Knockout of the circadian clock protein PER1 results in sex-dependent alterations of ET-1 production in mice in response to a high-salt diet plus mineralocorticoid treatment.

Previously, we showed that global knockout (KO) of the circadian clock transcription factor PER1 in male, but not female, mice fed a high-salt diet plus mineralocorticoid treatment (HS/DOCP) resulted in nondipping hypertension and decreased night/day ratio of sodium (Na) excretion. Additionally, we have shown that the endothelin-1 (ET-1) gene is targeted by both PER1 and aldosterone. We hypothesized that ET-1 would exhibit a sex-specific response to HS/DOCP treatment in PER1 KO. Here we show that male, but not female, global PER1 KO mice exhibit a decreased night/day ratio of urinary ET-1. Gene expression analysis revealed significant genotype differences in ET-1 and endothelin A receptor (ETA) expression in male, but not female, mice in response to HS/DOCP. Additionally, both wild-type and global PER1 KO male mice significantly increase endothelin B receptor (ETB) expression in response to HS/DOCP, but female mice do not. Finally, siRNA-mediated knockdown of PER1 in mouse cortical collecting duct cells (mpkCCDc14) resulted in increased ET-1 mRNA expression and peptide secretion in response to aldosterone treatment. These data suggest that PER1 is a negative regulator of ET-1 expression in response to HS/DOCP, revealing a novel mechanism for the regulation of renal Na handling in response to HS/DOCP treatment.

Female C57BL/6J mice lacking the circadian clock protein PER1 are protected from nondipping hypertension.

The circadian clock is integral to the maintenance of daily rhythms of many physiological outputs, including blood pressure. Our laboratory has previously demonstrated the importance of the clock protein period 1 (PER1) in blood pressure regulation in male mice. Briefly, a high-salt diet (HS; 4% NaCl) plus injection with the long-acting mineralocorticoid deoxycorticosterone pivalate (DOCP) resulted in nondipping hypertension [<10% difference between night and day blood pressure (BP) in Per1-knockout (KO) mice but not in wild-type (WT) mice]. To date, there have been no studies that have examined the effect of a core circadian gene KO on BP rhythms in female mice. The goal of the present study was to determine whether female Per1-KO mice develop nondipping hypertension in response to HS/DOCP treatment. For the first time, we demonstrate that loss of the circadian clock protein PER1 in female mice does not significantly change mean arterial pressure (MAP) or the BP rhythm relative to female C57BL/6 WT control mice. Both WT and Per1-KO female mice experienced a significant increase in MAP in response to HS/DOCP. Importantly, however, both genotypes maintained a >10% dip in BP on HS/DOCP. This effect is distinct from the nondipping hypertension seen in male Per1-KO mice, demonstrating that the female sex appears to be protective against PER1-mediated nondipping hypertension in response to HS/DOCP. Together, these data suggest that PER1 acts in a sex-dependent manner in the regulation of cardiovascular rhythms.

Renal Na-handling defect associated with PER1-dependent nondipping hypertension in male mice.

Many physiological functions have a circadian rhythm, including blood pressure (BP). BP is highest during the active phase, whereas during the rest period, BP dips 10-20%. Patients that do not experience this dip at night are termed "nondippers." Nondipping hypertension is associated with increased risk of cardiovascular disease. The mechanisms underlying nondipping hypertension are not understood. Without the circadian clock gene Per1, C57BL/6J mice develop nondipping hypertension on a high-salt diet plus mineralocorticoid treatment (HS/DOCP). Our laboratory has shown that PER1 regulates expression of several genes related to sodium (Na) transport in the kidney, including epithelial Na channel (ENaC) and Na chloride cotransporter (NCC). Urinary Na excretion also demonstrates a circadian pattern with a peak during active periods. We hypothesized that PER1 contributes to circadian regulation of BP via a renal Na-handling-dependent mechanism. Na-handling genes from the distal nephron were inappropriately regulated in KO mice on HS/DOCP. Additionally, the night/day ratio of Na urinary excretion by Per1 KO mice is decreased compared with WT (4 × vs. 7×, P < 0.001, n = 6 per group). Distal nephron-specific Per1 KO mice also show an inappropriate increase in expression of Na transporter genes αENaC and NCC. These results support the hypothesis that PER1 mediates control of circadian BP rhythms via the regulation of distal nephron Na transport genes. These findings have implications for the understanding of the etiology of nondipping hypertension and the subsequent development of novel therapies for this dangerous pathophysiological condition.

