Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: UNIPROT:P41181 (collecting duct)
5,183 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Epithelial Na+ channels (ENaCs) mediate sodium reabsorption in the cortical collecting duct (CCD), but the regulatory pathways that modulate the activity of these channels are incompletely understood. Here, we observed that endothelin-1 (ET-1) attenuates ENaC activity acutely by reducing the channel's open probability and chronically by decreasing the number of channels in the plasma membrane. To investigate whether beta1Pix, a signaling protein activated by ET-1, mediates ENaC activity, we reconstituted ENaC in CHO cells with or without coexpressed beta1Pix and found that beta1Pix negatively regulates ENaC. Knockdown of betaPix in native principal cells abolished the ET-1-induced decrease in ENaC channel number. Furthermore, we found that betaPix does not decrease ENaC activity through its guanine nucleotide exchange factor (GEF) activity for Rac1 and Cdc42. Instead, coexpression of beta1Pix mutant constructs revealed that beta1Pix affects ENaC activity through binding 14-3-3 proteins. Coimmunoprecipitation experiments supported a physical interaction between beta1Pix and 14-3-3beta in cultured principal cells. Coexpression of 14-3-3beta increased ENaC activity in CHO cells, but concomitant expression of beta1Pix attenuated this increase. Recruitment of 14-3-3beta by beta1Pix impaired the interaction of 14-3-3beta with the ubiquitin ligase Nedd4-2, thereby promoting ubiquitination and degradation of ENaC. Taken together, these results suggest that the inhibitory effects of chronic ET-1 on ENaC result from betaPix interacting with the 14-3-3/Nedd4-2 pathway.
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PMID:Endothelin-1 inhibits the epithelial Na+ channel through betaPix/14-3-3/Nedd4-2. 2033 96

Aldosterone-induced increases in apical membrane epithelial sodium channel (ENaC) density and Na transport involve the induction of 14-3-3 protein expression and their association with Nedd4-2, a substrate of serum- and glucocorticoid-induced kinase (SGK1)-mediated phosphorylation. A search for other 14-3-3 binding proteins in aldosterone-treated cortical collecting duct (CCD) cells identified the Rab-GAP, AS160, an Akt/PKB substrate whose phosphorylation contributes to the recruitment of GLUT4 transporters to adipocyte plasma membranes in response to insulin. In CCD epithelia, aldosterone (10 nM, 24 h) increased AS160 protein expression threefold, with a time-course similar to increases in SGK1 expression. In the absence of aldosterone, AS160 overexpression increased total ENaC expression 2.5-fold but did not increase apical membrane ENaC or amiloride-sensitive Na current (I(sc)). In AS160 overexpressing epithelia, however, aldosterone increased apical ENaC and I(sc) 2.5-fold relative to aldosterone alone, thus recruiting the accumulated ENaC to the apical membrane. Conversely, AS160 knockdown increased apical membrane ENaC and I(sc) under basal conditions to approximately 80% of aldosterone-stimulated values, attenuating further steroid effects. Aldosterone induced AS160 phosphorylation at five sites, predominantly at the SGK1 sites T568 and S751, and evoked AS160 binding to the steroid-induced 14-3-3 isoforms, beta and epsilon. AS160 mutations at SGK1 phospho-sites blocked its selective interaction with 14-3-3beta and epsilon and suppressed the ability of expressed AS160 to augment aldosterone action. These findings indicate that the Rab protein regulator, AS160, stabilizes ENaC in a regulated intracellular compartment under basal conditions, and that aldosterone/SGK1-dependent AS160 phosphorylation permits ENaC forward trafficking to the apical membrane to augment Na absorption.
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PMID:AS160 modulates aldosterone-stimulated epithelial sodium channel forward trafficking. 2041 Jan 34

