype differences were detected in the blood pressures of male mice with KS-BMAL1 KO displaying lower pressures than control mice. However, this difference was not present in female mice. Male KS-BMAL1 KO mice, but not female KS-BMAL1 KO mice, exhibited a lower Na⁺ balance in response to a K⁺-depleted diet compared with control mice. These data suggest that BMAL1-dependent mechanisms in the distal nephron and collecting duct contribute to blood pressure regulation and Na⁺ handling in a sex-specific manner.

METHODS

Animals and metabolic cages.

The mouse model used in this study was generated using floxed exon 8 BMAL1 mice [a gift from K. Esser (42)] crossed with kidney-specific cadherin Cre+ mice [The Jackson Laboratory (27)] to generate KS-BMAL1 KO mice. Floxed Cre- littermates were used as control mice. Experiments used virgin male and female young adult mice aged 22–29 wk old. Genomic DNA extracted from tail-clip samples was used for genotyping. Genomic DNA extracted from various tissues was used as the template in recombination PCR assays to confirm tissue-specific KO. Mice were housed in temperature (20–26°C)- and humidity-controlled, 12:12-h light-dark cycled rooms. Mice were provided ad libitum access to water and standard 18% protein rodent chow (no. 2918, Harlan Teklad). All experiments were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved and monitored by the University of Florida Institutional Animal Care and Use Committee and the North Florida/South Georgia Veterans Administration Institutional Animal Care and Use Committee.

Experiments used mice categorized into the following three groups:

1. Untreated mice under basal conditions (male control mice: n = 28, male KS-BMAL1 KO mice: n = 27, female control mice: n = 32, and female KS-BMAL1 KO mice: n = 30)

2. Mice administered treatment 1 (male control mice: n = 6 and male KS-BMAL1 KO mice: n = 6)

3. Mice administered treatment 2 (male control mice: n = 6, male KS-BMAL1 KO mice: n = 5), female control mice; n = 6, and female KS-BMAL1 KO mice: n = 6)

Tissues from untreated control and KS-BMAL1 KO randomly selected littermates were collected to measure basal conditions of physiological parameters or medullary mRNA expression. Separate sets of mice were randomly housed individually in metabolic cages (6 control and 6 KS-BMAL1 KO mice) and acclimated for 3 days using normal chow diet before beginning treatment regimens. Food administered in metabolic cages was prepared in gel form (1% agar) using powdered base diet.

For treatment 1, mice were placed on a gel control diet (0.25% NaCl and 0.6% K⁺, no. 99131, Envigo) for 3 days for baseline recordings. Next, mice were given a low-salt diet for 3 days (0% NaCl and 0.6% K⁺, no. 03582, Envigo). Mice were then placed on a HS diet (3.7% NaCl and 0.6% K⁺, no. 170601, Envigo) for 3 days. Finally, mice were given an injection of the mineralocorticoid analog desoxycorticosterone pivalate (DOCP; 75 µg/g body wt) and continued on the HS diet for 5 days.

For treatment 2, a separate set of mice was placed on a gel control diet (0.25% NaCl and 0.6% K⁺) for 3 days for baseline collections. A K⁺-deficient diet was then administered for 5 days (0.25% NaCl and 0.0001% K⁺, no. 99134, Envigo).

For both treatment groups, urine samples were collected every 12 h before each light-dark Zeitgeber (6:00 and 18:00 hours or 0 and 12 Zeitgeber time). Urine electrolyte concentrations were measured by a flame photometer (model 2655-00, Cole Parmer) according to the manufacturer’s instructions.

Telemetry.

An independent set of mice under basal conditions followed by treatment 1 was implanted with telemetry devices from Data Sciences International through the carotid artery and extended into the aortic arch as previously described (4, 33). After 7 days of recovery, systolic blood pressure, diastolic blood pressure, mean arterial pressure, pulse pressure, heart rate, and locomotor activity data were collected for 3 consecutive days. These mice were used exclusively for telemetry recordings.

Tissue collection.

Given the importance of factoring in time of day in the experimental design on top of the fact that this mouse model manipulates a circadian clock protein, we took a systematic approach by collecting tissue during the active (midnight) and inactive (noon) periods to determine basal mRNA expression. Due to the heterogeneity of the kidney, we isolated cortex and medulla tissue upon kidney removal for biochemical analysis. Serum from K⁺-depleted mice was collected at noon on the day following the final metabolic cage collection. An aldosterone ELISA kit was performed according to the manufacturer’s instructions to determine aldosterone levels (Enzo Life Sciences).

Recombination PCR.

Genomic DNA was extracted from the dissected renal inner medulla for recombination triplex PCR analysis using three different primers to detect the absence of exon 8 following Cre-mediated excision (32). Various extrarenal tissues were collected from male and female control and KS-BMAL1 KO mice to detect the absence of exon 8 following Cre-mediated excision.

Immunohistochemistry.

The efficacy of KS-BMAL1 KO was assessed using immunohistochemistry. Mice were anesthetized with inhalant isoflurane at noon. The kidneys were preserved by in vivo cardiac perfusion with PBS (pH 7.4) followed by periodate-lysine-2% paraformaldehyde, cut transversely into ~3-mm-thick slices from the middle of the kidney, and then immersed for 24–48 h at 4°C in the same fixative. Kidney samples from each animal were embedded in polyester wax made using polyethylene glycol 400 distearate (Polysciences) with 10% 1-hexadecanol, and 2-μm-thick sections were cut and mounted on gelatin-coated glass slides. Immunolocalization was performed using immunoperoxidase procedures. Briefly, sections were dewaxed in ethanol, rehydrated, and incubated in heated Trilogy (Cell Marque). Endogenous peroxidase activity was blocked by incubating the sections in 3% H₂O₂ for 45 min. After being blocked for 15 min (Protein Block, Dako), sections were incubated at 4°C overnight with BMAL1 primary antibody (Cell Signaling). Sections were washed in PBS and incubated for 30 min with polymer-linked peroxidase-conjugated secondary IgG (Vector ImmPress) and then 3,3′-diaminobenzidine chromogenic substrate for detection (Vector). The same process was repeated with a second primary antibody [phosphorylated Na⁺-Cl⁻ cotransporter (p-NCC), Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2), or aquaporin-2 (AQP2)] and blue-gray substrate (Vector SG). Sections were washed in distilled water, dehydrated with xylene, mounted, and observed by light microscopy [Nikon Eclipse Ti (16)]. Antibody information is shown in Table 1.