Effect of mineralocorticoid treatment in mice with collecting duct-specific knockout of endothelin-1.

Aldosterone increases blood pressure (BP) by stimulating sodium (Na) reabsorption within the distal nephron and collecting duct (CD). Aldosterone also stimulates endothelin-1 (ET-1) production that acts within the CD to inhibit Na reabsorption via a negative feedback mechanism. We tested the hypothesis that this renal aldosterone-endothelin feedback system regulates electrolyte balance and BP by comparing the effect of a high-salt (NaCl) diet and mineralocorticoid stimulation in control and CD-specific ET-1 knockout (CD ET-1 KO) mice. Metabolic balance and radiotelemetric BP were measured before and after treatment with desoxycorticosterone pivalate (DOCP) in mice fed a high-salt diet with saline to drink. CD ET-1 KO mice consumed more high-salt diet and saline and had greater urine output than controls. CD ET-1 KO mice exhibited increased BP and greater fluid retention and body weight than controls on a high-salt diet. DOCP with high-salt feeding further increased BP in CD ET-1 KO mice, and by the end of the study the CD ET-1 KO mice were substantially hypernatremic. Unlike controls, CD ET-1 KO mice failed to respond acutely or escape from DOCP treatment. We conclude that local ET-1 production in the CD is required for the appropriate renal response to Na loading and that lack of local ET-1 results in abnormal fluid and electrolyte handling when challenged with a high-salt diet and with DOCP treatment. Additionally, local ET-1 production is necessary, under these experimental conditions, for renal compensation to and escape from the chronic effects of mineralocorticoids.

Two rotary motors in F-ATP synthase are elastically coupled by a flexible rotor and a stiff stator stalk.

ATP is synthesized by ATP synthase (F(O)F(1)-ATPase). Its rotary electromotor (F(O)) translocates protons (in some organisms sodium cations) and generates torque to drive the rotary chemical generator (F(1)). Elastic power transmission between F(O) and F(1) is essential for smoothing the cooperation of these stepping motors, thereby increasing their kinetic efficiency. A particularly compliant elastic domain is located on the central rotor (c(10-15)/ε/γ), right between the two sites of torque generation and consumption. The hinge on the active lever on subunit ß adds further compliance. It is under contention whether or not the peripheral stalk (and the "stator" as a whole) also serves as elastic buffer. In the enzyme from Escherichia coli, the most extended component of the stalk is the homodimer b(2), a right-handed α-helical coiled coil. By fluctuation analysis we determined the spring constant of the stator in response to twisting and bending, and compared wild-type with b-mutant enzymes. In both deformation modes, the stator was very stiff in the wild type. It was more compliant if b was elongated by 11 amino acid residues. Substitution of three consecutive residues in b by glycine, expected to destabilize its α-helical structure, further reduced the stiffness against bending deformation. In any case, the stator was at least 10-fold stiffer than the rotor, and the enzyme retained its proton-coupled activity.

Endothelin-1 inhibits sodium reabsorption by ET(A) and ET(B) receptors in the mouse cortical collecting duct.

