Growing evidence implicates a key role for extracellular nucleotides in cellular regulation, including of ion channels and renal function, but the mechanisms for such actions are inadequately defined. We investigated purinergic regulation of the epithelial Na ؉ channel (ENaC) in mammalian collecting duct. We find that ATP decreases ENaC activity in both mouse and rat collecting duct principal cells. ATP and other nucleotides, including UTP, decrease ENaC activity via apical P2Y 2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y 2 couples to ENaC via PLC; direct activation of PLC mimics ATP action. Tonic regulation of ENaC in the collecting duct occurs via locally released ATP; scavenging endogenous ATP and inhibiting P2 receptors, in the absence of other stimuli, rapidly increases ENaC activity. Moreover, ENaC has greater resting activity in collecting ducts from P2Y 2 ؊/؊ mice. Loss of collecting duct P2Y 2 receptors in the knock-out mouse is the primary defect leading to increased ENaC activity based on the ability of direct PLC stimulation to decrease ENaC activity in collecting ducts from P2Y 2 ؊/؊ mice in a manner similar to ATP in collecting ducts from wild-type mice. These findings demonstrate that locally released ATP acts in an autocrine/paracrine manner to tonically regulate ENaC in mammalian collecting duct. Loss of this intrinsic regulation leads to ENaC hyperactivity and contributes to hypertension that occurs in P2Y 2 receptor ؊/؊ mice. P2Y 2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney. Systemic Naϩ balance influences blood pressure. Consequently, body Na ϩ content is under tight negative-feedback control by the renin-angiotensin-aldosterone system. Discretionary Na ϩ reabsorption in the aldosterone-sensitive distal renal nephron, including the collecting duct, fine-tunes plasma Na ϩ levels. Here, the activity of the luminal epithelial Na ϩ channel (ENaC) 2 is limiting for Na ϩ transport (1-3). ENaC is an end-effector of the renin-angiotensin-aldosterone system with aldosterone increasing ENaC activity. The importance of ENaC and its proper regulation to control of blood pressure is highlighted by several diseases associated with gain and loss of ENaC function (3, 4). For instance, gain of ENaC function results in inappropriate Na ϩ conservation and hypertension (e.g. Liddle syndrome). Conversely, loss of ENaC function results in renal salt wasting associated with hypotension (e.g. pseudohypoaldosteronism type 1).Although extrinsic regulation of ENaC in the distal nephron by hormones originating outside the kidney is considered pivotal to blood pressure control, complementary regulation of ENaC by autocrine/paracrine factors originating from intrarenal sources is just now becoming appreciated. ATP has been identified as a candidate signaling molecule possibly mediating intrinsic control of distal nephron Na ϩ reabsorption (5-14). ATP and other...

The mechanisms underlying "aldosterone escape," which refers to the excretion of sodium (Na ϩ ) during high Na ϩ intake despite inappropriately increased levels of mineralocorticoids, are incompletely understood. Because local purinergic tone in the aldosterone-sensitive distal nephron downregulates epithelial Na ϩ channel (ENaC) activity, we tested whether this mechanism mediates aldosterone escape. Here, urinary ATP concentration increased with dietary Na ϩ intake in mice. Physiologic concentrations of ATP decreased ENaC activity in a dosage-dependent manner. P2Y 2 Ϫ/Ϫ mice, which lack the purinergic receptor, had significantly less increased Na ϩ excretion than wild-type mice in response to high-Na ϩ intake. Exogenous deoxycorticosterone acetate and deletion of the P2Y 2 receptor each modestly increased the resistance of ENaC to changes in Na ϩ intake; together, they markedly increased resistance. Under the latter condition, ENaC could not respond to changes in Na ϩ intake. In contrast, as a result of aldosterone escape, wild-type mice had increased Na ϩ excretion in response to high-Na ϩ intake regardless of the presence of high deoxycorticosterone acetate. These data suggest that control of ENaC by purinergic signaling is necessary for aldosterone escape.

