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

Abstract

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

“…Casein kinase (CK)1-␦/ε inhibitor experiments were performed as previously described (30,31). For dose and time course, NCI-H295R cells were treated with either 0.1, 1, 10, or 100 M of the CK1-␦/ε inhibitor PF-670462 or vehicle (water) for 24, 48, or 72 h. For nuclear entry experiments, NCI-H295R cells were treated with 0.1 M PF-670462 or vehicle for 24 h.…”

“…Per and Cry dimerize and interact with Clock and Bmal1 to repress their transcriptional activity (3,11). Per1 may not act as a canonical repressor, however, as increasing evidence suggests that Per1 may activate gene expression through an unknown mechanism, and this may occur in a tissue-and gene-specific manner (8,16,18,31,36). However, our recent work (30) demonstrated that this possible mechanism could be through repression of the circadian repressor Cry2.…”

“…Surprisingly, no changes were observed in the expression of the aldosterone synthase gene (CYPB11), which encodes the ratelimiting step of aldosterone production. We (16,18,31,36) have previously shown that Per1 regulates both the basal and aldosterone-mediated regulation of the ␣-subunit of the renal epithelial Na ϩ channel (␣-ENaC). We (26) have also demonstrated that Per1 coordinately regulates the expression of multiple genes that contribute to the regulation of renal Na ϩ reabsorption.…”

Tick Tock

“…7 Our subsequent reports showed that Per1 regulates the expression of the a subunit of the epithelial sodium channel and the activity of epithelial sodium channel. 8,9 Consistent with these mechanistic molecular findings in renal models, studies in circadian KO mice have consistently demonstrated a role for each of the core clock proteins in BP control. [10][11][12][13] An important role for the kidney in these BP phenotypes has often been proposed, but the lack of renal cell type-specific KO models of circadian genes has prevented the use of a genetic model to directly test this hypothesis.…”

“…9 The Folic Acid for Vascular Outcomes Reduction in Transplantation trial failed to show a benefit of folic acid therapy on CV events in 4110 kidney transplant recipients. However, it generated a large dataset of carefully phenotyped transplant recipients, with follow-up and validated endpoints, which the investigators have used to study the role of BP on CVD.…”