The collecting duct (CD) is a major renal site for the hormonal regulation of Na homeostasis and is critical for systemic arterial blood pressure control. Our previous studies demonstrated that the endothelin-1 gene (edn1) is an early response gene to the action of aldosterone. Because aldosterone and endothelin-1 (ET-1) have opposing actions on Na reabsorption (JNa) in the kidney, we postulated that stimulation of ET-1 by aldosterone acts as a negative feedback mechanism, acting locally within the CD. Aldosterone is known to increase JNa in the CD, in part, by stimulating the epithelial Na channel (ENaC). In contrast, ET-1 increases Na and water excretion through its binding to receptors in the CD. To date, direct measurement of the quantitative effect of ET-1 on transepithelial JNa in the isolated in vitro microperfused mouse CD has not been determined. We observed that the CD exhibits substantial JNa in male and female mice that is regulated, in part, by a benzamil-sensitive pathway, presumably ENaC. ENaC-mediated JNa is greater in the cortical CD (CCD) than in the outer medullary CD (OMCD); however, benzamil-insensitive JNa is present in the CCD and not in the OMCD. In the presence of ET-1, ENaC-mediated JNa is significantly inhibited. Blockade of either ETA or ETB receptor restored JNa to control rates; however, only ETA receptor blockade restored a benzamil-sensitive component of JNa. We conclude 1) Na reabsorption is mediated by ENaC in the CCD and OMCD and also by an ENaC-independent mechanism in the CCD; and 2) ET-1 inhibits JNa in the CCD through both ETA and ETB receptor-mediated pathways.

Aldosterone: Renal Action and Physiological Effects.

Aldosterone exerts profound effects on renal and cardiovascular physiology. In the kidney, aldosterone acts to preserve electrolyte and acid-base balance in response to changes in dietary sodium (Na+ ) or potassium (K+ ) intake. These physiological actions, principally through activation of mineralocorticoid receptors (MRs), have important effects particularly in patients with renal and cardiovascular disease as demonstrated by multiple clinical trials. Multiple factors, be they genetic, humoral, dietary, or otherwise, can play a role in influencing the rate of aldosterone synthesis and secretion from the adrenal cortex. Normally, aldosterone secretion and action respond to dietary Na+ intake. In the kidney, the distal nephron and collecting duct are the main targets of aldosterone and MR action, which stimulates Na+ absorption in part via the epithelial Na+ channel (ENaC), the principal channel responsible for the fine-tuning of Na+ balance. Our understanding of the regulatory factors that allow aldosterone, via multiple signaling pathways, to function properly clearly implicates this hormone as central to many pathophysiological effects that become dysfunctional in disease states. Numerous pathologies that affect blood pressure (BP), electrolyte balance, and overall cardiovascular health are due to abnormal secretion of aldosterone, mutations in MR, ENaC, or effectors and modulators of their action. Study of the mechanisms of these pathologies has allowed researchers and clinicians to create novel dietary and pharmacological targets to improve human health. This article covers the regulation of aldosterone synthesis and secretion, receptors, effector molecules, and signaling pathways that modulate its action in the kidney. We also consider the role of aldosterone in disease and the benefit of mineralocorticoid antagonists. © 2023 American Physiological Society. Compr Physiol 13:4409-4491, 2023.

Manipulations in the peripheral stalk of the Saccharomyces cerevisiae F1F0-ATP synthase.

The Saccharomyces cerevisiae F(1)F(0)-ATP synthase peripheral stalk is composed of the OSCP, h, d, and b subunits. The b subunit has two membrane-spanning domains and a large hydrophilic domain that extends along one side of the enzyme to the top of F(1). In contrast, the Escherichia coli peripheral stalk has two identical b subunits, and subunits with substantially altered lengths can be incorporated into a functional F(1)F(0)-ATP synthase. The differences in subunit structure between the eukaryotic and prokaryotic peripheral stalks raised a question about whether the two stalks have similar physical and functional properties. In the present work, the length of the S. cerevisiae b subunit has been manipulated to determine whether the F(1)F(0)-ATP synthase exhibited the same tolerances as in the bacterial enzyme. Plasmid shuffling was used for ectopic expression of altered b subunits in a strain carrying a chromosomal disruption of the ATP4 gene. Wild type growth phenotypes were observed for insertions of up to 11 and a deletion of four amino acids on a nonfermentable carbon source. In mitochondria-enriched fractions, abundant ATP hydrolysis activity was seen for the insertion mutants. ATPase activity was largely oligomycin-insensitive in these mitochondrial fractions. In addition, very poor complementation was seen in a mutant with an insertion of 14 amino acids. Lengthier deletions yielded a defective enzyme. The results suggest that although the eukaryotic peripheral stalk is near its minimum length, the b subunit can be extended a considerable distance.