We used patch-clamp electrophysiology to investigate regulation of the epithelial Na ϩ channel (ENaC) by endothelin-1 (ET-1) in isolated, split-open rat collecting ducts. ET-1 significantly decreases ENaC open probability by about threefold within 5 min. ET-1 decreases ENaC activity through basolateral membrane ETB but not ETA receptors. In rat collecting duct, we find no role for phospholipase C or protein kinase C in the rapid response of ENaC to ET-1. ET-1, although, does activate src family tyrosine kinases and their downstream MAPK1/2 effector cascade in renal principal cells. Both src kinases and MAPK1/2 signaling are necessary for ET-1-dependent decreases in ENaC open probability in the split-open collecting duct. We conclude that ET-1 in a physiologically relevant manner rapidly suppresses ENaC activity in native, mammalian principal cells. These findings may provide a potential mechanism for the natriuresis observed in vivo in response to ET-1, as well as a potential cause for the salt-sensitive hypertension found in animals with impaired endothelin signaling.salt-sensitive hypertension; systemic blood pressure ENDOTHELIN-1 (ET-1) is a powerful vasoconstricting peptide hormone that is an important regulator of systemic blood pressure (53). Independent of its vascular effects, ET-1 also affects renal Na ϩ and water handling favoring natriuresis and diuresis. While circulating ET-1 arises from endothelial cells, local ET-1 systems also exist. For instance in the kidney, the collecting duct produces significant amounts of 25,38,51). ET-1 targets cells through two distinct receptor subtypes, ET A and ET B (32, 41). Renal collecting duct cells have both types of receptors and are able to bind 49,50). Thus, collecting duct-derived ET-1, acting in a paracrine/ autocrine manner, is an important regulator of renal Na ϩ handling (2,20,26,42).Regulated Na ϩ reabsorption in the renal collecting duct, in part, controls blood pressure. Here, activity of the aldosteronesensitive epithelial Na ϩ channel (ENaC) is limiting for Na ϩ transport (reviewed in Refs. 19,30,31). Dysfunction and inappropriate regulation of ENaC consequently result in improper renal Na ϩ handling and thus, blood pressure disorders. For instance, gain of ENaC function in rodents and humans is causative for hypertension associated with the hallmarks of low plasma renin activity and aldosterone levels (1,22,23,45,46). Amiloride, an ENaC blocker, ameliorates this hypertension.Spotting lethal (sl) rats have a naturally occurring null mutation of ET B (17). These rats, when rescued from lethal intestinal aganglionosis by directed ET B transgene expression in the enteric nervous system, are particularly sensitive to DOCA and salt-induced hypertension (18,33,34). Similarly, mice with collecting duct-specific knockout of the ET B receptor have elevated blood pressure that further increases with high salt feeding (20). Collecting duct-specific ET-1 knockout, moreover, leads to hypertension exacerbated by high salt (2, 42). Plasma renin activity and aldoste...

We tested whether ATP release through Connexin 30 (Cx30) is part of a local purinergic regulatory system intrinsic to the aldosterone-sensitive distal nephron (ASDN) important for proper control of sodium excretion; if changes in sodium intake influence ATP release via Cx30; and if this allows a normal ENaC response to changes in systemic sodium levels. In addition, we define the consequences of disrupting ATP regulation of ENaC in Cx30 Blood pressure is under negative feedback control. A key factor setting blood pressure is systemic sodium balance. Under normal conditions, as sodium intake increases renal sodium excretion increases to maintain sodium balance and thus, blood pressure. The renin-angiotensin-aldosterone system (RAAS) 2 plays a central role in negative feedback regulation of blood pressure. This system responds to changes in blood pressure and effective circulating volume: both of which are tied to systemic sodium levels. A key end-effector of RAAS, specifically the mineralocorticoid, aldosterone, is the epithelial Na ϩ channel (ENaC) localized to the apical membrane of principal cells in the aldosterone-sensitive distal nephron (ASDN; (1-3). Plasma sodium levels are fine-tuned in the ASDN through discretionary sodium reabsorption mediated chiefly by the activity of ENaC. Thus, ENaC activity and regulation of this channel are critical to control of blood pressure. This is best appreciated by considering that gain and loss of ENaC function increase and decrease, respectively, blood pressure by impacting renal sodium excretion (4 -7).Emerging evidence supports that local control of ENaC by autocrine/paracrine regulation also plays an important role in setting channel activity in response to changing sodium intake (8 -12). For instance, local control by a purinergic system intrinsic to the ASDN modulates the open probability of ENaC and thus, sodium excretion, in response to changes in sodium intake (8, 9, 13). There are two types of purinergic (P2) receptors: classic seven-transmembrane G protein-coupled receptors, metabotropic P2Y receptors; and ionotropic P2X receptors, which function as ligand-gated non-selective ion channels (14 -16). The P2Y 2 receptor important for control of ENaC is in the apical plasma membrane of principal cells and coupled to PLC via G q/11 (13, 16 -18).Mice lacking the P2Y 2 receptor have impaired sodium excretion and increases in blood pressure associated with decreased plasma renin and aldosterone, as well as potassium (8). Gain of ENaC function similarly leads to hypertension associated with decreased plasma renin, aldosterone, and potassium levels (4 -7). Plasma potassium levels are decreased, in part, because K ϩ secretion increases in the ASDN as Na ϩ is reabsorbed here via ENaC. Plasma renin and aldosterone levels decrease as part of a feedback response attempting to mitigate inappropriate sodium retention. In the P2Y 2 knock-out mouse, ENaC is hyperactive, having increased open probability, and unable to respond normally to changes in sodium intake due to disruptio...

