The mineralocorticoid aldosterone is a major regulator of sodium transport in target epithelia and contributes to the control of blood pressure and cardiac function. It specifically functions to increase renal absorption of sodium from tubular fluid via regulation of the α subunit of the epithelial sodium channel (αENaC). We previously used microarray technology to identify the immediate transcriptional targets of aldosterone in a mouse inner medullary collecting duct cell line and found that the transcript induced to the greatest extent was the circadian clock gene Period 1. Here, we investigated the role of Period 1 in mediating the downstream effects of aldosterone in renal cells. Aldosterone treatment stimulated expression of Period 1 (Per1) mRNA in renal collecting duct cell lines and in the rodent kidney. RNA silencing of Period 1 dramatically decreased expression of mRNA encoding αENaC in the presence or absence of aldosterone. Furthermore, expression of αENaC-encoding mRNA was attenuated in the renal medulla of mice with disruption of the Per1 gene, and these mice exhibited increased urinary sodium excretion. Renal αENaC-encoding mRNA was expressed in an apparent circadian pattern, and this pattern was dramatically altered in mice lacking functional Period genes. These results suggest a role for Period 1 in the regulation of the renal epithelial sodium channel and more broadly implicate the circadian clock in control of sodium balance.

Aldosterone and endothelin-1 (ET-1) act on collecting duct cells of the kidney and are important regulators of renal sodium transport and cardiovascular physiology. We recently identified the ET-1 gene (edn1) as a novel aldosterone-induced transcript. However, aldosterone action on edn1 has not been characterized at the present time. In this report, we show that aldosterone stimulated edn1 mRNA in acutely isolated rat inner medullary collecting duct cells ex vivo and ET-1 peptide in rat inner medulla in vivo. Aldosterone induction of edn1 mRNA occurred in cortical, outer medullary, and inner medullary collecting duct cells in vitro. Inspection of the edn1 promoter revealed two putative hormone response elements. Levels of heterogeneous nuclear RNA synthesis demonstrated that edn1 mRNA stimulation occurred at the level of transcription. RNA knockdowns corroborated pharmacological studies and demonstrated both mineralocorticoid receptor and glucocorticoid receptor participated in this response. Aldosterone resulted in dose-dependent nuclear translocation and binding of mineralocorticoid receptor and glucocorticoid receptor to the edn1 hormone response elements. Hormone receptors mediated the association of chromatin remodeling complexes, histone modification, and RNA polymerase II at the edn1 promoter. Direct interaction between aldosterone and ET-1 has important implications for renal and cardiovascular function.The steroid hormone aldosterone is critical for sodium homeostasis and blood pressure control. Aldosterone works by modulating the fine regulation of sodium reabsorption in the distal nephron and collecting duct of the kidney. Classic aldosterone action is mediated through the mineralocorticoid receptor (MR), 3 a member of the nuclear receptor family of proteins that function as ligand-dependent transcription factors (1). MR acts on cells of the distal nephron and collecting duct to stimulate transcription of genes involved in transepithelial sodium transport, including the epithelial sodium channel ␣ subunit (scnn1a, ␣ENaC), the sodium, potassium-ATPase ␣1 subunit (atp1a1), and the serum-and glucocorticoid-regulated kinase-1 (sgk1) (2). The increase in expression of genes involved in sodium transport results in net sodium reabsorption followed by in increase in extracellular fluid volume and a consequent increase in blood pressure. Indeed, MR antagonists such as spironolactone and eplerenone are used clinically as diuretic and anti-hypertensive agents (3). The mechanism of MR action is consistent with a classic steroid receptor mechanism (4). Prior to activation MR resides in the cytosol. Ligand binding induces a conformational change that releases chaperone proteins and reveals a nuclear localization signal. Nuclear MR binds directly to DNA at hormone response elements (HREs) in target genes to modulate their transcription. A typical HRE for MR consists of two receptor binding half-sites with the consensus sequence 5Ј-TGTTCT-3Ј, arranged as an inverted palindrome (1, 5). These HREs facilitate binding of...

Two classes of H pumps, H-K-ATPase and H-ATPase, contribute to luminal acidification and HCO(3) transport in the collecting duct (CD). At least two H-K-ATPase alpha-subunits are expressed in the CD: HKalpha(1) and HKalpha(2). Both exhibit K dependence but have different inhibitor sensitivities. The HKalpha(1) H-K-ATPase is Sch-28080 sensitive, whereas the pharmacological profile of the HKalpha(2) H-K-ATPase is not completely understood. The present study used a nonpharmacological, genetic approach to determine the contribution of HKalpha(1) and HKalpha(2) to cortical CD (CCD) intercalated cell (IC) proton transport in mice fed a normal diet. Intracellular pH (pH(i)) recovery was determined in ICs using in vitro microperfusion of CCD after an acute intracellular acid load in wild-type mice and mice of the same strain lacking expression of HKalpha(1), HKalpha(2), or both H-K-ATPases (HKalpha(1,2)). A-type and B-type ICs were differentiated by luminal loading with BCECF-AM and peritubular chloride removal from CO(2)/HCO(3)-buffered solutions to identify the membrane locations of Cl/HCO(3) exchange activity. H-ATPase- and Na/H exchange-mediated H transport were inhibited with bafilomycin A(1) (100 nM) and EIPA (10 microM), respectively. Here, we report 1) initial pH(i) and buffering capacity were not significantly altered in the ICs of HKalpha-deficient mice, 2) either HKalpha(1) or HKalpha(2) deficiency resulted in slower acid extrusion, and 3) A-type ICs from HKalpha(1,2)-deficient mice had significantly slower acid extrusion compared with A-type ICs from HKalpha(1)-deficient mice alone. These studies are the first nonpharmacological demonstration that both HKalpha(1) and HKalpha(2) contribute to H secretion in both A-type and B-type ICs in animals fed a normal diet.

Inwardly rectifying K؉ channels or Kirs are a large gene family and have been predicted to have two transmembrane segments, M1 and M2, intracellular N and C termini, and two extracellular loops, E1 and E2, separated by an intramembranous pore-forming segment, H5. H5 contains a stretch of eight residues that are similar in voltage-dependent K ؉ channels, Kvs, and this stretch is called the signature sequence of K ؉ channels. Because mutations in this sequence altered selectivity in Kvs, it has been designated as the selectivity filter. Previously, we used N-glycosylation substitution mutants to map the extracellular topology of a weak inwardly rectifying K ؉ channel, Kir1.1 or ROMK1, and found that the entire H5 segment was extracellular. We now report utilization of introduced N-glycosylation