Endothelin-1 gene regulation.

Over two decades of research have demonstrated that the peptide hormone endothelin-1 (ET-1) plays multiple, complex roles in cardiovascular, neural, pulmonary, reproductive, and renal physiology. Differential and tissue-specific production of ET-1 must be tightly regulated in order to preserve these biologically diverse actions. The primary mechanism thought to control ET-1 bioavailability is the rate of transcription from the ET-1 gene (edn1). Studies conducted on a variety of cell types have identified key transcription factors that govern edn1 expression. With few exceptions, the cis-acting elements bound by these factors have been mapped in the edn1 regulatory region. Recent evidence has revealed new roles for some factors originally believed to regulate edn1 in a tissue or hormone-specific manner. In addition, other mechanisms involved in epigenetic regulation and mRNA stability have emerged as important processes for regulated edn1 expression. The goal of this review is to provide a comprehensive overview of the specific factors and signaling systems that govern edn1 activity at the molecular level.

Mineralocorticoids stimulate the activity and expression of renal H+,K+-ATPases.

In the renal collecting duct, mineralocorticoids drive Na(+) reabsorption, K(+) secretion, and H(+) secretion through coordinated actions on apical and basolateral transporters. Whether mineralocorticoids act through H(+),K(+)-ATPases to maintain K(+) and acid-base homeostasis is unknown. Here, treatment of mice with the mineralocorticoid desoxycorticosterone pivalate (DOCP) resulted in weight gain, a decrease in blood [K(+)] and [Cl(-)], and an increase in blood [Na(+)] and [HCO(3)(-)]. DOCP treatment increased the rate of H(+),K(+)-ATPase-mediated H(+) secretion in intercalated cells of the inner cortical collecting duct. mRNA expression of the catalytic subunit HKα(1) did not significantly change, whereas HKα(2) mRNA expression dramatically increased in the outer and inner medulla of DOCP-treated mice. A high-K(+) diet abrogated this increase in renal HKα(2) expression, showing that DOCP-mediated stimulation of HKα(2) expression depends on dietary K(+) intake. DOCP treatment of mice lacking HKα(1) (HKα(1)(-/-)) resulted in greater urinary Na(+) retention than observed in either wild-type mice or mice lacking both HKα(1) and HKα(2) (HKα(1,2)(-/-)). DOCP-treated HKα(1,2)(-/-) mice exhibited a lower blood [HCO(3)(-)] and less Na(+) and K(+) retention than either wild-type or HKα(1)(-/-) mice. Taken together, these results indicate that H(+),K(+)-ATPases-especially the HKα(2)-containing H(+),K(+)-ATPases-play an important role in the effects of mineralocorticoids on K(+), acid-base, and Na(+) balance.

Regulation of αENaC expression by the circadian clock protein Period 1 in mpkCCD(c14) cells.

The epithelial sodium channel (ENaC) mediates the fine-tuned regulation of external sodium (Na) balance. The circadian clock protein Period 1 (Per1) is an aldosterone-induced gene that regulates mRNA expression of the rate-limiting alpha subunit of ENaC (αENaC). In the present study, we examined the effect of Per1 on αENaC in the cortex, the site of greatest ENaC activity in the collecting duct, and examined the mechanism of Per1 action on αENaC. Compared to wild type mice, Per1 knockout mice exhibited a 50% reduction of steady state αENaC mRNA levels in the cortex. Importantly, siRNA-mediated knockdown of Per1 decreased total αENaC protein levels in mpkCCD(c14) cells, a widely used model of the murine cortical collecting duct (CCD). Per1 regulated basal αENaC expression and participated in the aldosterone-mediated regulation of αENaC in mpkCCD(c14) cells. Because circadian clock proteins mediate their effects as part of multi-protein complexes at E-box response elements in the promoters of target genes, the ability of Per1 to interact with these sequences from the αENaC promoter was tested. For the first time, we show that Per1 and Clock are present at an E-box response element found in the αENaC promoter. Together these data support an important role for the circadian clock protein Per1 in the direct regulation of αENaC transcription and have important implications for understanding the role of the circadian clock in the regulation of renal function.

