Charles S. Wingo

The circadian clock protein Period 1 regulates expression of the renal epithelial sodium channel in mice

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 alpha subunit of the epithelial sodium channel (alphaENaC). 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 alphaENaC in the presence or absence of aldosterone. Furthermore, expression of alphaENaC-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 alphaENaC-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.

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 (ednl). Studies conducted on a variety of cell types have identified key transcription factors that govern ednl expression. With few exceptions, the cts‐acting elements bound by these factors have been mapped in the ednl regulatory region. Recent evidence has revealed new roles for some factors originally believed to regulate ednl 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 ednl expression. The goal of this review is to provide a comprehensive overview of the specific factors and signaling systems that govern ednl activity at the molecular level.—Stow, L. R., Jacobs, M. E., Wingo, C. S., Cain, B. D. Endothelin‐1 gene regulation. FASEB J. 25, 16–28 (2011). www.fasebj.org

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.

An Integrated View of Potassium Homeostasis.

The plasma potassium level is normally maintained within narrow limits by multiple mechanisms. This article reviews the mechanisms that regulate potassium homeostasis and describes the important role that the circadian clock exerts on these processes.

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.

Narrative Review: Evolving Concepts in Potassium Homeostasis and Hypokalemia

Hypokalemia is a common disorder that can result from potassium redistribution between plasma and intracellular fluid (ICF), inadequate potassium intake, or excessive potassium excretion (1). When hypokalemia reflects true potassium depletion, the body activates several mechanisms, especially in the kidney, to conserve potassium (Figure 1). Although short periods of mild potassium depletion are typically well tolerated in healthy individuals, severe potassium depletion can result in glucose intolerance (2) and serious cardiac (3), renal (4), and neurologic (5) dysfunction, including death. Prolonged potassium depletion of even modest proportion can provoke or exacerbate kidney injury or hypertension (6, 7). Indeed, reduced potassium intake correlates directly with higher blood pressure in both normotensive and hypertensive individuals (8). Figure 1. Integrated model of the regulation of body potassium balance. CNS = central nervous system. Left. Classic mechanisms. Right. Additional putative mechanisms. Normal Potassium Balance and Renal Potassium Excretion To set the context, we first summarize key concepts of steady-state potassium handling. Total body potassium is roughly 55 mmol/kg of body weight, with 98% distributed to the ICF (primarily in muscle, the liver, and erythrocytes) and 2% in the extracellular fluid (1). Na,K-ATPase isoforms (see Glossary) actively pump potassium into the cell and maintain and restore the electrochemical gradient between the normal extracellular potassium concentration of 3.5 to 5.0 mmol/L and the intracellular potassium concentration of approximately 150 mmol/L, which is particularly important for normal functioning of excitable cells. -Catecholamines, aldosterone, insulin, pH, and osmolality influence the transcellular potassium distribution and thus how well cells buffer changes in plasma potassium concentration (9). Physicians exploit this fact in the emergency management of severe hyperkalemia when they use insulin or -catecholamines to drive plasma potassium into cells. Normal persons who consume a typical Western diet ingest approximately 70 mmol of potassium per day (10). The intestine absorbs virtually all of the ingested potassium and delivers it to the liver for processing by means of the hepatoportal circulation. Minimal amounts of potassium are excreted in the feces. Renal potassium excretion, the principal defense against chronic potassium imbalances, depends on free filtration at the glomerulus, extensive proximal tubule reabsorption, and a highly regulated secretory process of the distal convoluted tubule and segments of the collecting duct in the cortex and outer medulla (the cortical collecting duct and the outer medullary collecting duct, respectively) (Figure 2). The cortical collecting duct and outer medullary collecting duct consist of at least 2 very different cell types, termed principal cells and intercalated cells (Figure 2). Principal cells, which comprise approximately 70% to 75% of collecting duct cells, mediate sodium reabsorption and potassium secretion and are targets for angiotensin II (11, 12), aldosterone, aldosterone receptor antagonists, and potassium-sparing diuretics (Figure 2). Principal cells exploit the electrochemical gradient established by sodium entry into the cell through a sodium channel at the luminal membrane (the molecular target of amiloride) and the basolateral membrane Na,K-ATPase to drive potassium secretion through 2 classes of luminal membrane potassium channels (13). One of these, the renal outer medullary potassium (known among renal physiologists as ROMK) channels, secrete potassium under normal tubular fluid flow conditions and are inserted into or internalized from the luminal membrane, depending on the demand for potassium secretion. The other class of potassium channels are the big conductance channels (known as BK channels), which are relatively inactive under normal conditions but exhibit increased activity during high tubular flow or high-potassium conditions (13). Factors that regulate principal cell potassium secretion include previous potassium