Nedd4-2, a E3 ubiquitin ligase, regulates epithelial sodium channel-mediated transcellular Na(+) transport in the collecting duct. We investigated the effect of Nedd4-2 on the junctional complex and paracellular conductance in mpkCCD(c14) cells, a collecting duct cell line. We demonstrate that Nedd4-2 coimmunoprecipitated with and reduced the expression of transfected occludin in HEK293 cells. This interaction was mediated via a conserved PY motif in the COOH terminus of occludin and mutation of this PY motif increased the half-life of transfected occludin in HEK293 cells from 6.4 to 11.4 h. We demonstrate that Nedd4-2 ubiquitinates occludin, which was not seen when a catalytically inactive form of Nedd4-2 was used. Overexpression of Nedd4-2 in mpkCCD(c14) cells reduced occludin at the tight junction and transiently increased paracellular conductance in a Ca(2+) switch assay consistent with a delay in the formation of tight junctions. Conversely, siRNA-mediated knockdown of Nedd4-2 increased occludin levels and reduced paracellular conductance. In summary, we demonstrate that Nedd4-2 plays a role in tight junction assembly and the regulation of paracellular conductance in the collecting duct.
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PMID:Nedd4-2 interacts with occludin to inhibit tight junction formation and enhance paracellular conductance in collecting duct epithelia. 2050 82

The KCNQ1 K(+) channel plays a key role in the regulation of several physiological functions, including cardiac excitability, cardiovascular tone, and body electrolyte homeostasis. The metabolic sensor AMP-activated protein kinase (AMPK) has been shown to regulate a growing number of ion transport proteins. To determine whether AMPK regulates KCNQ1, we studied the effects of AMPK activation on KCNQ1 currents in Xenopus laevis oocytes and collecting duct epithelial cells. AMPK activation decreased KCNQ1 currents and channel surface expression in X. laevis oocytes, but AMPK did not phosphorylate KCNQ1 in vitro, suggesting an indirect regulatory mechanism. As it has been recently shown that the ubiquitin-protein ligase Nedd4-2 inhibits KCNQ1 plasma membrane expression and that AMPK regulates epithelial Na(+) channels via Nedd4-2, we examined the role of Nedd4-2 in the AMPK-dependent regulation of KCNQ1. Channel inhibition by AMPK was blocked in oocytes coexpressing either a dominant-negative or constitutively active Nedd4-2 mutant, or a Nedd4-2 interaction-deficient KCNQ1 mutant, suggesting that Nedd4-2 participates in the regulation of KCNQ1 by AMPK. KCNQ1 is expressed at the basolateral membrane in mouse polarized kidney cortical collecting duct (mpkCCD(c14)) cells and in rat kidney. Treatment with the AMPK activators AICAR (2 mM) or metformin (1 mM) reduced basolateral KCNQ1 currents in apically permeabilized polarized mpkCCD(c14) cells. Moreover, AICAR treatment of rat kidney slices ex vivo induced AMPK activation and intracellular redistribution of KCNQ1 from the basolateral membrane in collecting duct principal cells. AICAR treatment also induced increased ubiquitination of KCNQ1 immunoprecipitated from kidney slice homogenates. These results indicate that AMPK inhibits KCNQ1 activity by promoting Nedd4-2-dependent channel ubiquitination and retrieval from the plasma membrane.
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PMID:AMP-activated protein kinase inhibits KCNQ1 channels through regulation of the ubiquitin ligase Nedd4-2 in renal epithelial cells. 2086 Oct 72

Regulation of renal Na(+) transport is essential for controlling blood pressure, as well as Na(+) and K(+) homeostasis. Aldosterone stimulates Na(+) reabsorption by the Na(+)-Cl(-) cotransporter (NCC) in the distal convoluted tubule (DCT) and by the epithelial Na(+) channel (ENaC) in the late DCT, connecting tubule, and collecting duct. Aldosterone increases ENaC expression by inhibiting the channel's ubiquitylation and degradation; aldosterone promotes serum-glucocorticoid-regulated kinase SGK1-mediated phosphorylation of the ubiquitin-protein ligase Nedd4-2 on serine 328, which prevents the Nedd4-2/ENaC interaction. It is important to note that aldosterone increases NCC protein expression by an unknown post-translational mechanism. Here, we present evidence that Nedd4-2 coimmunoprecipitated with NCC and stimulated NCC ubiquitylation at the surface of transfected HEK293 cells. In Xenopus laevis oocytes, coexpression of NCC with wild-type Nedd4-2, but not its catalytically inactive mutant, strongly decreased NCC activity and surface expression. SGK1 prevented this inhibition in a kinase-dependent manner. Furthermore, deficiency of Nedd4-2 in the renal tubules of mice and in cultured mDCT(15) cells upregulated NCC. In contrast to ENaC, Nedd4-2-mediated inhibition of NCC did not require the PY-like motif of NCC. Moreover, the mutation of Nedd4-2 at either serine 328 or 222 did not affect SGK1 action, and mutation at both sites enhanced Nedd4-2 activity and abolished SGK1-dependent inhibition. Taken together, these results suggest that aldosterone modulates NCC protein expression via a pathway involving SGK1 and Nedd4-2 and provides an explanation for the well-known aldosterone-induced increase in NCC protein expression.
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PMID:Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway. 2185 80

