Oleh Pochynyuk

Paracrine Regulation of the Epithelial Na+ Channel in the Mammalian Collecting Duct by Purinergic P2Y2 Receptor Tone

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 P2Y2 receptors. ENaC in collecting ducts isolated from mice lacking this receptor have blunted responses to ATP. P2Y2 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 P2Y2-/- mice. Loss of collecting duct P2Y2 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 P2Y2-/- 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 P2Y2 receptor-/- mice. P2Y2 receptor activation by nucleotides thus provides physiologically important regulation of ENaC and electrolyte handling in mammalian kidney.

Angiotensin II increases activity of the epithelial Na+ channel (ENaC) in distal nephron additively to aldosterone.

Background: Mineralocorticoid aldosterone controls ENaC-mediated Na+ reabsorption in the distal nephron. Results: Ang II stimulates production of reactive oxygen species to stimulate ENaC, and this effect is preserved when mineralocorticoids are high. Conclusion: Ang II directly controls ENaC activity and expression in murine distal nephrons. Significance: Ang II may have a specific role in regulation of sodium handling in the distal nephron during variations in dietary Na+ intake. Dietary salt intake controls epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in the distal nephron by affecting status of the renin-angiotensin-aldosterone system (RAAS). Whereas regulation of ENaC by aldosterone is generally accepted, little is known about whether other components of RAAS, such as angiotensin II (Ang II), have nonredundant to aldosterone-stimulatory actions on ENaC. We combined patch clamp electrophysiology and immunohistochemistry in freshly isolated split-opened distal nephrons of mice to determine the mechanism and molecular signaling pathway of Ang II regulation of ENaC. We found that Ang II acutely increases ENaC Po, whereas prolonged exposure to Ang II also induces translocation of α-ENaC toward the apical membrane in situ. Ang II actions on ENaC Po persist in the presence of saturated mineralocorticoid status. Moreover, aldosterone fails to stimulate ENaC acutely, suggesting that Ang II and aldosterone have different time frames of ENaC activation. AT1 but not AT2 receptors mediate Ang II actions on ENaC. Unlike its effect in vasculature, Ang II did not increase [Ca2+]i in split-opened distal nephrons as demonstrated using ratiometric Fura-2-based microscopy. However, application of Ang II to mpkCCDc14 cells resulted in generation of reactive oxygen species, as probed with fluorescent methods. Consistently, inhibiting NADPH oxidase with apocynin abolished Ang II-mediated increases in ENaC Po in murine distal nephron. Therefore, we concluded that Ang II directly regulates ENaC activity in the distal nephron, and this effect complements regulation of ENaC by aldosterone. We propose that stimulation of AT1 receptors with subsequent activation of NADPH oxidase signaling pathway mediates Ang II actions on ENaC.

Regulation of the epithelial Na+ channel by endothelin-1 in rat collecting duct

We used patch-clamp electrophysiology to investigate regulation of the epithelial Na+ 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.

Insight toward epithelial Na+ channel mechanism revealed by the acid-sensing ion channel 1 structure

The epithelial Na+ channel/degenerin (ENaC/DEG) protein family includes a diverse group of ion channels, including nonvoltage‐gated Na+ channels of epithelia and neurons, and the acid‐sensing ion channel 1 (ASIC1). In mammalian epithelia, ENaC helps regulate Na+ and associated water transport, making it a critical determinant of systemic blood pressure and pulmonary mucosal fluidity. In the nervous system, ENaC/DEG proteins are related to sensory transduction. While the importance and physiological function of these ion channels are established, less is known about their structure. One hallmark of the ENaC/DEG channel family is that each channel subunit has only two transmembrane domains connected by an exceedingly large extracellular loop. This subunit structure was recently confirmed when Jasti and colleagues determined the crystal structure of chicken ASIC1, a neuronal acid‐sensing ENaC/DEG channel. By mapping ENaC to the structural coordinates of cASIC1, as we do here, we hope to provide insight toward ENaC structure. ENaC, like ASIC1, appears to be a trimeric channel containing 1α, 1β, and 1γ subunit. Heterotrimeric ENaC and monomeric ENaC subunits within the trimer possibly contain many of the major secondary, tertiary, and quaternary features identified in cASIC1 with a few subtle but critical differences. These differences are expected to have profound effects on channel behavior. In particular, they may contribute to ENaC insensitivity to acid and to its constitutive activity in the absence of time‐ and ligand‐dependent inactivation. Experiments resulting from this comparison of cASIC1 and ENaC may help clarify unresolved issues related to ENaC architecture, and may help identify secondary structures and residues critical to ENaC function.