intake; intracellular potassium level; sodium delivery to the cells; urine flow rate; and hormones, such as aldosterone and -catecholamines (14). The other collecting duct cell type, intercalated cells, mediate acidbase transport but upregulate expression of luminal H,K-ATPases (see Glossary) during potassium depletion to enhance potassium reabsorption (1) (Figure 2). Figure 2. Segmental handling of potassium excretion along the nephron and collecting duct cell types under normal conditions or conditions of potassium excess or deficiency. We present a simplified model of potassium handling by collecting duct cell types. Principal cells in the collecting duct are responsible for secretion of excess potassium in the circulation into the tubule lumen and thus into the urine. This secretion is accomplished by luminal membrane potassium channels responding primarily to the electrochemical gradient for potassium generated by the combined actions of the basolateral membrane Na,K-ATPase and a luminal membrane sodium channel (the target of the potassium-sparing diuretic amiloride). In states of potassium depletion, potassium secretion by the principal cells is inhibited and the luminal membrane H,K-ATPase is activated in the intercalated cells to reclaim the potassium that remains in the tubular fluid, thereby limiting urinary potassium wasting. ADP = adenosine diphosphate; ATP = adenosine triphosphate; CCD = cortical collecting duct; DT = distal tubule; Glom = glomerulus; IMCD = inner medullary collecting duct; MTAL = medullary thick ascending limb of Henle loop; OMCD = outer medullary collecting duct; Pi = inorganic phosphate. To summarize, mammalian cells require a steep concentration gradient of potassium between ICF and extracellular fluid to function properly, which requires primary active transport by Na,K-ATPase. The kidney excretes sufficient amounts of potassium to maintain total body homeostasis. Although the proximal nephron reabsorbs the bulk of the potassium filtered at the glomerulus, the collecting duct fine-tunes potassium excretion and is subject to several regulatory influences. Feedback Control of Potassium Balance The use of thermostats to adjust heating or cooling is a common example of feedback control, a control mechanism in a homeostatic system that uses the consequences or outputs of a process to feed back and regulate the process itself. The thermostat detects the error (for example, the room is too hot) and signals for the air conditioner to provide cool air. Once the room reaches the temperature set at the thermostat (the room becomes cool enough), the air conditioner turns off. This example of feedback control also applies to potassium homeostasis. Although feedback control of potassium balance has been recognized for decades, only recently have some of its secrets been discovered. In response to a high-potassium meal that includes glucose, pancreatic insulin secretion activates skeletal muscle and liver Na,K-ATPase, which pumps potassium from the plasma to the ICF of these cells. This mechanism minimizes the postprandial increase in plasma potassium concentration (15). With muscle activity, potassium is released into the plasma and filtered at the glomerulus. To maintain balance, the amount of potassium consumed in the meal (minus the small amount lost in the feces) is secreted into the urine. When potassium consumption increases plasma potassium concentration enough, it triggers aldosterone synthesis and release from the adrenals, which stimulates the activity and synthesis of Na,K-ATPase and luminal potassium channels in collecting duct principal cells to secrete the excess potassium (16) (Figures 1 and 2). Aldosterone also enhances potassium secretion in the distal colon (17), which can be especially important when renal function is compromised. Conversely, if potassium intake is very low or its output is very high, plasma potassium concentration decreases and feedback regulation redistributes potassium from ICF to plasma and minimizes renal potassium excretion. Skeletal muscle becomes insulin-resistant to potassium (but not glucose) uptake even before plasma potassium concentration decreases, which blunts the shift of potassium from plasma into the cell (18). After hypokalemia ensues, the expression of skeletal muscle Na,K-ATPase 2 isoform decreases, which allows a net potassium leak from ICF to the plasma (19). The low plasma potassium concentration suppresses adrenal aldosterone release so that the kidney can reclaim all but about 1% of the filtered potassium (Figure 2). This renal potassium conservation involves downregulation of potassium secretion by means of the ROMK channels in cortical collecting duct principal cells. Chronic potassium depletion activates a renal NADPH oxidase (a relative of the enzyme that produces the respiratory burst in neutrophils) that produces reactive oxygen species to signal for the ROMK channels to be internalized and thus incompetent to conduct membrane transport of potassium (20, 21). In addition, an H,K-ATPase (a relative of the proton pump active in the gastric mucosa) is activated in outer medullary collecting duct intercalated cells to reclaim any remaining filtered potassium into the plasma. Figure 2 summarizes the responses of the different nephron segments to accommodate changes in potassium intake. Feedforward Control of Potassium Balance Feedforward control refers to a pathway in a homeostatic system that responds to a signal in the environment in a predetermined manner, without responding to how the system subsequently reacts (that is, without responding to feedback). The most famous example of feedforward control is the conditioned salivation of Pavlovs dogs in anticipation of food (22). Pavlov implanted small stomach pouches in dogs to measure salivation. He and his assistants would ring bell