The expression of the serum- and glucocorticoid-regulated kinase 1 (Sgk1) is induced by mineralocorticoids and, in turn, upregulates the renal epithelial Na(+) channel (ENaC). Total inactivation of Sgk1 has been associated with transient urinary Na(+) wasting with a low-Na(+) diet, while the aldosterone-mediated ENaC channel activation was unchanged in the collecting duct. Since Sgk1 is ubiquitously expressed, we aimed to study the role of renal Sgk1 and generated an inducible kidney-specific knockout (KO) mouse. We took advantage of the previously described TetOn/CreLoxP system, in which rtTA is under the control of the Pax8 promotor, allowing inducible inactivation of the floxed Sgk1 allele in the renal tubules (Sgk1fl/fl/Pax8/LC1 mice). We found that under a standard Na(+) diet, renal water and Na(+)/K(+) excretion had a tendency to be higher in doxycycline-treated Sgk1 KO mice compared with control mice. The impaired ability of Sgk1 KO mice to retain Na(+) increased significantly with a low-salt diet despite higher plasma aldosterone levels. On a low-Na(+) diet, the Sgk1 KO mice were also hyperkaliuric and lost body weight. This phenotype was accompanied by a decrease in systolic and diastolic blood pressure. At the protein level, we observed a reduction in phosphorylation of the ubiquitin protein-ligase Nedd4-2 and a decrease in the expression of the Na(+)-Cl(-)-cotransporter (NCC) and to a lesser extent of ENaC.
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PMID:Inducible kidney-specific Sgk1 knockout mice show a salt-losing phenotype. 2230 19

Sgk1 is a relatively unstable kinase that regulates epithelial Na(+) transport in the distal nephron of the kidney. We identified a 5' variant alternate transcript of human Sgk1 (Sgk1_v3) that is expressed in the connecting tubule and collecting duct, is regulated by aldosterone and insulin, and is predicted to encode an NH(2)-terminal variant Sgk1 isoform, Sgk1_i3. Sgk1_i3 contains a polybasic motif, KKR, in its NH(2) terminus that regulates ubiquitination and stability of the expressed protein in HEK293 cells. In Fisher rat thyroid, and mpkCCD(c14) cells, Sgk1_i3 had a significantly greater effect on Na(+) transport compared with Sgk1 and its stimulatory effect was dependent on the kinase domain. Sgk1_i3 increased the abundance of cleaved epithelial Na(+) channel (ENaC) subunits at the cell surface, which was inhibited by coexpression of Nedd4-2. Together, the data demonstrate that a renally expressed Sgk1 isoform, Sgk1_i3, shows improved stability, is regulated by insulin and aldosterone, and stimulates ENaC activity when heterologously expressed in collecting duct cells.
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PMID:A regulated NH2-terminal Sgk1 variant with enhanced function is expressed in the collecting duct. 2303 40

In the renal collecting duct, binding of AVP to the V2 receptor triggers signaling changes that regulate osmotic water transport. Short-term regulation of water transport is dependent on vasopressin-induced phosphorylation of aquaporin-2 (AQP2) at Ser256. The protein kinase that phosphorylates this site is not known. We use Bayes' theorem to rank all 521 rat protein kinases with regard to the likelihood of a role in Ser256 phosphorylation on the basis of prior data and new experimental data. First, prior probabilities were estimated from previous transcriptomic and proteomic profiling data, kinase substrate specificity data, and evidence for kinase regulation by vasopressin. This ranking was updated using new experimental data describing the effects of several small-molecule kinase inhibitors with known inhibitory spectra (H-89, KN-62, KN-93, and GSK-650394) on AQP2 phosphorylation at Ser256 in inner medullary collecting duct suspensions. The top-ranked kinase was Ca2+/calmodulin-dependent protein kinase II (CAMK2), followed by protein kinase A (PKA) and protein kinase B (AKT). Liquid chromatography-tandem mass spectrometry (LC-MS/MS)-based in vitro phosphorylation studies compared the ability of three highly ranked kinases to phosphorylate AQP2 and other inner medullary collecting duct proteins, PKA, CAMK2, and serum/glucocorticoid-regulated kinase (SGK). All three proved capable of phosphorylating AQP2 at Ser256, although CAMK2 and PKA were more potent than SGK. The in vitro phosphorylation experiments also identified candidate protein kinases for several additional phosphoproteins with likely roles in collecting duct regulation, including Nedd4-2, Map4k4, and 3-phosphoinositide-dependent protein kinase 1. We conclude that Bayes' theorem is an effective means of integrating data from multiple data sets in physiology.
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PMID:Use of LC-MS/MS and Bayes' theorem to identify protein kinases that phosphorylate aquaporin-2 at Ser256. 2508 63

