Jacob Richards

Advances in understanding the peripheral circadian clocks

In the past decade, it has become increasingly evident that the circadian clock system plays an important role in many physiological processes. The circadian clock can be divided into 2 parts: the central clock, residing in the suprachiasmatic nucleus of the hypothalamus, which receives light cues, and the peripheral clocks that reside in various tissues throughout the body. The peripheral clocks play an integral and unique role in each of their respective tissues, driving the circadian expression of specific genes involved in a variety of physiological functions. The goal of this review is to provide an introduction to and overview of the peripheral clocks, including potential mechanisms, targets, and implications for disease states. The peripheral clocks include the cardiovascular, metabolic, endocrine, immune, and reproductive systems.— Richards, J., Gumz, M. L. Advances in understanding the peripheral circadian clocks. FASEB J. 26, 3602–3613 (2012). www.fasebj.org

The Circadian Protein Period 1 Contributes to Blood Pressure Control and Coordinately Regulates Renal Sodium Transport Genes

The circadian clock protein period 1 (Per1) contributes to the regulation of expression of the &agr; subunit of the renal epithelial sodium channel at the basal level and in response to the mineralocorticoid hormone aldosterone. The goals of the present study were to define the role of Per1 in the regulation of additional renal sodium handling genes in cortical collecting duct cells and to evaluate blood pressure (BP) in mice lacking functional Per1. To determine whether Per1 regulates additional genes important in renal sodium handling, a candidate gene approach was used. Immortalized collecting duct cells were transfected with a nontarget small interfering RNA or a Per1-specific small interfering RNA. Expression of the genes for &agr;-epithelial sodium channel and Fxyd5, a positive regulator of Na, K-ATPase activity, decreased in response to Per1 knockdown. Conversely, mRNA expression of caveolin 1, Ube2e3, and ET-1, all negative effectors of epithelial sodium channel, was induced after Per1 knockdown. These results led us to evaluate BP in Per1 KO mice. Mice lacking Per1 exhibit significantly reduced BP and elevated renal ET-1 levels compared with wild-type animals. Given the established role of renal ET-1 in epithelial sodium channel inhibition and BP control, elevated renal ET-1 is one possible explanation for the lower BP observed in Per1 KO mice. These data support a role for the circadian clock protein Per1 in the coordinate regulation of genes involved in renal sodium reabsorption. Importantly, the lower BP observed in Per1 KO mice compared with wild-type mice suggests a role for Per1 in BP control as well.

A role for the circadian clock protein Per1 in the regulation of the NaCl co-transporter (NCC) and the with-no-lysine kinase (WNK) cascade in mouse distal convoluted tubule cells.

Background: The role of the circadian protein Per1 in the regulation of sodium reabsorption in the distal convoluted tubule (DCT) is unknown. Results: Per1 transcriptionally regulates the sodium transporter NCC and the WNK kinase cascade. Conclusion: Per1 regulates sodium reabsorption in the DCT through NCC and the WNK cascade. Significance: These data demonstrate a role for Per1 in the regulation of renal sodium transporters. It has been well established that blood pressure and renal function undergo circadian fluctuations. We have demonstrated that the circadian protein Per1 regulates multiple genes involved in sodium transport in the collecting duct of the kidney. However, the role of Per1 in other parts of the nephron has not been investigated. The distal convoluted tubule (DCT) plays a critical role in renal sodium reabsorption. Sodium is reabsorbed in this segment through the actions of the NaCl co-transporter (NCC), which is regulated by the with-no-lysine kinases (WNKs). The goal of this study was to test if Per1 regulates sodium transport in the DCT through modulation of NCC and the WNK kinases, WNK1 and WNK4. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of NCC and WNK1 but increased expression of WNK4 in the renal cortex of mice. These findings were confirmed by using Per1 siRNA and pharmacological blockade of Per1 nuclear entry in mDCT15 cells, a model of the mouse distal convoluted tubule. Transcriptional regulation was demonstrated by changes in short lived heterogeneous nuclear RNA. Chromatin immunoprecipitation experiments demonstrated interaction of Per1 and CLOCK with the promoters of NCC, WNK1, and WNK4. This interaction was modulated by blockade of Per1 nuclear entry. Importantly, NCC protein expression and NCC activity, as measured by thiazide-sensitive, chloride-dependent 22Na uptake, were decreased upon pharmacological inhibition of Per1 nuclear entry. Taken together, these data demonstrate a role for Per1 in the transcriptional regulation of NCC, WNK1, and WNK4.