Table 1.

Antibody information

Antibody Target	Manufacturer	Catalog Number	Reference	Description	Dilution Used	RRID
AQP2	Stressmarq	SPC-503D	35	Rabbit, polyclonal	IHC: 1:400	AB_10965071
BMAL1	Cell Signaling	D2L7G	18	Rabbit, polyclonal	IHC: 1:3,000; Western blot analysis: 1:1,000	AB_2728705
NKCC2	Stressmarq	SPC-401D	19	Rabbit, polyclonal	IHC: 1:10,000	AB_10640877
T53 p-NCC	Previously characterized		30	Rabbit, polyclonal	IHC: 1:1,000,000	Not applicable

Antibodies were used in this study as previously described (18, 19, 30, 35). AQP2, aquaporin 2; BMAL1, brain and muscle ARNT-like 1; NKCC2, Na⁺-K⁺-2Cl⁻ cotransporter; T53 p-NCC, T53 phosphorylated Na⁺-Cl⁻ cotransporter; IHC, immunohistochemistry.

Quantitative PCR.

Total RNA was isolated and quantitative PCR was performed as previously described (29). The probes used are shown in Table 2.

Table 2.

Quantitative PCR probes

Gene	Species	Gene Expression Assay Identification
ACTB	Mouse	Mm00607939_s1
ATP4A	Mouse	Mm00444417_m1
ATP12A	Mouse	Mm01318023_m1
KCNJ1	Mouse	Mm00444727_s1
KCNJ15	Mouse	Mm02020346_s1
KCNMA1	Mouse	Mm00516078_m1
SCNN1A	Mouse	Mm00803386_m1
SCNN1B	Mouse	Mm00441215_m1
SCNN1G	Mouse	Mm00441228_m1
SGK1	Mouse	Mm00441387_g1
SLC12A1	Mouse	Mm01275821_m1
SLC26A4	Mouse	Mm01258316_m1
WNK1	Mouse	Mm01184014_m1
WNK4	Mouse	Mm00841400_m1

ACTB, β-actin; ATP4A and ATP12A, gastric and nongastric subunits of H⁺/K⁺-ATPase; KCNJ1, gene that encodes renal outer medullary K⁺ channel protein; KCNJ15, inwardly rectifying K⁺ channel; KCNMA1, large-conductance channel subunit coding gene; SCNN1A, SCNN1B, and SCNN1G, α-, β-, and γ-subunits of the epithelial Na⁺ channel, respectively; SGK1, serum/glucocorticoid-regulated kinase 1; SLC12A1, Na⁺-K⁺-2Cl⁻ cotransporter gene; SLC26A4, pendrin gene; WNK, with no lysine protein kinase.

Western blot analysis.

Renal inner medullary samples from male mice were homogenized using T-PER Tissue Protein Extraction Reagent and a protease/phosphatase inhibitor cocktail (Thermo Scientific). The protein concentration for each sample was determined by BCA (Pierce). Protein (25 µg) was separated on a 4–20% Tris·HCl precast gel (Bio-Rad) and transferred to a polyvinylidene difluoride membrane. The membrane was stained with Ponceau for 5 min and imaged before being washed with Tris-buffered saline. The membrane was blocked overnight at 4°C with 5% BSA in Tris-buffered saline plus 0.1% Tween (TBS-T) and then incubated overnight at 4°C with anti-BMAL1 (1:1,000). The membrane was washed for 30 min in TBS-T and then incubated with horseradish peroxidase-conjugated anti-rabbit secondary antibody. After an additional wash with TBS-T for 30 min, detection was performed using SignalFire ECL reagent (Cell Signaling Technology) with a 30-s exposure time for imaging. Densitometry was performed using Fiji, and protein abundance was normalized to Ponceau staining.

Statistical analyses.

Graphical data are presented as means ± SE. Two-way ANOVA was used to analyze differences between the four groups with repeated measures when necessary. Tukey multiple comparisons were used for post hoc analysis. A Student’s t test was used to compare two groups. For quantitative PCR data sets that did not pass the D’Agostino-Pearson omnibus (K2) normality test, nonparametric Mann-Whitney t tests were performed to compare groups. Three-way ANOVA was used to compare quantitative PCR data between sexes. Analysis was performed using GraphPad Prism version 7.0 software (GraphPad Software). Statistical significance was defined as P < 0.05. Rhythms in blood pressure from each mouse were analyzed with Cosinor software (freely available at http://www.circadian.org/softwar.html). Period, mesor, and amplitude values were calculated. Mesor refers to the midline estimating statistic of rhythm, amplitude represents the extent of predictable change within a cycle, and period refers to the duration of a cycle from peak to peak.

RESULTS

Verification of the KS-BMAL1 KO model.

A potential issue of using genetically modified mice that implement the Cre/lox method are off-target deletions (2). This problem is particularly important in blood pressure studies as many systems and organs contribute to blood pressure control. Ksp-cadherin Cre was used in this study to maximize the specificity of the KO to the kidney. A thorough examination of the mouse model used in this study was carried out to distinguish which tissues were affected by the kidney-specific cadherin Cre/lox method used in creating KS-BMAL1 KO. Floxed Cre- littermates were used as control mice. Here, the term ‘floxed’ represents the gene with the two loxP sites (Fig. 1A). Recombination PCR for BMAL1 on male and female renal inner medullary tissue from KS-BMAL1 KO mice yielded two bands, one being the product of the recombined BMAL1 gene (570 bp, denoted by * in Fig. 1B) and the other, smaller band being the product of the intact floxed gene that is present in cells that do not express kidney-specific cadherin Cre recombinase (Fig. 1, A and B). The same procedure performed using genomic DNA from several nonrenal tissues provided no evidence for recombination except for a faint band that was detected in the ovaries (Fig. 1C).

Fig. 1.

