The circadian clock plays an important role in the regulation of physiological processes, including renal function and blood pressure. We have previously shown that the circadian protein period (Per)1 regulates the expression of multiple Na+ transport genes in the collecting duct, including the α-subunit of the renal epithelial Na+ channel. Consistent with this finding, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. We have also recently demonstrated the potential opposing actions of cryptochrome (Cry)2 on Per1 target genes. Recent work by others has demonstrated that Cry1/2 regulates aldosterone production through increased expression of the adrenal gland-specific rate-limiting enzyme 3β-dehydrogenase isomerase (3β-HSD). Therefore, we tested the hypothesis that Per1 plays a role in the regulation of aldosterone levels and renal Na+ retention. Using RNA silencing and pharmacological blockade of Per1 nuclear entry in the NCI-H295R human adrenal cell line, we showed that Per1 regulates 3β-HSD expression in vitro. These results were confirmed in vivo: mice with reduced levels of Per1 had decreased levels of plasma aldosterone and decreased mRNA expression of 3β-HSD. We postulated that mice with reduced Per1 would have a renal Na+-retaining defect. Indeed, metabolic cage experiments demonstrated that Per1 heterozygotes excreted more urinary Na+ compared with wild-type mice. Taken together, these data support the hypothesis that Per1 regulates aldosterone levels and that Per1 plays an integral role in the regulation of Na+ retention.

Mounting evidence suggests that the circadian clock plays an integral role in the regulation of many physiological processes including blood pressure, renal function, and metabolism. The canonical molecular clock functions via activation of circadian target genes by Clock/Bmal1 and repression of Clock/Bmal1 activity by Per1–3 and Cry1/2. However, we have previously shown that Per1 activates genes important for renal sodium reabsorption, which contradicts the canonical role of Per1 as a repressor. Moreover, Per1 knockout (KO) mice exhibit a lowered blood pressure and heavier body weight phenotype similar to Clock KO mice, and opposite that of Cry1/2 KO mice. Recent work has highlighted the potential role of Per1 in repression of Cry2. Therefore, we postulated that Per1 potentially activates target genes through a Cry2-Clock/Bmal1-dependent mechanism, in which Per1 antagonizes Cry2, preventing its repression of Clock/Bmal1. This hypothesis was tested in vitro and in vivo. The Per1 target genes αENaC and Fxyd5 were identified as Clock targets in mpkCCDc14 cells, a model of the renal cortical collecting duct. We identified PPARα and DEC1 as novel Per1 targets in the mouse hepatocyte cell line, AML12, and in the liver in vivo. Per1 knockdown resulted in upregulation of Cry2 in vitro, and this result was confirmed in vivo in mice with reduced expression of Per1. Importantly, siRNA-mediated knockdown of Cry2 and Per1 demonstrated opposing actions for Cry2 and Per1 on Per1 target genes, supporting the potential Cry2-Clock/Bmal1-dependent mechanism underlying Per1 action in the liver and kidney.

The circadian clock plays an integral role in blood pressure (BP) control. In the canonical model of the circadian clock, the transcription factors Clock and Bmal1 activate circadian target genes, while Period 1 (Per1) and Cryptochrome (Cry 1 or 2) repress this action. Others have shown that mice lacking Clock or Bmal1 are hypotensive, while Cry 1/2 double knockout (KO) mice exhibit salt‐sensitive hypertension. Interestingly, our data show that Per1 KO mice exhibit lower BP than wild type (WT) mice and that Per1 positively regulates expression of the alpha subunit of the renal epithelial sodium channel (αENaC). These results suggest a non‐canonical role for Per1 in the kidney and in the regulation of BP. To address this finding, we first evaluated expression of Bmal1, Clock and Cry2 in renal cortical collecting duct (CCD) cells stably transfected with Per1 shRNA and in nuclear extracts from the renal cortex of WT or Per1 KO mice. Whereas loss of Per1 had no effect on Clock or Bmal1 protein expression, Cry2 protein levels were consistently upregulated in the absence of Per1 in vitro and in vivo. Knockdown of Clock using siRNA resulted in decreased αENaC expression in CCD cells in vitro, mimicking the effect seen with Per1 knockdown. Conversely, Cry2 knockdown appeared to cause increased αENaC expression. Taken together, these data suggest that Per1 action on αENaC regulation and BP may involve the circadian repressor Cry 2.

Regulation of sodium balance by the kidney is an important determinant of long term blood pressure (BP) control. We have previously shown that the circadian clock protein Per1 regulates expression of the alpha subunit of the renal epithelial sodium channel (αENaC). Together with our recent finding that Per1 knockout (KO) mice exhibit dramatically lower BP than wild type (WT) mice, these data led to the hypothesis that the BP phenotype involves a renal Na handling mechanism. Consistent with our hypothesis, metabolic balance studies showed that Per1 KO mice excreted more urinary Na compared to WT mice. We measured plasma aldosterone (Aldo) levels in WT and Per1 KO mice at noon and midnight. WT mice experienced the expected circadian surge in Aldo. Importantly, the circadian Aldo surge did not occur and Aldo levels were significantly lower in Per1 KO mice. Thus we examined the expression of the circadian target gene Hsd3b6, an adrenal glomerulosa cell‐specific enzyme in the Aldo synthesis pathway. Whereas Hsd3b6 was expressed in a circadian manner in WT mice, that pattern was lost in Per1 KO mice. Hsd3b6 expression was significantly less in Per1 KO mice, providing one possible explanation for the lower plasma Aldo levels observed in Per1 KO mice. Taken together, these data support the hypothesis that the BP phenotype observed in mice lacking Per1 may involve a primary adrenal defect in addition to a renal defect in Na conservation.

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