Clock Genes in Hypertension: Novel Insights from Rodent Models

The circadian clock plays an integral role in the regulation of physiological processes, including the regulation of blood pressure. However, deregulation of the clock can lead to pathophysiological states including hypertension. Recent work has implicated the circadian clock genes in the regulation of processes in the heart, kidney, vasculature, and the metabolic organs, which are all critical in the regulation of the blood pressure. The goal of this review is to provide an introduction and general overview into the role of circadian clock genes in the regulation of blood pressure with a focus on their deregulation in the etiology of hypertension. This review will focus on the core circadian clock genes CLOCK, BMAL1, Per, and Cry.

Inhibition of αENaC expression and ENaC activity following blockade of the circadian clock-regulatory kinases CK1δ/ε

Increasing evidence suggests that the circadian clock plays an important role in the control of renal function and blood pressure. We previously showed that the circadian clock protein Period (Per)1, positively regulates the expression of the rate limiting subunit of the renal epithelial sodium channel (αENaC), which contributes to blood pressure regulation. Casein kinases 1δ and 1ε (CK1δ/ε) are critical regulators of clock proteins. CK1δ/ε must phosphorylate the circadian clock protein Per1 in order for the latter to enter the nucleus. We used a commercially available CK1δ/ε inhibitor, PF670462, to test the effect of CK1δ/ε blockade and inhibited Per1 nuclear entry on αENaC in a model of the renal cortical collecting duct (mpkCCD(c14) cells). CK1δ/ε blockade prevented Per1 and Clock from interacting with an E-box from the αENaC promoter. CK1δ/ε inhibition reduced αENaC mRNA levels by <60%. A similar decrease in αENaC mRNA was observed following siRNA-mediated CK1δ/ε knock-down. Inhibition of CK1δ/ε effectively prevented the transcriptional response of αENaC to aldosterone, suggesting an interaction between the circadian clock and aldosterone-mediated regulation of αENaC. CK1δ/ε inhibition significantly reduced αENaC but increased Caveolin-1 membrane protein levels; transepithelial current, a measure of ENaC activity, was decreased. Importantly, single channel analysis in amphibian renal cells demonstrated a dramatic decrease in the number of patches with observable ENaC current following CK1δ/ε inhibition. The present study shows for the first time that CK1δ/ε inhibition and impaired Per1 nuclear entry results in decreased αENaC expression and ENaC activity, providing further support for direct control of ENaC by the circadian clock.

A role for the circadian clock protein Per1 in the regulation of aldosterone levels and renal Na+ retention

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.

Transcriptional regulation of NHE3 and SGLT1 by the circadian clock protein Per1 in proximal tubule cells

