Male kidney-specific BMAL1 knockout mice are protected from K+-deficient, high-salt diet-induced blood pressure increases.

The circadian clock protein basic helix-loop-helix aryl hydrocarbon receptor nuclear translocator-like protein 1 (BMAL1) is a transcription factor that impacts kidney function, including blood pressure (BP) control. Previously, we have shown that male, but not female, kidney-specific cadherin Cre-positive BMAL1 knockout (KS-BMAL1 KO) mice exhibit lower BP compared with littermate controls. The goal of this study was to determine the BP phenotype and immune response in male KS-BMAL1 KO mice in response to a low-K+ high-salt (LKHS) diet. BP, renal inflammatory markers, and immune cells were measured in male mice following an LKHS diet. Male KS-BMAL1 KO mice had lower BP following the LKHS diet compared with control mice, yet their circadian rhythm in pressure remained unchanged. Additionally, KS-BMAL1 KO mice exhibited lower levels of renal proinflammatory cytokines and immune cells following the LKHS diet compared with control mice. KS-BMAL1 KO mice were protected from the salt-sensitive hypertension observed in control mice and displayed an attenuated immune response following the LKHS diet. These data suggest that BMAL1 plays a role in driving the BP increase and proinflammatory environment that occurs in response to an LKHS diet.NEW & NOTEWORTHY We show here, for the first time, that kidney-specific BMAL1 knockout mice are protected from blood pressure (BP) increases and immune responses to a salt-sensitive diet. Other kidney-specific BMAL1 knockout models exhibit lower BP phenotypes under basal conditions. A salt-sensitive diet exacerbates this genotype-specific BP response, leading to fewer proinflammatory cytokines and immune cells in knockout mice. These data demonstrate the importance of distal segment BMAL1 in BP and immune responses to a salt-sensitive environment.

Adrenal-Specific KO of the Circadian Clock Protein BMAL1 Alters Blood Pressure Rhythm and Timing of Eating Behavior.

Brain and muscle ARNT-like 1 (BMAL1) is a core circadian clock protein and transcription factor that regulates many physiological functions, including blood pressure (BP). Male global Bmal1 knockout (KO) mice exhibit ∼10 mmHg reduction in BP, as well as a blunting of BP rhythm. The mechanisms of how BMAL1 regulates BP remains unclear. The adrenal gland synthesizes hormones, including glucocorticoids and mineralocorticoids, that influence BP rhythm. To determine the role of adrenal BMAL1 on BP regulation, adrenal-specific Bmal1 (ASCre/+ ::Bmal1) KO mice were generated using aldosterone synthase Cre recombinase to KO Bmal1 in the adrenal gland zona glomerulosa. We confirmed the localization and efficacy of the KO of BMAL1 to the zona glomerulosa. Male ASCre/+ ::Bmal1 KO mice displayed a shortened BP and activity period/circadian cycle (typically 24 h) by ∼1 h and delayed peak of BP and activity by ∼2 and 3 h, respectively, compared with littermate Cre- control mice. This difference was only evident when KO mice were in metabolic cages, which acted as a stressor, as serum corticosterone was increased in metabolic cages compared with home cages. AS Cre/+ ::Bmal1 KO mice also displayed altered diurnal variation in serum corticosterone. Furthermore, these mice have altered eating behaviors where they have a blunted night/day ratio of food intake, but no change in overall food consumed compared with controls. Overall, these data suggest that adrenal BMAL1 has a role in the regulation of BP rhythm and eating behaviors.

The circadian clock protein PER1 is important in maintaining endothelin axis regulation in Dahl salt-sensitive rats.