Characterization of kidney-specific brain and muscle ARNT-like 1 (BMAL1) knockout (KS-BMAL1 KO) mice. A: model showing exon 8 of BMAL1 excision by Ksp-cadherin Cre. Solid gray arrows represent primers used for recombination PCR. *Band produced from the recombined floxed gene. B: recombination PCR assay showing the expected 570-bp product (*) in renal inner medullary tissue of KS-BMAL1 KO mice but not control (CNTL) mice. C: additional assays assessing nonrenal tissue revealing the lack of the KO band. D−I: representative images of BMAL1 protein expression in renal segments. D, F, and H: colabeling with BMAL1 (brown) in CNTL mice; E, G, and I: colabeling with BMAL1 (brown) in KS-BMAL1 KO mice. D and E: blue staining with Na⁺-K⁺-2Cl⁻ cotransporter 2 (NKCC2) representing the cortical thick ascending limb (TAL). F and G: blue staining with phosphorylated Na⁺-Cl⁻ cotransporter (p-NCC) to identify distal convoluted tubules (DCTs). H and I: blue staining with aquaporin 2 representing medullary collecting duct (CD) cells. Both PCR and immunohistochemistry images are from male tissue except for ovary tissue from female mice. Arrows indicate BMAL1-positive stained nuclei; arrowheads indicate nuclei with negative BMAL1 staining. J: immunoblots of renal inner medullary homogenates from male mice to demonstrate BMAL1 protein expression and total protein from Ponceau staining. The predicted size of BMAL1 band (78 kDa) is noted. K: quantification determined by normalizing BMAL1 values with total protein. PT, proximal tubule. Values are means ± SE; n = 4. Student’s t test was used to compare genotypes. Scale bar = 0.05 mm.

The BMAL1 KO efficiency of renal protein expression was assessed using double-labeling immunohistochemistry from tissues collected at noon in male and female mice. Kidney sections dual labeled using NKCC2 established that BMAL1 expression was virtually absent in thick ascending limb cells in KS-BMAL1 KO compared with control mice (Fig. 1, D and E). Costaining with p-NCC antibody determined that KS-BMAL1 KO mice had BMAL1 expression in fewer distal convoluted tubule cells than control mice (Fig. 1, F and G). AQP2 costaining showed complete KO of BMAL1 expression in principal cells of kidney sections (Fig. 1, H and I). Western blot analysis of BMAL1 in inner medullary tissue, containing collecting ducts, thin limbs, and vasa recta, demonstrated that KS-BMAL1 KO mice had approximately half the protein expression of control mice (Fig. 1, J and K). Further assessment of dual-labeled kidney sections demonstrated efficient KO of BMAL1 in intercalated cells of KS-BMAL1 KO mice compared with control mice (Fig. 2, A and B). Expression of BMAL1 remained intact in vasa recta capillaries (Fig. 2, C and D), macula densa cells (Fig. 2, E and F), and glomerular capillaries and afferent arterioles of KS-BMAL1 KO mice (Fig. 2, G and H). Several basal physiological parameters are shown in Table 3. It is important to note when describing the KO model used in this study, BMAL1 expression was clearly absent or decreased in the distal nephron and collecting duct segments.

Fig. 2.

Representative images of brain and muscle ARNT-like 1 (BMAL1) expression in different regions of the kidney from male kidney-specific BMAL1 knockout (KS-BMAL1 KO) mouse sections. A–H: colabeling with BMAL1 (brown) and Na⁺-K⁺-2Cl⁻ cotransporter (NKCC2; A–D) or aquaporin 2 (E–H) in blue stain. NKCC2- and aquaporin 2-positive stained cells efficiently lacked BMAL1 expression in KS-BMAL1 KO mice. G: afferent and efferent arterioles (arrowheads) of a glomerulus. H: medullary vascular bed containing vasa recta capillaries (VR). The macula densa (MD) was positive for BMAL1. CD, collecting duct; IC, intercalated cells; PT, proximal tubule; TAL, thick ascending limb. Scale bar = 0.05 mm.

Table 3.

Basal physiological parameters in young adult control and KS-BMAL1 KO mice measured from samples collected at noon (Zeitgeber 6)

Parameter (Noon)	Control Mice	KS-BMAL1 KO Mice
Body weight, g
Male	31.7 ± 0.8 (12)	32.2 ± 0.7 (12)
Female	21.7 ± 0.6 (12)‡	22.2 ± 0.7 (12)‡
Blood Na+, mmol/L
Male	145.5 ± 0.4 (8)	145.9 ± 0.6 (4)
Female	146.5 ± 0.7 (4)	145.6 ± 0.6 (7)
Blood K+, mmol/L
Male	4.9 ± 0.09 (6)	4.5 ± 0.11 (5)
Female	5.8 ± 0.2 (4)‡	5.8 ± 0.3 (6)‡
Blood bicarbonate, mmol/L
Male	15.5 ± 0.4 (8)	17.2 ± 1.2 (4)†
Female	16.1 ± 0.7 (4)	19.3 ± 0.6 (7)*†
Blood pH
Male	7.4 ± 0.02 (8)	7.4 ± 0.02 (4)
Female	7.4 ± 0.03 (4)	7.4 ± 0.02 (7)
Hematocrit
Male	38.6 ± 0.3 (8)	42.0 ± 4.4 (4)
Female	53.75 ± 1.7 (4)‡	54.4 ± 0.9 (7)‡
Urine volume, g/24 h
Male	4.2 ± 0.5 (6)	4.9 ± 0.3 (5)
Female	1.8 ± 0.2 (6)‡	2.5 ± 0.4 (6)‡

Values are means ± SE with numbers of animals per group shown in parentheses. Arterial blood samples were run through a blood gas analyzer to determine Na⁺, K⁺, bicarbonate, pH, and hematocrit. Serum K⁺ was measured via flame photometry. KS-BMAL1 KO mice, kidney-specific brain and muscle ARNT-like 1 knockout mice. Two-way ANOVA with Tukey multiple-comparisons test was used for statistical analysis.

^*Significantly differenct vs. control mice of the same sex (Tukey);

^†significantly different vs. control mice;

^‡significantly different vs. male mice.

Blood pressure under basal conditions in male mice.

