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

Abstract

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.

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

“…Chromatin Immunoprecipitation (ChIP)-mDCT15 cells were grown to ϳ80% confluence and then treated with vehicle (water) or 10 M PF670462 for 48 h. ChIP was performed using the ChIP-IT TM Express Enzymatic Kit (Active Motif) according to the manufacturer's instructions and as described previously (28). Chromatin concentrations were calculated, and equal amounts of vehicle-treated and 10 M PF670462-treated chromatin were used per pull-down.…”

Circadian regulation of renal function

“…Per1 was found to positively regulate the expression of αENaC at baseline and after aldosterone stimulation in mouse CCD, OMCD and IMCD cells [8,71]. In the CCD, this regulation occurs through direct binding with an E-box domain on the αENaC promoter along with the mineralocorticoid receptor (MR) in the presence of aldosterone [71,72]. This direct regulation of αENaC by Per1 was also shown to be inhibited by Casein kinase 1δ/ε, a regulator of the core clock mechanism via phosphorylation of Per proteins and inhibition of Per1 nuclear translocation [73].…”

The Circadian Clock in the Regulation of Renal Rhythms

“…Aldosterone-mediated transcriptional regulation of the Scnn1a gene, encoding αENaC, occurs through the mineralocorticoid receptor (MR). PER1 appears to be involved in this regulation (Richards et al 2013d). In one of the first reports to link the molecular clock to renal sodium handling, Gumz and colleagues demonstrated that knockdown of PER1 in several renal CD cell lines resulted in decreased expression of αENaC (Gumz et al 2009).…”