Endothelin-1 (ET-1) is a peptide hormone that acts on its receptors to regulate sodium handling in the kidney's collecting duct. Dysregulation of the endothelin axis is associated with various diseases, including salt-sensitive hypertension and chronic kidney disease. Previously, our lab has shown that the circadian clock gene PER1 regulates ET-1 levels in mice. However, the regulation of ET-1 by PER1 has never been investigated in rats. Therefore, we used a novel model where knockout of Per1 was performed in Dahl salt-sensitive rat background (SS Per1 -/-) to test a hypothesis that PER1 regulates the ET-1 axis in this model. Here, we show increased renal ET-1 peptide levels and altered endothelin axis gene expression in several tissues, including the kidney, adrenal glands, and liver in SS Per1 -/- compared with control SS rats. Edn1 antisense lncRNA Edn1-AS, which has previously been suggested to be regulated by PER1, was also altered in SS Per1 -/- rats compared with control SS rats. These data further support the hypothesis that PER1 is a negative regulator of Edn1 and is important in the regulation of the endothelin axis in a tissue-specific manner.

Kidney-specific KO of the circadian clock protein PER1 alters renal Na+ handling, aldosterone levels, and kidney/adrenal gene expression.

PERIOD 1 (PER1) is a circadian clock transcription factor that is regulated by aldosterone, a hormone that increases blood volume and Na+ retention to increase blood pressure. Male global Per1 knockout (KO) mice develop reduced night/day differences in Na+ excretion in response to a high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP), a model of salt-sensitive hypertension. In addition, global Per1 KO mice exhibit higher aldosterone levels on a normal-salt diet. To determine the role of Per1 in the kidney, male kidney-specific Per1 KO (KS-Per1 KO) mice were generated using Ksp-cadherin Cre recombinase to remove exons 2-8 of Per1 in the distal nephron and collecting duct. Male KS-Per1 KO mice have increased Na+ retention but have normal diurnal differences in Na+ excretion in response to HS + DOCP. The increased Na+ retention is associated with altered expression of glucocorticoid and mineralocorticoid receptors, increased serum aldosterone, and increased medullary endothelin-1 compared with control mice. Adrenal gland gene expression analysis revealed that circadian clock and aldosterone synthesis genes have altered expression in KS-Per1 KO mice compared with control mice. These results emphasize the importance of the circadian clock not only in maintaining rhythms of physiological functions but also for adaptability in response to environmental cues, such as HS + DOCP, to maintain overall homeostasis. Given the prevalence of salt-sensitive hypertension in the general population, these findings have important implications for our understanding of how circadian clock proteins regulate homeostasis.NEW & NOTEWORTHY For the first time, we show that knockout of the circadian clock transcription factor PERIOD 1 using kidney-specific cadherin Cre results in increased renal Na+ reabsorption, increased aldosterone levels, and changes in gene expression in both the kidney and adrenal gland. Diurnal changes in renal Na+ excretion were not observed, demonstrating that the clock protein PER1 in the kidney is important for maintaining homeostasis and that this effect may be independent of time of day.

Knockout of the circadian clock protein PER1 results in sex-dependent alterations of ET-1 production in mice in response to a high-salt diet plus mineralocorticoid treatment.

Previously, we showed that global knockout (KO) of the circadian clock transcription factor PER1 in male, but not female, mice fed a high-salt diet plus mineralocorticoid treatment (HS/DOCP) resulted in nondipping hypertension and decreased night/day ratio of sodium (Na) excretion. Additionally, we have shown that the endothelin-1 (ET-1) gene is targeted by both PER1 and aldosterone. We hypothesized that ET-1 would exhibit a sex-specific response to HS/DOCP treatment in PER1 KO. Here we show that male, but not female, global PER1 KO mice exhibit a decreased night/day ratio of urinary ET-1. Gene expression analysis revealed significant genotype differences in ET-1 and endothelin A receptor (ETA) expression in male, but not female, mice in response to HS/DOCP. Additionally, both wild-type and global PER1 KO male mice significantly increase endothelin B receptor (ETB) expression in response to HS/DOCP, but female mice do not. Finally, siRNA-mediated knockdown of PER1 in mouse cortical collecting duct cells (mpkCCDc14) resulted in increased ET-1 mRNA expression and peptide secretion in response to aldosterone treatment. These data suggest that PER1 is a negative regulator of ET-1 expression in response to HS/DOCP, revealing a novel mechanism for the regulation of renal Na handling in response to HS/DOCP treatment.