The contribution of BMAL1 in distal segments on the regulation of blood pressure was determined in male mice. Basal systolic blood pressure in male KS-BMAL1 KO mice was ~8 mmHg lower than control mice as measured by telemetry (n = 4–5, Fig. 3, A and C). KS-BMAL1 KO mice also displayed a significantly lower mean arterial pressure of ~7 mmHg compared with control mice (Fig. 3E). Diastolic blood pressure tended to be lower in KS-BMAL1 KO mice compared with control mice, but this difference did not reach statistical significance with a P value of 0.07 (Fig. 3F). Three-day 24-h mean values are shown in Table 4. Although there were differences in blood pressure magnitude, the circadian rhythm was maintained in male KS-BMAL1 KO mice as determined by cosinor analysis (Table 5). Neither heart rate nor activity contributed to blood pressure differences as both genotypes exhibited similar values (Fig. 3, I and J).

Fig. 3.

Basal systolic blood pressures measured via telemetry of male (A and C) and female (B and D) control and kidney-specific brain and muscle ARNT-like 1 knockout (KS-BMAL1 KO) mice. E−L: basal mean arterial pressures of male (E) and female (G) mice, diastolic blood pressures of male (F) and female (H) mice, heart rates of male (I) and female (K) mice, and locomotor activity of male (J) and female (L) mice. Values are expressed as means ± SE from 3 consecutive days of recording and were plotted by hourly averages in Zeitgeber time (A, B, and E−L) and by 24-h averages (C and D); n = 4–7. The shaded backgrounds indicate the active periods (night); the white backgrounds indicate the inactive period (day). P values indicate comparison of genotypes by two-way ANOVA with repeated measures or Student’s t test.

Table 4.

Twenty-four hour averages of telemetry parameters measured for 3 continuous days

Telemeter Recording	Control	KS-BMAL1 KO
Systolic blood pressure, mmHg
Male	127.1 ± 0.7 (4)	119.0 ± 2.3 (5)*†
Female	120.3 ± 1.6 (5)‡	118.5 ± 1.5 (7)†‡
Mean arterial pressure, mmHg
Male	110.2 ± 0.2 (4)	103.5 ± 2.2 (5)*†
Female	107.0 ± 1.0 (5)	104.1 ± 1.3 (7)†
Diastolic blood pressure, mmHg
Male	91.5 ± 0.8 (4)	86.3 ± 2.0 (5)†
Female	91.9 ± 1.3 (5)	87.8 ± 1.6 (7)†
Heart rate, beats/min
Male	585 ± 3 (4)	568 ± 9 (5)
Female	627 ± 12 (5)‡	614 ± 3 (7)‡
Pulse pressure, mmHg
Male	35.5 ± 1.4 (4)	32.7 ± 1.0 (5)
Female	28.3 ± 2.1 (5)‡	30.7 ± 1.6 (7)‡
Activity
Male	10.6 ± 0.7 (4)	10.2 ± 1.6 (5)
Female	10.4 ± 1.7 (5)	10.1 ± 0.5 (7)

Values are expressed as means ± SE with numbers of animals per group shown in parentheses. KS-BMAL1 KO mice, kidney-specific brain and muscle ARNT-like 1 knockout mice. Two-way ANOVA with Tukey multiple-comparisons test was used for statistical analysis.

^*Significantly differenct vs. control mice of the same sex (Tukey);

^†significantly different vs. control mice;

^‡significantly different vs. male mice.

Table 5.

Cosinor analysis of systolic blood pressure, mean arterial pressure, and diastolic blood pressure measured via telemetry from male and female mice under basal conditions

Cosinor Analysis	Control Mice	KS-BMAL1 KO Mice
Systolic blood pressure
Mesor§
Male	127 ± 0.7 (4)	119 ± 2 (5)*†
Female	120 ± 2 (5)‡	119 ± 1 (7)†‡
Amplitude
Male	12 ± 0.5 (4)	11 ± 0.7 (5)
Female	12 ± 0.7 (5)	13 ± 0.8 (7)
Period
Male	24 ± 0.02 (4)	24 ± 0.2 (5)
Female	24 ± 0.1 (5)	24 ± 0.1 (7)
Mean arterial pressure
Mesor
Male	110 ± 0.2 (4)	102 ± 2 (5)*†
Female	107 ± 1 (5)	103 ± 1 (7)†
Amplitude
Male	11 ± 1 (4)	10 ± 1 (5)
Female	11 ± 0.6 (5)	12 ± 0.7 (7)
Period
Male	24 ± 0.1 (4)	24 ± 0.2 (5)
Female	24 ± 0.1 (5)	24 ± 0.2 (7)
Diastolic blood pressure
Mesor
Male	91 ± 1 (4)	86 ± 2 (5)†
Female	92 ± 1 (5)	88 ± 2 (7)†
Amplitude§
Male	11 ± 1 (4)	9 ± 1 (5)
Female	10 ± 0.5 (5)	12 ± 0.8 (7)
Period
Male	24 ± 0.2 (4)	24 ± 0.2 (5)
Female	24 ± 0.1 (5)	24 ± 0.2 (7)

Values are expressed as means ± SE with numbers of animals per group shown in parentheses. The period, mesor, and amplitude were calculated for each parameter. KS-BMAL1 KO mice, kidney-specific brain and muscle ARNT-like 1 knockout mice. Two-way ANOVA with Tukey multiple-comparisons test was used for statistical analysis.

^*Significantly different vs. control mice of the same sex (Tukey);

^†significantly different vs. control mice;

^‡significantly different vs. male mice;

^§statistical difference in interaction.

Blood pressure under basal conditions in female mice.

The effect of BMAL1 in renal distal segments on blood pressure control in female mice was assessed by telemetry. Female control and KS-BMAL1 KO mice did not express a difference in systolic blood pressure (n = 5–7; Fig. 3, B and D), mean arterial pressure (Fig. 3G), or diastolic blood pressure (Fig. 3H). Three-day 24-h mean of blood pressure measurements are shown in Table 4. The circadian rhythm of blood pressure was not affected by KS-BMAL1 KO in female mice as demonstrated by cosinor analysis (Table 5). There was no difference in activity or heart rate between genotypes (Fig. 3, K and L).

Basal gene expression of factors important in solute handling in the male renal medulla.