Pharmacological profiles of the murine gastric and colonic H,K-ATPases.

BACKGROUND: The H,K-ATPase, consisting of α and ß subunits, belongs to the P-type ATPase family. There are two isoforms of the α subunit, HKα1 and HKα2 encoded by different genes. The ouabain-resistant gastric HKα1-H,K-ATPase is Sch28080-sensitive. However, the colonic HKα2-H,K-ATPase from different species shows poor primary structure conservation of the HKα2 subunit between species and diverse pharmacological sensitivity to ouabain and Sch28080. This study sought to determine the contribution of each gene to functional activity and its pharmacological profile using mouse models with targeted disruption of HKα1, HKα2, or HKbeta genes. METHODS: Membrane vesicles from gastric mucosa and distal colon in wild-type (WT), HKα1, HKα2, or HKß knockout (KO) mice were extracted. K-ATPase activity and pharmacological profiles were examined. RESULTS: The colonic H,K-ATPase demonstrated slightly greater affinity for K(+) than the gastric H,K-ATPase. This K-ATPase activity was not detected in the colon of HKα2 KO but was observed in HKß KO with properties indistinguishable from WT. Neither ouabain nor Sch28080 had a significant effect on the WT colonic K-ATPase activity, but orthovanadate abolished this activity. Amiloride and its analogs benzamil and 5-N-ethyl-N-isopropylamiloride inhibited K-ATPase activity of HKα1-containing H,K-ATPase; the dose dependence of inhibition was similar for all three inhibitors. In contrast, the colonic HKα2-H,K-ATPase was not inhibited by these compounds. CONCLUSIONS: These data demonstrate that the mouse colonic H,K-ATPase exhibits a ouabain- and Sch28080-insensitive, orthovanadate-sensitive K-ATPase activity. Interestingly, pharmacological studies suggested that the mouse gastric H,K-ATPase is sensitive to amiloride. GENERAL SIGNIFICANCE: Characterization of the pharmacological profiles of the H,K-ATPases is important for understanding the relevant knockout animals and for considering the specificity of the inhibitors.

Aldosterone modulates steroid receptor binding to the endothelin-1 gene (edn1).

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 renal H+-K+-ATPases: physiology, regulation, and structure.

The H(+)-K(+)-ATPases are ion pumps that use the energy of ATP hydrolysis to transport protons (H(+)) in exchange for potassium ions (K(+)). These enzymes consist of a catalytic alpha-subunit and a regulatory beta-subunit. There are two catalytic subunits present in the kidney, the gastric or HKalpha(1) isoform and the colonic or HKalpha(2) isoform. In this review we discuss new information on the physiological function, regulation, and structure of the renal H(+)-K(+)-ATPases. Evaluation of enzymatic functions along the nephron and collecting duct and studies in HKalpha(1) and HKalpha(2) knockout mice suggest that the H(+)-K(+)-ATPases may function to transport ions other than protons and potassium. These reports and recent studies in mice lacking both HKalpha(1) and HKalpha(2) suggest important roles for the renal H(+)-K(+)-ATPases in acid/base balance as well as potassium and sodium homeostasis. Molecular modeling studies based on the crystal structure of a related enzyme have made it possible to evaluate the structures of HKalpha(1) and HKalpha(2) and provide a means to study the specific cation transport properties of H(+)-K(+)-ATPases. Studies to characterize the cation specificity of these enzymes under different physiological conditions are necessary to fully understand the role of the H(+)-K(+) ATPases in renal physiology.

The renal H,K-ATPases.