The epithelial Na + channel (ENaC) in the aldosterone-sensitive distal nephron (ASDN) is under negative-feedback regulation by the renin-angiotensin-aldosterone system in protection of sodium balance and blood pressure. We test here whether aldosterone is necessary and sufficient for ENaC expression and activity in the ASDN. Surprisingly, ENaC expression and activity are robust in adrenalectomized (Adx) mice. Exogenous mineralocorticoid increases ENaC activity equally well in control and Adx mice. Plasma [AVP] is significantly elevated in Adx vs. control mice. Vasopressin (AVP) stimulates ENaC. Inhibition of the V 2 AVP receptor represses ENaC activity in Adx mice. The absence of aldosterone combined with elevated AVP release compromises normal feedback regulation of ENaC in Adx mice in response to changes in sodium intake. These results demonstrate that aldosterone is sufficient but not necessary for ENaC activity in the ASDN. Aldosterone-independent stimulation by AVP shifts the role of ENaC in the ASDN from protecting Na + balance to promoting water reabsorption. This stimulation of ENaC likely contributes to the hyponatremia of adrenal insufficiency.epithelial transport | hypertension | sodium excretion | diabetes insipidus | sodium wasting R enal sodium excretion is fine-tuned in the aldosterone-sensitive distal nephron (ASDN). Here, the activity of the epithelial Na + channel (ENaC) is limiting for sodium reabsorption (reviewed in refs. 1 and 2). ENaC serves as the apical entry pathway for electrogenic Na + reabsorption through principal cells. Normal ENaC function is required for proper sodium balance and, thus, normal blood pressure. Gain-of-function mutations in ENaC cause inappropriate renal sodium retention and consequent increases in mean arterial pressure (2, 3). Inhibition of ENaC corrects the renal and blood pressure phenotypes resulting from such mutations. Loss-of-function mutations in ENaC, in contrast, cause renal sodium wasting and corresponding decreases in blood pressure (2, 4).The activity of ENaC is under negative-feedback regulation by the renin-angiotensin-aldosterone system (RAAS; ref. 1). The mineralocorticoid, aldosterone, is the final hormone in this cascade. This antinatriuretic factor is essential for proper Na + balance (5, 6). Decreases in blood pressure evoke via reninAngII signaling secretion of aldosterone from the adrenal gland. Aldosterone through the mineralocorticoid receptor (MR) stimulates ENaC in the ASDN to minimize renal sodium excretion in protection of Na + balance and vascular volume (2, 4). Pathological increases in aldosterone elevate blood pressure by promoting inappropriate renal sodium retention (7,8). Inhibition of ENaC ameliorates inappropriate renal sodium retention. In contrast, pathological decreases in aldosterone result in sodium wasting arising from inappropriate increases in renal sodium excretion (4,8,9). MR agonism and antagonism increase and decrease ENaC activity, respectively (10-12). There is strong support for a tight positive relation between...

The physiological significance of the renal tubular prorenin receptor (PRR) has been difficult to elucidate due to developmental abnormalities associated with global or renal-specific PRR knockout (KO). We recently developed an inducible renal tubule-wide PRR KO using the Pax8/LC1 transgenes and demonstrated that disruption of renal tubular PRR at 1 mo of age caused no renal histological abnormalities. Here, we examined the role of renal tubular PRR in blood pressure (BP) regulation and Na(+) excretion and investigated the signaling mechanisms by which PRR regulates Na(+) balance. No detectable differences in BP were observed between control and PRR KO mice fed normal- or low-Na(+) diets. However, compared with controls, PRR KO mice had elevated plasma renin concentration and lower cumulative Na(+) balance with normal- and low-Na(+) intake. PRR KO mice had an attenuated hypertensive response and reduced Na(+) retention following angiotensin II (ANG II) infusion. Furthermore, PRR KO mice had significantly lower epithelial Na(+) channel (ENaC-α) expression. Treatment with mouse prorenin increased, while PRR antagonism decreased, ENaC activity in isolated split-open collecting ducts (CD). The prorenin effect was prevented by protein kinase A and Akt inhibition, but unaffected by blockade of AT1, ERK1/2, or p38 MAPK pathways. Taken together, these data indicate that renal tubular PRR, likely via direct prorenin/renin stimulation of PKA/Akt-dependent pathways, stimulates CD ENaC activity. Absence of renal tubular PRR promotes Na(+) wasting and reduces the hypertensive response to ANG II.

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Collecting duct (CD)-derived endothelin-1 (ET-1) acting via endothelin B (ETB) receptors promotes Na(+) excretion. Compromise of ET-1 signaling or ETB receptors in the CD cause sodium retention and increase blood pressure. Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the CD. To test for ETB receptor regulation of ENaC, we combined patch-clamp electrophysiology with CD-specific knockout (KO) of endothelin receptors. We also tested how ET-1 signaling via specific endothelin receptors influences ENaC activity under differing dietary Na(+) regimens. ET-1 significantly decreased ENaC open probability in CD isolated from wild-type (WT) and CD ETA KO mice but not CD ETB KO and CD ETA/B KO mice. ENaC activity in WT and CD ETA but not CD ETB and CD ETA/B KO mice was inversely related to dietary Na(+) intake. ENaC activity in CD ETB and CD ETA/B KO mice tended to be elevated under all dietary Na(+) regimens compared with WT and CD ETA KO mice, reaching significance with high (2%) Na(+) feeding. These results show that the bulk of ET-1 inhibition of ENaC activity is mediated by the ETB receptor. In addition, they could explain the Na(+) retention and elevated blood pressure observed in CD ET-1 KO, CD ETB KO, and CD ETA/B KO mice consistent with ENaC regulation by ET-1 via ETB receptors contributing to the antihypertensive and natriuretic effects of the local endothelin system in the mammalian CD.