BMAL1 is an important transcription factor that regulates the expression of a large fraction of genes. The majority of the renal cortex consists of proximal tubules, whereas the majority of the medulla comprises distal tubules. Given the specificity of Ksp-cadherin Cre to distal tubule segments, as demonstrated by our characterization of KS-BMAL1 KO, BMAL1 expression in KS-BMAL1 KO mice was likely affected to a greater extent in the medulla rather than the cortex. We took a candidate gene approach to perform quantitative PCR and measure mRNA from dissected medullary samples collected at noon and midnight to gain a better understanding of what pathways might be affected by KS-BMAL1 KO mice. We focused our efforts on genes encoding products that function in Na⁺ and K⁺ transport. Several genes exhibited genotype- and time of day-dependent changes in male mice, as shown in Fig. 4. mRNA expression of KCNJ1, a gene that encodes renal outer medullary K⁺ channel protein, was higher at midnight than at noon. mRNA expression of genes that encode for gastric and nongastric subunits of H⁺/K⁺-ATPase (ATP4A and ATP12A), inwardly rectifying K⁺ channel (KCNJ15), epithelial Na⁺ channel (ENaC) β- and γ-subunits (SCNN1B and SCNN1G), and a member of the with no lysine protein kinase (WNK) family (WNK1) was significantly lower at midnight compared with noon. ATP4A, KCNJ15, serum/glucocorticoid-regulated kinase 1 protein coding gene (SGK1), and pendrin protein coding gene (SLC26A4) were significantly lower in KS-BMAL1 KO mice compared with control mice. WNK4 mRNA levels were higher in KS-BMAL1 KO mice versus control mice. Table 6 shows the relative effect size of the percent change in mRNA expression from time of day and between genotypes in male mice.

Fig. 4.

Relative mRNA expression in medullary tissue collected at noon and midnight from male control mice and kidney-specific brain and muscle ARNT-like 1 knockout (KS-BMAL1 KO) mice. The genes chosen have been previously described as important factors in solute handling. Values were normalized to control samples at noon. Values are expressed as means ± SE; n = 4–7. Two-way ANOVA was used to compare groups for each gene. Mann-Whitney t tests were used on data sets that failed to pass the normality test. ATP4A and ATP12A, gastric and nongastric subunits of H⁺/K⁺-ATPase; KCNJ1, gene that encodes renal outer medullary K⁺ channel protein; KCNJ15, inwardly rectifying K⁺ channel; KCNMA1, large-conductance channel subunit coding gene; SCNN1A, SCNN1B, and SCNN1G, α-, β-, and γ-subunits of the epithelial Na⁺ channel, respectively; SGK1, serum/glucocorticoid-regulated kinase 1; SLC12A1, Na⁺-K⁺-2Cl⁻ cotransporter gene; SLC26A4, pendrin gene; WNK, with no lysine protein kinase.

Table 6.

Percent change of quantitative PCR data from Figs. 4 and 5

	Time (Percent Change), %		Genotype (Percent Change), %
Gene	Male	Female	Male	Female
ATP4A	−59*	−51*	−32*	−35*
ATP12A	−74*	−71*	25	67
KCNJ1	119*	79*	41	24*
KCNJ15	−43*	−78*	−30*	13
KCNMA1	−10	−72*	−17	−2
SCNN1A	−12	−8	0.5	7
SCNN1B	−41*	−57*	−6	37*
SCNN1G	−27*	−38*	−11	23*
SGK1	−4	−27	−26*	−15
SLC12A1	−11	68*	46	3
SLC26A4	−33	−90*	−40*	11
WNK1	−52*	−43*	6	−1
WNK4	−11	19*	22*	−3

Values are shown as percentages. The relative percent change of gene expression for midnight (vs. noon) (time) and knockout (vs. control) (genotype) are given for each sex. ATP4A and ATP12A, gastric and nongastric subunits of H⁺/K⁺-ATPase; KCNJ1, gene that encodes renal outer medullary K⁺ channel protein; KCNJ15, inwardly rectifying K⁺ channel; KCNMA1, large-conductance channel subunit coding gene; SCNN1A, SCNN1B, and SCNN1G, α-, β-, and γ-subunits of the epithelial Na⁺ channel, respectively; SGK1, serum/glucocorticoid-regulated kinase 1; SLC12A1, Na⁺-K⁺-2Cl⁻ cotransporter gene; SLC26A4, pendrin gene; WNK, with no lysine protein kinase.

^*Comparisons that were significant using original values.

Basal gene expression of factors important in solute handling in the female renal medulla.

Genes of select solute handling factors were used to measure mRNA expression in female medullary samples collected at noon and midnight (Fig. 5). KCNJ1, NKCC2 protein coding gene (SLC12A1), and WNK4 exhibited higher expression at midnight than at noon. ATP4A, ATP12A, KCNJ15, large-conductance channel subunit coding gene (KCNMA1), SCNN1B, SCNN1G, SLC26A4, and WNK1 mRNA expression was lower at midnight compared with noon. ATP4A expression was lower in KS-BMAL1 KO mice than in control mice. KCNJ1, and SCNN1G displayed higher expression in KS-BMAL1 KO mice than in control mice. mRNA expression of SCNN1B was significantly higher in KS-BMAL1 KO mice at noon compared with control mice; however, levels were comparable between the genotypes at midnight. Table 6 shows the relative effect size of the percent change in mRNA expression from time of day and between genotypes in female mice. When gene expression data from both male and female mice were combined, three-way ANOVA statistics were used to determine if there were any sex differences. Female mice had less ATP4A expression than male mice, particularly at noon. Female mice had less KCNJ15 and KCNMA1 mRNA expression than male mice, particularly at midnight. Female mice had greater KCNJ1 and SGK1 expression than male mice. Female mice exhibited greater SCNN1G expression than male mice, particularly in KS-BMAL1 KO mice. Female mice had higher WNK4 expression than male mice, particularly at midnight. Female KS-BMAL1 KO mice had higher expression of SCNN1B than male KS-BMAL1 KO mice; control mice were comparable. Female tissue collected at midnight had greater SLC12A1 expression than male tissue collected at midnight; however, noon samples were comparable.

Fig. 5.