PURPOSE OF REVIEW: We integrate recent evidence that demonstrates the importance of the gastric (HKalpha1) and nongastric (HKalpha2)-containing hydrogen potassium adenosine triphosphatases (H,K-ATPases) on physiological function and their role in potassium (K), sodium (Na), and acid-base balance. RECENT FINDINGS: Previous studies focused on the primary role of H,K-ATPases as a mechanism of K conservation during states of K deprivation. Both isoforms function in H secretion and K absorption in vivo during K deprivation, but recent findings show that these pumps also function in acid secretion in animals fed normal K-replete diets. The complicated pharmacological inhibition of both pumps is reviewed. Interestingly, HKalpha2-null mice have a reduced expression and activity of the renal epithelial Na channel alpha subunit in the colon. When the human nongastric isoform was studied in a heterologous expression system with its cognate beta subunit (NaKbeta1), the pump exhibited substantial Na affinity at the 'K'-binding site. Evidence cited herein raises the possibility that either directly or indirectly the renal HKalpha2-containing H,K-ATPase may affect Na balance. SUMMARY: Both H,K-ATPase isoforms are active in normal animals and not just under conditions of K depletion. The possibility that either one or both isoforms contribute to Na absorption, particularly in humans, raises important clinical implications for these pumps in the kidney.

EDN1-AS, A Novel Long Non-coding RNA Regulating Endothelin-1 in Human Proximal Tubule Cells.

Endothelin-1 (ET-1) is a peptide hormone that functions as a vasoconstrictor in the vasculature, whereas in the collecting duct of the kidney it exerts blood pressure-lowering effects via natriuretic actions. Aberrant ET-1 signaling is associated with several pathological states including hypertension and chronic kidney disease. ET-1 expression is regulated largely through transcriptional control of the gene that encodes ET-1, EDN1. Here we report a long, non-coding RNA (lncRNA) that appears to be antisense to the EDN1 gene, called EDN1-AS. Because EDN1-AS represents a potential novel mechanism to regulate ET-1 expression, we examined the regulation of EDN1-AS expression and action. A putative glucocorticoid receptor response (GR) element upstream of the predicted EDN1-AS transcription start site was identified using the ENCODE database and the UCSC genome browser. Two homozygous deletion clones of the element were generated using CRISPR/Cas9. This deletion resulted in a significant increase in the expression of EDN1-AS, which was associated with increased secretion of ET-1 peptide from HK-2 cells (two-fold increase in KO cells vs. CNTL, n = 7, P < 0.05). Phenotypic characterization of these CRISPR clones revealed a difference in cell growth rates. Using a standard growth assay, we determined that the KO1 clone exhibited a three-fold increase in growth over 8 days compared to control cells (n = 4, P < 0.01) and the KO2 clone exhibited a two-fold increase (n = 4, P < 0.01). These results support a role for EDN1-AS as a novel regulatory mechanism of ET-1 expression and cellular proliferation.

Characterization of the rabbit HKalpha2 gene promoter.

The HKalpha2 gene directs synthesis of the HKalpha2 subunit of the H(+), K(+)-ATPase. In the kidney and colon, the gene is highly expressed and is thought to play a role in potassium (K(+)) conservation. The rabbit has been an important experimental system for physiological studies of ion transport in the kidney, so the rabbit HKalpha2 gene has been cloned and characterized. The genomic clones and the previously reported HKalpha2a and HKalpha2c subunit cDNAs provided a means to address several issues regarding the structure and expression of the HKalpha2 gene. First, the genomic organization established that the rabbit HKalpha2 gene was unambiguously homologous to the mouse HKalpha2 gene and the human ATP1AL1 gene. Second, the mapping of the transcription start site for the alternate transcript, HKalpha2c, confirmed that it was an authentic rabbit transcript. Finally, isolation of DNA from the 5' end of the HKalpha2 gene enabled us to initiate studies on its regulation in the rabbit cortical collecting duct. The promoter and two putative negative regulatory regions were identified and the effect of cell confluency on gene expression was studied.

Aldosterone alters the chromatin structure of the murine endothelin-1 gene.