Acute Regulation of the Epithelial Na+ Channel by Phosphatidylinositide 3-OH Kinase Signaling in Native Collecting Duct Principal Cells

Activity of the epithelial Na(+) channel (ENaC) is limiting for Na(+) reabsorption in the aldosterone-sensitive distal nephron. Hormones, including aldosterone and insulin, increase ENaC activity, in part by stimulating phosphatidylinositide 3-OH kinase (PI3-K) signaling. Recent studies in heterologous expression systems reveal a close spatiotemporal coupling between PI3-K signaling and ENaC activity with the phospholipid product of this kinase, PI(3,4,5)P(3), in some cases, directly binding the channel and increasing open probability (P(o)). This study tested whether this tight coupling plays a physiologic role in modulating ENaC activity in native tissue and polarized epithelial cells. IGF-I was found to increase Na(+) reabsorption across mpkCCD(c14) principal cell monolayers in a PI3-K-sensitive manner. Inhibition of PI3-K signaling, moreover, rapidly decreased Na(+) reabsorption and ENaC activity in mpkCCD(c14) cells that were treated with corticosteroids and IGF-I. These decreases paralleled changes in apical membrane PI(3,4,5)P(3) levels, demonstrating tight spatiotemporal coupling between ENaC activity and PI3-K/PI(3,4,5)P(3) signaling within this membrane. For further probing of the mechanism underpinning this coupling, cortical collecting ducts (CCD) were isolated from rat and split open to expose the apical membrane for patch-clamp analysis. Inhibition of PI3-K signaling with wortmannin and LY294002 but not its inactive analogue rapidly and markedly decreased the P(o) of ENaC. Moreover, IGF-I acutely increased P(o) of ENaC in CCD principal cells in a PI3-K-sensitive manner. Together, these observations stress the importance of tight spatiotemporal coupling between PI3-K signaling and ENaC within the apical membrane of principal cells to the physiologic control of this ion channel.

Dietary Na+ inhibits the open probability of the epithelial sodium channel in the kidney by enhancing apical P2Y2-receptor tone

Apical release of ATP and UTP can activate P2Y2 receptors in the aldosterone‐sensitive distal nephron (ASDN) and inhibit the open probability (Po) of the epithelial sodium channel (ENaC). Little is known, however, about the regulation and physiological relevance of this system. Patch‐clamp studies in freshly isolated ASDN provide evidence that increased dietary Na+ intake in wild‐type mice lowers ENaC Po, consistent with a contribution to Na+ homeostasis, and is associated with increased urinary concentrations of UTP and the ATP hydrolytic product, ADP. Genetic deletion of P2Y2 receptors in mice (P2Y2 −/−; litter‐mates to wild‐type mice) or inhibition of apical P2Y‐receptor activation in wild‐type mice prevents dietary Na+‐induced lowering of ENaC Po. Although they lack suppression of ENaC Po by dietary NaCl, P2Y2−/− mice do not exhibit NaCl‐sensitive blood pressure, perhaps as a consequence of compensatory down‐regulation of aldosterone levels. Consistent with this hypothesis, clamping mineralocorticoid activity at high levels unmasks greater ENaC activity and NaCl sensitivity of blood pressure in P2Y2−/− mice. The studies indicate a key role of the apical ATP/UTP‐P2Y2‐receptor system in the inhibition of ENaC Po in the ASDN in response to an increase in Na+ intake, thereby contributing to NaCl homeostasis and blood pressure regulation.—Pochynyuk, O., Rieg, T., Bugaj, V., Schroth, J., Fridman, A., Boss, G. R., Insel, P. A., Stockand, J. D., Vallon, V. Dietary Na+ inhibits the open probability of the epithelial sodium channel in the kidney by enhancing apical P2Y2‐receptor tone. FASEB J. 24, 2056–2065 (2010). www.fasebj.org

Activation of the epithelial Na+ channel in the collecting duct by vasopressin contributes to water reabsorption

We used patch-clamp electrophysiology on isolated, split-open murine collecting ducts (CD) to test the hypothesis that regulation of epithelial sodium channel (ENaC) activity is a physiologically important effect of vasopressin. Surprisingly, this has not been tested directly before. We ask whether vasopressin affects ENaC activity distinguishing between acute and chronic effects, as well as, parsing the cellular signaling pathway and molecular mechanism of regulation. In addition, we quantified possible synergistic regulation of ENaC by vasopressin and aldosterone associating this with a requirement for distal nephron Na+ reabsorption during water conservation vs. maintenance of Na+ balance. We find that vasopressin significantly increases ENaC activity within 2-3 min by increasing open probability (P(o)). This activation was dependent on adenylyl cyclase (AC) and PKA. Water restriction (18-24 h) and pretreatment of isolated CD with vasopressin (approximately 30 min) resulted in a similar increase in P(o). In addition, this also increased the number (N) of active ENaC in the apical membrane. Similar to P(o), increases in N were sensitive to inhibitors of AC. Stressing animals with water and salt restriction separately and jointly revealed an important effect of vasopressin: conservation of water and Na+ each independently increased ENaC activity and jointly had a synergistic effect on channel activity. These results demonstrate a quantitatively important action of vasopressin on ENaC suggesting that distal nephron Na+ reabsorption mediated by this channel contributes to maintenance of water reabsorption. In addition, our results support that the combined actions of vasopressin and aldosterone are required to achieve maximally activated ENaC.