Relative mRNA expression in medullary tissue collected at noon and midnight from female control mice and kidney-specific brain and muscle ARNT-like 1 knockout (KS-BMAL1 KO) mice. The genes chosen have been previously described as important factors in solute handling. Values were normalized to control samples at noon. Values are expressed as means ± SE; n = 4–7. Two-way ANOVA was used to compare groups for each gene. Mann-Whitney t tests were used for data sets that failed to pass the normality test. ATP4A and ATP12A, gastric and nongastric subunits of H⁺/K⁺-ATPase; KCNJ1, gene that encodes renal outer medullary K⁺ channel protein; KCNJ15, inwardly rectifying K⁺ channel; KCNMA1, large-conductance channel subunit coding gene; SCNN1A, SCNN1B, and SCNN1G, α-, β-, and γ-subunits of the epithelial Na⁺ channel, respectively; SGK1, serum/glucocorticoid-regulated kinase 1; SLC12A1, Na⁺-K⁺-2Cl⁻ cotransporter gene; SLC26A4, pendrin gene; WNK, with no lysine protein kinase.

Solute handling in response to HS/DOCP treatment in male mice.

HS/DOCP treatment has been demonstrated to induce nondipping hypertension in global PER1 KO mice, which is associated with changes in renal Na⁺ handling (7, 29). We sought to determine if there were genotype differences in solute handling in response to HS/DOCP in male KS-BMAL1 KO mice and if this treatment affects the normal dip in blood pressure, which is ≥10% in normal mice and humans. There was no difference in K⁺ balance (dietary intake minus urinary output) between groups throughout the HS/DOCP treatment regimen (Fig. 6A). Mice return to baseline levels of K⁺ balance after 5 days of HS/DOCP treatment. The Na⁺ balance throughout treatment was also the same between genotypes (Fig. 6B). Both KS-BMAL1 KO and control mice retained more Na⁺ following HS/DOCP treatment compared with baseline. Additionally, both groups had similar cumulative Na⁺ retention (Fig. 6C). It is important to note that genotype differences in solute handling were not detected during the low salt or HS treatments. KS-BMAL1 KO mice did not display a change in diurnal rhythm of blood pressure. Following HS/DOCP treatment, control and KS-BMAL1 KO mice displayed a normal blood pressure dip. Additionally, the dip was similar between the genotypes (P = 0.9; Fig. 6D).

Fig. 6.

Male control (CNTL) and kidney-specific brain and muscle ARNT-like 1 knockout (KS-BMAL1 KO) mice following high-salt diet (HS) and desoxycorticosterone pivalate (DOCP). A and B: 24-h K⁺ retention (intake minus output; A) and Na⁺ retention (B) in response to a diet regimen of low salt (LS), HS, and HS/DOCP compared with the baseline normal diet (N). C: cumulative Na⁺ balance throughout the entire treatment was quantified. D: differences between active and inactive mean arterial pressure during HS/DOCP treatment expressed as a percentage of the day value. Values are means ± SE; n = 6 for solute handling, n = 4 for control mean arterial pressure; n = 5 for KS-BMAL1 KO mean arterial pressure. Two-way ANOVA with repeated measures was used to compare groups. Student’s t test to compare night and day.

Solute handling response to K⁺ depletion in male mice.

An established consequence of low dietary K⁺ intake is increased Na⁺ reabsorption (37). We administered a K⁺-deficient diet to male mice to determine if there were differences in solute handling between genotypes. Within the first 24 h of K⁺ depletion, urinary K⁺ excretion decreased to near 0 meq and then remained at that level throughout the treatment period in both genotypes (Fig. 7A). However, renal Na⁺ handling in response to dietary K⁺ depletion was different between genotypes. Male KS-BMAL1 KO mice excreted less urinary Na⁺ than control mice by days 4 and 5 of the K⁺-depleted diet. Control mice initially decreased urinary Na⁺ excretion, which remained lower than baseline throughout the treatment period. KS-BMAL1 KO mice also had an initial decrease in urinary Na⁺; however, Na⁺ excretion returned to baseline levels by day 4 of treatment (Fig. 7B). Effects were similar when excretion was subtracted from solute intake (data not shown). Changes in food intake and body weight throughout the experiment were similar between both groups (Fig. 7, C and D). Dietary K⁺ depletion resulted in a significant decrease in urinary aldosterone in both genotypes, as expected (Fig. 7E). There was no genotype difference in serum aldosterone levels after 5 days of K⁺ depletion (Fig. 7F).

Fig. 7.

Night and day rates of K⁺ excretion (A) and Na⁺ excretion (B) during 5 days of K⁺ depletion compared with normal diet in male control and kidney-specific brain and muscle ARNT-like 1 knockout (KS-BMAL1 KO) mice. Gray backgrounds represent the active period (nighttime); white backgrounds represent the inactive period (daytime). C and D: food intake (C) and change in body weight (D) throughout the treatment. E: urinary aldosterone levels during the active period (nighttime) for baseline and days 1, 3, and 5 of K⁺-depleted diet treatment. F: serum aldosterone concentrations from male mice following K⁺ depletion. Values are means ± SE; n = 5–6. Two-way ANOVA with repeated measures was used to compare groups over time, and a t test was used to compare serum aldosterone levels.

Solute handling response to K⁺ depletion in female mice.

To determine if female mice had a similar response to male mice, we also administered a K⁺-deficient diet to female control and KS-BMAL1 KO mice. As seen with male mice, K⁺ excretion in female mice responded similarly between genotypes by quickly decreasing and remaining near 0 meq for the 5 days of treatment (Fig. 8A). In contrast to male control mice, neither female control nor KS-BMAL1 KO mice exhibited a decrease in urinary Na⁺ excretion throughout treatment (Fig. 8B). There was no genotype difference when solute excretion was subtracted from intake. Additionally, food intake and body weight were similar between genotypes throughout the 5 days of treatment (Fig. 8, C and D).

Fig. 8.

Night and day rates of K⁺ excretion (A) and Na⁺ excretion (B) during 5 days of K⁺ depletion compared with normal diet in female control and kidney-specific brain and muscle ARNT-like 1 knockout (KS-BMAL1 KO) mice. Gray backgrounds represent the active period (nighttime); white backgrounds represent the inactive period (daytime). C and D: food intake (C) and change in body weights (D) throughout the treatment. Values are means ± SE; n = 5–6. Two-way ANOVA with repeated measures was used to compare groups over time.

Cumulative Na⁺ levels in male and female mice following a K⁺-depleted diet.