UNLABELLED: Aldosterone increases sodium reabsorption in the renal collecting duct and systemic blood pressure. Paradoxically, aldosterone also induces transcription of the endothelin-1 (Edn1) gene to increase protein (ET-1) levels, which inhibits sodium reabsorption. AIMS: Here we investigated changes in the chromatin structure of the Edn1 gene of collecting duct cell lines in response to aldosterone treatment. The Edn1 gene has a CpG island that encompasses the transcription start site and four sites in the 5' regulatory region previously linked to transcriptional regulation. MATERIALS AND METHODS: The chromatin structure of the Edn1 gene was investigated using a quantitative PCR-based DNaseI hypersensitivity assay in murine hepatocyte (AML12), renal cortical collecting duct (mpkCCDC14), outer medullary collecting duct1 (OMCD1), and inner medullary collecting duct-3 (IMCD-3) cell lines. KEY FINDINGS: The CpG island was uniformly accessible. One calcium-responsive NFAT element remained at low chromatin accessibility in all cell lines under all conditions tested. However, the second calcium responsive NFAT element located at -1563bp upstream became markedly more accessible in IMCD-3 cells exposed to aldosterone. Importantly, one established aldosterone hormone response element HRE at -671bp relative to the transcription start site was highly accessible, and another HRE (-551bp) became more accessible in aldosterone-treated IMCD-3 and OMCD1 cells. SIGNIFICANCE: The evidence supports a model in which aldosterone activation of the mineralocorticoid receptor (MR) results in the MR-hormone complex binding at HRE at -671bp to open chromatin structure around other regulatory elements in the Edn1 gene.

Molecular regulation of the HKalpha2 subunit of the H+,K(+)-ATPases.

H+, K(+)-Adenosine triphosphatases (H+, K(+)-ATPase) located in the mammalian kidney participate in maintaining ion and acid/base balance. The molecular mechanisms by which physiological conditions lead to an upregulation of H+, K(+)-ATPase activity are poorly understood. However, studies in recent years have provided tangible progress towards understanding the role of the renal H+, K(+)-ATPases. The cloning of cDNAs from the kidneys of several mammalian species has provided evidence for the presence of several H+, K(+)-ATPase isoforms. The different H+, K(+)-ATPases may make distinct contributions to ion and acid/base balance. Studies of mRNA, protein and H+, K(+)ATPase activity levels in the kidney under a variety of physiological conditions have revealed that the HKalpha2 (colonic) subunit of the H+, K(+)ATPase is highly regulated. The pump is responsive to potassium, sodium and probably hormones. Recent developments have focused on defining the HKa2 gene promoter. Analysis of the 5' end of the HKalpha2 gene from human, rat and rabbit has identified conserved elements that may serve as core promoter and regulatory elements. This review will summarize recent data related to the molecular regulation of the HKalpha2 subunit of the H+, K(+)-ATPase.

MicroRNA regulation of endothelin-1 mRNA in renal collecting duct cells.

AIMS: Recently, microRNAs (miRNAs) have been implicated in control of Edn1 mRNA in several tissues. Here we examined the role of miRNA action on Edn1 mRNA expression in renal distal collecting duct cells. MAIN METHODS: A microarray study was conducted to provide a comprehensive assessment of miRNAs present in a murine inner medullary collecting duct (mIMCD-3) cell line. The experiment was designed as a comparison between mIMCD-3 cells grown in the presence and absence of aldosterone. Argonaute (Ago) immunoprecipitation experiments were used to investigate binding of the RNA induced silencing complex (RISC) to Edn1 mRNA. KEY FINDINGS: Thirty-four miRNAs were detected in very high abundance in mIMCD-3 cells, and a large number of others were present at lower levels. The microarray experiments were validated by quantitative PCR analysis of selected miRNAs. The microarray data, in combination with in silico examination of the Edn1 3' UTR provided a panel of candidate miRNAs that could act upon the Edn1 expression. Edn1 mRNA was co-immunoprecipitated with an Argonaute protein antibody, and this interaction was blocked by anti-miR-709 oligonucleotides. SIGNIFICANCE: These results define the miRNA landscape of the mIMCD-3 cell line. Moreover, Edn1 was shown to interact with Argonaute protein suggesting that it is a target of the RNA induced silencing complex (RISC).