The present study investigates the role of small G-proteins of the Ras family in the epidermal growth factor (EGF)-activated cellular signalling pathway that downregulates activity of the epithelial Na+ channel (ENaC). We found that H-Ras is a key component of this EGF-activated cellular signalling mechanism in M1 mouse collecting duct cells. Expression of a constitutively active H-Ras mutant inhibited the amiloride-sensitive current. The H-Ras-mediated signalling pathway that inhibits activity of ENaC involves c-Raf, and that the inhibitory effect of H-Ras on ENaC is abolished by the MEK1/2 inhibitor, PD98059. The inhibitory effect of H-Ras is not mediated by Nedd4-2, a ubiquitin protein ligase that regulates the abundance of ENaC at the cell surface membrane, or by a negative effect of H-Ras on proteolytic activation of the channel. The inhibitory effects of EGF and H-Ras on ENaC, however, were not observed in cells in which expression of caveolin-1 (Cav-1) had been knocked down by siRNA. These findings suggest that the inhibitory effect of EGF on ENaC-dependent Na+ absorption is mediated via the H-Ras/c-Raf, MEK/ERK signalling pathway, and that Cav-1 is an essential component of this EGF-activated signalling mechanism. Taken together with reports that mice expressing a constitutive mutant of H-Ras develop renal cysts, our findings suggest that H-Ras may play a key role in the regulation of renal ion transport and renal development.
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PMID:H-Ras mediates the inhibitory effect of epidermal growth factor on the epithelial Na+ channel. 2577 17

Adaptation of the organism to potassium (K⁺) deficiency requires precise coordination among organs involved in K⁺ homeostasis, including muscle, liver, and kidney. How the latter performs functional and molecular changes to ensure K⁺ retention is not well understood. Here, we investigated the role of ubiquitin-protein ligase NEDD4-2, which negatively regulates the epithelial sodium channel (ENaC), Na⁺/Cl^- cotransporter (NCC), and with no-lysine-kinase 1 (WNK1). After dietary K⁺ restriction for 2 weeks, compared with control littermates, inducible renal tubular NEDD4-2 knockout (Nedd4L^Pax8/LC1 ) mice exhibited severe hypokalemia and urinary K⁺ wasting. Notably, expression of the ROMK K⁺ channel did not change in the distal convoluted tubule and decreased slightly in the cortical/medullary collecting duct, whereas BK channel abundance increased in principal cells of the connecting tubule/collecting ducts. However, K⁺ restriction also enhanced ENaC expression in Nedd4L^Pax8/LC1 mice, and treatment with the ENaC inhibitor, benzamil, reversed excessive K⁺ wasting. Moreover, K⁺ restriction increased WNK1 and WNK4 expression and enhanced SPAK-mediated NCC phosphorylation in Nedd4L^Pax8/LC1 mice, with no change in total NCC. We propose a mechanism in which NEDD4-2 deficiency exacerbates hypokalemia during dietary K⁺ restriction primarily through direct upregulation of ENaC, whereas increased BK channel expression has a less significant role. These changes outweigh the compensatory antikaliuretic effects of diminished ROMK expression, increased NCC phosphorylation, and enhanced WNK pathway activity in the distal convoluted tubule. Thus, NEDD4-2 has a crucial role in K⁺ conservation through direct and indirect effects on ENaC, distal nephron K⁺ channels, and WNK signaling.
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PMID:Renal Tubular Ubiquitin-Protein Ligase NEDD4-2 Is Required for Renal Adaptation during Long-Term Potassium Depletion. 2828 84


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