The effects of potassium depletion and supplementation on blood pressure: a clinical review.

Nonpharmacologic treatment currently is recognized as an important part in the treatment of hypertension, and the role of dietary potassium intake in blood pressure (BP) control is becoming quite evident. Clinical studies have examined the mechanism by which hypokalemia can increase BP and the benefit of a large potassium intake on BP control. Epidemiologic data suggest that potassium intake and BP are correlated inversely. In normotensive subjects, those who are salt sensitive or who have a family history of hypertension appear to benefit most from the hypotensive effects of potassium supplementation. The greatest hypotensive effect of potassium supplementation occurs in patients with severe hypertension. This effect is pronounced with prolonged potassium supplementation. The antihypertensive effect of increased potassium intake appears to be mediated by several factors, which include enhancing natriuresis, modulating baroreflex sensitivity, direct vasodilation, or lowering cardiovascular reactivity to norepinephrine or angiotensin II. Potassium repletion in patients with diuretic-induced hypokalemia improves BP control. An increase in potassium intake should be included in the nonpharmacologic management of patients with uncomplicated hypertension.

Dietary Modulation of Active Potassium Secretion in the Cortical Collecting Tubule of Adrenalectomized Rabbits

Addisonian patients can maintain potassium homeostasis despite the absence of mineralocorticoid. The present in vitro microperfusion studies examine what role the cortical collecting tubule might play in this process. All studies were performed on tubules harvested from adrenalectomized rabbits, which were maintained on 0.15 M NaCl drinking water and dexamethasone 50 mug/d. Perfusion and bath solutions were symmetrical Ringers bicarbonate with [K] of 5 meq/liter. Initial studies on cortical collecting tubules from adrenalectomized animals ingesting a high potassium chow (9 meq K/kg body wt) demonstrated net potassium secretion against an electrochemical gradient (mean collected fluid [K] 16.5+/-2.6 meq/liter with an observed transepithelial voltage of -6.3+/-4.1 mV; predicted voltage for passive distribution of potassium being -28.2 mV). To examine whether this active potassium secretion could be modulated by dietary potassium, independent of mineralocorticoid, two diets identical in all respects except for potassium content were formulated. Potassium secretion was compared in cortical collecting tubules harvested from adrenalectomized animals on low (0.1 meq K) and high (10 meq K) potassium intake. Mean net potassium secretion by cortical collecting tubules was 2.02+/-0.54 peq mm(-1) min(-1) in the low potassium diet group and 5.34+/-.74 peq.mm(-1).min(-1) in the high potassium group. The mean transepithelial voltages of the collecting tubules did not differ between the two dietary groups. While net Na reabsorption was significantly greater in tubules from the high K group, this could not account for the differences in K secretion. These data demonstrate that: (a) the cortical collecting tubule can actively secrete potassium and that the magnitude of this potassium secretion correlates with potassium intake; (b) this active potassium secretory process in independent of mineralocorticoid. These findings support the hypothesis that the cortical collecting tubule may contribute to K homeostasis in Addisons disease.

Regulation of αENaC expression by the circadian clock protein Period 1 in mpkCCDc14 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.

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.