To calculate the overall effect of the K⁺-deficient diet on renal Na⁺ handling, cumulative daily Na⁺ retention (Na⁺ intake minus urinary Na⁺ output) was calculated for male and female mice over the course of the study. The cumulative Na⁺ balance in male mice was significantly different between genotypes with male KS-BMAL1 KO mice having lower cumulative Na⁺ balance compared with control mice in response to the low-K⁺ diet (Fig. 9A). As expected, male control mice exhibited a more positive Na⁺ balance in response to the low-K⁺ diet. In contrast, female control mice did not increase to the level seen in mice mice (Fig. 9B compared with Fig. 9A). Moreover, in female mice, cumulative Na⁺ was the same between genotypes (Fig. 9B).

Fig. 9.

Cumulative Na⁺ balance in male (A) and female (B) control and kidney-specific brain and muscle ARNT-like 1 knockout (KS-BMAL1 KO) mice following treatment with a K⁺-depleted diet. Gray backgrounds represent the active period (nighttime); white backgrounds represent the inactive period (daytime). Values are means ± SE; n = 6. Two-way ANOVA with repeated measures was used to compare genotypes over treatment period.

DISCUSSION

The major finding from this study is that BMAL1 in the kidney functions in a sex-dependent manner, affecting blood pressure regulation and the renal response to dietary K⁺ restriction in male, but not female, mice. BMAL1 in the distal nephron and collecting duct of male mice regulates Na⁺ handling during dietary K⁺ deprivation. Interestingly, female mice do not display this genotype difference in Na⁺ handling, indicating that the BMAL1-dependent mechanisms in distal segments are sex specific. Furthermore, BMAL1 in distal segments contributes to a substantial (~7 mmHg) reduction in basal mean arterial pressure in male, but not female, mice. The lower basal blood pressure exhibited by male KS-BMAL1 KO mice compared with control mice is consistent with previous reports using other KO models of BMAL1 and suggests that a substantial component of this blood pressure regulation may be due to Na⁺ transport in the aldosterone-sensitive distal nephron and collecting duct.

The lower blood pressure phenotype in male KS-BMAL1 KO mice is consistent with previous studies that used other KO models of BMAL1 (5, 20, 38, 44). Indeed, the reproducibility of this effect is striking and clearly demonstrates the importance of BMAL1 to blood pressure regulation. Not only does the KO genotype in all of these studies have lower pressures, but the magnitude of the difference is similar as well, ranging from 7 to 10 mmHg lower than controls. In the present study, the use of Ksp-cadherin Cre demonstrates that the major contributor to the blood pressure phenotype in KS-BMAL1 KO mice is the kidney. In healthy humans and mice, blood pressure follows a circadian rhythm with an ∼10−20% dip in blood pressure during the inactive period (3). KO of the circadian clock protein BMAL1 in renal cells does not have an effect on circadian rhythm in mice (20, 38, 44). Notably, global BMAL1 KO lost its diurnal variation in blood pressure and did not display the typical rise that occurs in the active period (5). Xie et al. (42) demonstrated that smooth muscle cell-specific KO of BMAL1 led to a dampened rise of blood pressure during the active period compared with wild-type mice. This effect is independent of normal light/dark conditions as KO mice held in constant darkness also displayed lower blood pressures than controls (42). Data from the present study suggest BMAL1 in distal segments contributes to substantial blood pressure changes only in male mice and that this occurs independent of changes in circadian rhythms.

Studies in mice and humans have consistently shown that dietary K⁺ depletion leads to an increase in Na⁺ retention (9, 14, 15, 37). Briefly, K⁺ depletion leads to an increase of Na⁺ reabsorption in the distal nephron, decreasing Na⁺ delivered to the collecting duct and limiting the loss of K⁺ in the urine. Data in this study are consistent with previous literature where male control mice excrete less K⁺ and Na⁺ following K⁺ depletion. Although the K⁺ handling response is similar in male KS-BMAL1 KO mice, less Na⁺ is retained compared with control mice during the K⁺-depleted diet treatment. This response appears to be specific to K⁺ depletion as there was no genotype difference when male mice were treated with low salt, HS, or HS/DOCP. Similarly, in another study (44), collecting duct-specific BMAL1 KO mice were challenged with HS for 6 days and this was not associated with genotype differences. Together with the data presented in this report, these data indicate that BMAL1 in distal segments plays a role in regulating Na⁺ handling in a state of K⁺ depletion but apparently not under HS conditions alone.

Previous studies have provided some insights into how renal BMAL1 contributes to Na⁺ homeostasis and blood pressure control. Of note, BMAL1 regulates the expression of Na⁺/H⁺ exchanger 3 in the rodent kidney (26, 40), but this was not tested in the present study since proximal tubule BMAL1 expression was unaffected by Ksp-cadherin-driven Cre recombinase. Speed et al. (31) showed that BMAL1 mRNA expression in the kidney of rats was regulated by endothelin type B receptor activity in a salt-dependent manner. Renal endothelin in rats has been reported to increase following K⁺ depletion; however, this effect was seen after 12 wk of treatment compared with the 5 days used in this study (34). Additionally, treatment with HS and endothelin type B receptor antagonist had no effect on genotype differences in collecting duct-specific BMAL1 KO mice (44). Chronic corticosterone administration for 4 wk in mice led to increased BMAL1 expression in whole kidney homogenates, which was associated with changes in solute handling markers such as increased p-NCC (13). The HS/DOCP treatment period used in this study was acute, measuring changes over 5 days, and genotype differences in Na⁺ handling were not observed in this model. Gene expression data from this study are consistent with the lower Na⁺ retention exhibited by male KS-BMAL1 KO mice compared with control mice in that lower levels of pendrin and SGK1 mRNA were observed in KS-BMAL1 KO mice. In support of this, regardless of genotype, female mice lacked the Na⁺ response differences seen in male mice and had similar levels of pendrin and SGK1. Additionally, the higher mRNA expression of β- and γ-subunts of ENaC in female KS-BMAL1 KO mice compared with control mice may represent a compensatory response, which would be consistent with the lack of difference in Na⁺ retention following a K⁺-depleted diet, as seen with male mice.