Thiazolidinedione-Induced Fluid Retention Is Independent of Collecting Duct αENaC Activity

Thiazolidinediones are agonists of peroxisome proliferator-activated receptor gamma (PPARgamma) that can induce fluid retention and weight gain through unclear mechanisms. To test a proposed role for the epithelial sodium channel ENaC in thiazolidinedione-induced fluid retention, we used mice with conditionally inactivated alphaENaC in the collecting duct (Scnn1a(loxloxCre) mice). In control mice, rosiglitazone did not alter plasma aldosterone levels or protein expression of ENaC subunits in the kidney, but did increase body weight, plasma volume, and the fluid content of abdominal fat pads, and decreased hematocrit. Scnn1a(loxloxCre) mice provided functional evidence for blunted Na+ uptake in the collecting duct, but still exhibited rosiglitazone-induced fluid retention. Moreover, treatment with rosiglitazone or pioglitazone did not significantly alter the open probability or number of ENaC channels per patch in isolated, split-open cortical collecting ducts of wild-type mice. Finally, patch-clamp studies in primary mouse inner medullary collecting duct cells did not detect ENaC activity but did detect a nonselective cation channel upregulated by pioglitazone. These data argue against a primary and critical role of ENaC in thiazolidinedione-induced fluid retention.

Rapid Translocation and Insertion of the Epithelial Na+ Channel in Response to RhoA Signaling

Activity of the epithelial Na+ channel (ENaC) is limiting for Na+ absorption across many epithelia. Consequently, ENaC is a central effector impacting systemic blood volume and pressure. Two members of the Ras superfamily of small GTPases, K-Ras and RhoA, activate ENaC. K-Ras activates ENaC via a signaling pathway involving phosphatidylinositol 3-kinase and production of phosphatidylinositol 3,4,5-trisphosphate with the phospholipid directly interacting with the channel to increase open probability. How RhoA increases ENaC activity is less clear. Here we report that RhoA and K-Ras activate ENaC through independent signaling pathways and final mechanisms of action. Activation of RhoA signaling rapidly increases the membrane levels of ENaC likely by promoting channel insertion. This process dramatically increases functional ENaC current, resulting in tight spatial-temporal control of these channels. RhoA signals to ENaC via a transduction pathway, including the downstream effectors Rho kinase and phosphatidylinositol-4-phosphate 5-kinase. Phosphatidylinositol 4,5-biphosphate produced by activated phosphatidylinositol 4-phosphate 5-kinase may play a role in targeting vesicles containing ENaC to the plasma membrane.

Identification of a Functional Phosphatidylinositol 3,4,5-Trisphosphate Binding Site in the Epithelial Na+ Channel

Membrane phospholipids, such as phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5)P3), are signaling molecules that can directly modulate the activity of ion channels, including the epithelial Na+ channel (ENaC). Whereas PI(3,4,5)P3 directly activates ENaC, its binding site within the channel has not been identified. We identify here a region of γ-mENaC just following the second trans-membrane domain (residues 569–583) important to PI(3,4,5)P3 binding and regulation. Deletion of this track decreases activity of ENaC heterologously expressed in Chinese hamster ovary cells. K-Ras and its first effector phosphoinositide 3-OH kinase (PI3-K), as well as RhoA and its effector phosphatidylinositol 4-phosphate 5-kinase increase ENaC activity. Whereas the former, via generation of PI(3,4,5)P3, increases ENaC open probability, the latter increases activity by increasing membrane levels of the channel. Deletion of the region just distal to the second trans-membrane domain disrupted regulation by K-Ras and PI3-K but not RhoA and phosphatidylinositol 4-phosphate 5-kinase. Moreover, PI(3,4,5)P3 binds ENaC with deletion of the region following the second transmembrane domain disrupting this interaction and disrupting direct activation of the channel by PI(3,4,5)P3. Mutation analysis revealed the importance of conserved positive and negative charged residues as well as bulky amino acids within this region to modulation of ENaC by PI3-K. The current results identify the region just distal to the second trans-membrane domain within γ-mENaC as being part of a functional PI(3,4,5)P3 binding site that directly impacts ENaC activity. Phospholipid binding to this site is probably mediated by the positively charged amino acids within this track, with negatively charged and bulky residues also influencing specificity of interactions.