We have previously demonstrated that the circadian clock protein period (Per)1 coordinately regulates multiple genes involved in Na(+) reabsorption in renal collecting duct cells. Consistent with these results, Per1 knockout mice exhibit dramatically lower blood pressure than wild-type mice. The proximal tubule is responsible for a majority of Na(+) reabsorption. Previous work has demonstrated that expression of Na(+)/H(+) exchanger 3 (NHE3) oscillates with a circadian pattern and Na(+)-glucose cotransporter (SGLT)1 has been demonstrated to be a circadian target in the colon, but whether these target genes are regulated by Per1 has not been investigated in the kidney. The goal of the present study was to determine if Per1 regulates the expression of NHE3, SGLT1, and SGLT2 in the kidney. Pharmacological blockade of nuclear Per1 entry resulted in decreased mRNA expression of SGLT1 and NHE3 but not SGLT2 in the renal cortex of mice. Per1 small interfering RNA and pharmacological blockade of Per1 nuclear entry in human proximal tubule HK-2 cells yielded the same results. Examination of heterogeneous nuclear RNA suggested that the effects of Per1 on NHE3 and SGLT1 expression occurred at the level of transcription. Per1 and the circadian protein CLOCK were detected at promoters of NHE3 and SGLT1. Importantly, both membrane and intracellular protein levels of NHE3 and SGLT1 were decreased after blockade of nuclear Per1 entry. This effect was associated with reduced activity of Na(+)-K(+)-ATPase. These data demonstrate a role for Per1 in the transcriptional regulation of NHE3 and SGLT1 in the kidney.

Tissue-specific and time-dependent regulation of the endothelin axis by the circadian clock protein Per1

Aims The present study is designed to consider a role for the circadian clock protein Per1 in the regulation of the endothelin axis in mouse kidney, lung, liver and heart. Renal endothelin-1 (ET-1) is a regulator of the epithelial sodium channel (ENaC) and blood pressure (BP), via activation of both endothelin receptors, ETA and ETB. However, ET-1 mediates many complex events in other tissues. Main methods Tissues were collected in the middle of murine rest and active phases, at noon and midnight, respectively. ET-1, ETA and ETB mRNA expressions were measured in the lung, heart, liver, renal inner medulla and renal cortex of wild type and Per1 heterozygous mice using real-time quantitative RT-PCR. Key findings The effect of reduced Per1 expression on levels of mRNAs and the time-dependent regulation of expression of the endothelin axis genes appeared to be tissue-specific. In the renal inner medulla and the liver, ETA and ETB exhibited peaks of expression in opposite circadian phases. In contrast, expressions of ET-1, ETA and ETB in the lung did not appear to vary with time, but ET-1 expression was dramatically decreased in this tissue in Per1 heterozygous mice. Interestingly, ET-1 and ETA, but not ETB, were expressed in a time-dependent manner in the heart. Significance Per1 appears to regulate expression of the endothelin axis genes in a tissue-specific and time-dependent manner. These observations have important implications for our understanding of the best time of day to deliver endothelin receptor antagonists.

Opposing actions of Per1 and Cry2 in the regulation of Per1 target gene expression in the liver and kidney

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.

Role of Per1 and the mineralocorticoid receptor in the coordinate regulation of αENaC in renal cortical collecting duct cells.

Renal function and blood pressure (BP) exhibit a circadian pattern of variation, but the molecular mechanism underlying this circadian regulation is not fully understood. We have previously shown that the circadian clock protein Per1 positively regulates the basal and aldosterone-mediated expression of the alpha subunit of the renal epithelial sodium channel (αENaC). The mechanism of this regulation has not been determined however. To further elucidate the mechanism of mineralocorticoid receptor (MR) and Per1 action, site-directed mutagenesis, DNA pull-down assays and chromatin immunoprecipitation (ChIP) methods were used to investigate the coordinate regulation of αENaC by Per1 and MR. Mutation of two circadian response E-boxes in the human αENaC promoter abolished both basal and aldosterone-mediated promoter activity. DNA pull down assays demonstrated the interaction of both MR and Per1 with the E-boxes from the αENaC promoter. These observations were corroborated by ChIP experiments showing increased occupancy of MR and Per1 on an E-box of the αENaC promoter in the presence of aldosterone. This is the first report of an aldosterone-mediated increase in Per1 on a target gene promoter. Taken together, these results demonstrate the novel finding that Per1 and MR mediate the aldosterone response of αENaC through DNA/protein interaction in renal collecting duct cells.