The sex-specific differences in renal mRNA expression found in the present study highlight the importance of including both male and female subjects when investigating gene regulatory pathways, particularly with blood pressure control. Genetic rodent models have often been used to study sex differences in blood pressure regulation (10). Additionally, sex chromosomes have been linked to altering molecular pathways that affect blood pressure (6). As evident from the mRNA data in this study, female mice exhibited greater night/day differences in gene expression for the majority of genes tested. Given the paucity of studies examining circadian gene expression in female rodent models, this effect is notable. Whether the greater magnitude of night/day differences in gene expression observed in female mice is related to the lack of phenotype observed in the present study remains to be determined. Future intervention studies are needed to clarify the functional implications of these gene expression differences. These include thiazide treatment, which has been demonstrated to be more beneficial in male subjects (36), or ENaC inhibition, which has been shown to be more effective in female subjects (28).

Through characterization of the mouse model in this study, there is evidence of extrarenal Ksp-cadherin Cre recombinase activity in the ovaries. Recent studies have demonstrated that Aqp2-Cre recombinase activity exhibits extrarenal activity in the brain (23, 24, 44). Thus, it is interesting to compare and contrast the KS-BMAL1 KO model in this study to the collecting duct BMAL1 KO mouse model recently reported (44). Both models demonstrate that BMAL1 in distal segments regulates blood pressure independent of circadian rhythms, which is specific to male and not female animals (44). These data suggest the importance of the collecting duct in the phenotype of KS-BMAL1 KO mice. It is interesting to note that the genotype difference in blood pressure of collecting duct-specific BMAL1 KO mice was primarily seen at diastole, whereas KS-BMAL1 KO mice was at systole. We and others have shown that female circadian clock gene mutant mice exhibit a protective phenotype in a hypertensive setting (1, 8, 44). Although the lack of genotype effect in female mice from this study is consistent with previous literature, a possible reason that female KS-BMAL1 KO mice do not exhibit a lower blood pressure versus control mice could be attributed to a “floor effect,” in that their blood pressure is already low to begin with. Induction of hypertension in these female mice could yield more insight into this effect.

Differences in glomerular filtration rate could contribute to the differences in Na⁺ retention observed in male KS-BMAL1 KO mice. Nikolaeva et al. (20) demonstrated that there was no change in glomerular filtration rate in an inducible tubule-specific BMAL1 KO mouse model. Visual inspection of the macula densa of KS-BMAL1 KO mice via immunohistochemistry indicated that there was no change in BMAL1 expression. However, the pool of macula densa cells assessed was small, and KO of BMAL1 in the thick ascending limb suggests possible disruption of Na⁺ intake in this segment leading to altered Na⁺ delivery to the macula densa. For these reasons, we cannot rule out a role for glomerular filtration rate or tubuloglomerular feedback in this model.

To the best of our knowledge, this is the first report of a difference in response to a K⁺-depleted diet between male and female mice. Our data indicate that distal segment-specific BMAL1 plays a role in Na⁺ handling during a state of K⁺ deficiency and that this action is sex specific. Previous characterization of basal Na⁺ handling factors demonstrated that female mice possess higher expression of distal Na⁺ reabsorptive transporters compared with male mice (39). Similarly, kidneys from female mice have a higher proportion of collecting duct-mediated Na⁺ reabsorption (12). These findings suggest that female mice obtain a greater capacity for adaptation to a K⁺-depleted state; however, additional studies more directly comparing the response in each sex need to be conducted. The ability of female mice to adapt to dietary K⁺ restriction may account for the absence of a genotype effect in female mice in this study. In contrast, there is evidence that male mice better adapt to a high-K⁺ diet compared with female mice (17). Interestingly, women have been shown to be more likely to develop hypokalemia following thiazide administration (36). Altogether, there is strong evidence that regulation of K⁺ homeostasis appears to be sex specific in nature.

In conclusion, renal BMAL1 plays a role in blood pressure control and Na⁺ handling in response to a K⁺-depleted diet. Absence of BMAL1 in the male kidney results in an alteration of renal mechanisms that lowers blood pressure in a sex-dependent manner and reduces Na⁺ retention during K⁺ depletion. The finding that KS-BMAL1 KO does not alter the circadian rhythm of blood pressure underscores the importance of this protein for normal renal physiology, independent of its role as a circadian clock component. Indeed, the ability of a single core circadian protein in the distal segments of the kidney to affect blood pressure and the renal response to dietary K⁺ depletion highlights the importance of BMAL1 for normal renal function. Female KS-BMAL KO mice do not exhibit a blood pressure phenotype, nor do they show differences in Na⁺ handling following K⁺ depletion, contrary to what is seen in male mice. Of note, female mice, regardless of genotype, appear to retain less Na⁺ than male mice during K⁺ deprivation. Sex-specific changes in gene expression, such as lower SGK1 and pendrin, provide insight as what processes BMAL1 is affecting in male mice. Ultimately, our data demonstrate that distal segment-specific BMAL1 plays an important role in solute handling and blood pressure control in a sex-specific manner.

GRANTS

This work was supported by the Department of Physiology at the University of Florida (T32 Trainee Grant 5T32-HL-083810-10 to G. R. Crislip), the Division of Nephrology at the University of Florida (T32 Trainee Grant 5-T32-DK-104721 to L. G. Douma), American Heart Association Postdoctoral Fellowship 19POST34450134 (to G. R. Crislip) and Grant-in-Aid Award P0133392 (to M. L. Gumz), Gatorade Trust through the University of Florida Department of Medicine, and National Institutes of Health Grant 1-R01-DK-109570-01A1 (to M. L. Gumz).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the author(s).

AUTHOR CONTRIBUTIONS

G.R.C., L.G.D., I.J.L., C.S.W., and M.L.G. conceived and designed research; G.R.C., L.G.D., S.H.M., K.-Y.C., I.J.L., J.G.J., D.B., K.B.G., and M.R.H. performed experiments; G.R.C., L.G.D., S.H.M., K.-Y.C., I.J.L., J.G.J., D.B., K.B.G., M.R.H., J.W.V., and M.L.G. analyzed data; G.R.C., L.G.D., I.J.L., J.W.V., C.S.W., and M.L.G. interpreted results of experiments; G.R.C., S.H.M., and D.B. prepared figures; G.R.C. and M.L.G. drafted manuscript; G.R.C., L.G.D., S.H.M., I.J.L., D.B., K.B.G., M.R.H., J.W.V., C.S.W., and M.L.G. edited and revised manuscript; G.R.C. and M.L.G. approved final version of manuscript.