Abstract P204: Epithelial Sodium Channel Stimulation by Glucose

Hypertension ◽  
2016 ◽  
Vol 68 (suppl_1) ◽  
Author(s):  
Parijat S Joy ◽  
Peter M. Snyder
Keyword(s):  
Diabetes Mellitus ◽  
Cell Surface ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Endocytic Pathway ◽  
Sodium Absorption ◽  
Dependent Manner ◽  
Elevated Glucose ◽  
Underlying Mechanisms

There is a link between diabetes mellitus and hypertension, but the underlying mechanisms are poorly understood. The epithelial Na + channel ENaC plays an important role in blood pressure control; ENaC mutations cause Liddle’s syndrome, an inherited form of hypertension. Previous work suggests that ENaC abundance is increased in diabetes mellitus, but the underlying mechanisms are unclear. Here we tested the effect of glucose on ENaC regulation. In Ussing chamber experiments using mouse kidney collecting duct cells (mCCD) and primary cultures of human lung epithelia, elevated glucose increased ENaC-mediated short-circuit current by 2-3 times in a dose-dependent manner from 100mg/dl to 400mg/dl of glucose. This was caused by an increase in ENaC abundance at the cell surface. We hypothesized that hyperglycemia might enhance ENaC cell surface abundance by altering activity of Nedd4-2, an E3 ubiquitin-protein ligase that binds to PY motifs within ENaC. Consistent with this hypothesis, we found that mutation of the PY motifs abolished ENaC stimulation by elevated glucose. Moreover, using a biotinylation assay, we found that elevated glucose (300 mg/dl) slowed ENaC endocytosis and reduced its degradation in the endocytic pathway. These changes in trafficking are explained by our finding that glucose reduced ENaC binding to Nedd4-2, and hence, reduced ENaC ubiquitination. O-GlcNAcylation plays a role in insulin signaling and glucose toxicity due to increased O-GlcNAcylation of target proteins. To test a role for O-GlcNAcylation in ENaC stimulation by glucose, we used 6-Diazo-5-oxo-l-norleucine (DON) to inhibit O-GlcNAcylation. DON abolished ENaC stimulation by elevated glucose. Using anti-O-GlcNAc antibody, we found that Nedd4-2 is a substrate for O-GlcNAcylation, and this modification was increased by elevated glucose. DON also reversed the reduction in binding of Nedd4-2 to ENaC at high glucose levels. Together, our data suggest a model in which hyperglycemia stimulates ENaC through O-GlcNAcylation of Nedd4-2, increasing ENaC abundance at cell surface thus increasing epithelial sodium absorption.

Modulation of aldosterone-induced stimulation of ENaC synthesis by changing the rate of apical Na+ entry

2001 ◽  
Vol 281 (4) ◽  
pp. F687-F692 ◽  
Author(s):  
Lisette Dijkink ◽  
Anita Hartog ◽  
Carel H. Van Os ◽  
René J. M. Bindels
Keyword(s):  
Feedback Inhibition ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Inhibitory Effect ◽  
Cortical Collecting Duct ◽  
Entry Rate ◽  
Protein Levels ◽  
Stimulation Of

Primary cultures of immunodissected rabbit connecting tubule and cortical collecting duct cells were used to investigate the effect of apical Na+ entry rate on aldosterone-induced transepithelial Na+ transport, which was measured as benzamil-sensitive short-circuit current ( I sc). Stimulation of the apical Na+ entry, by long-term short-circuiting of the monolayers, suppressed the aldosterone-stimulated benzamil-sensitive I sc from 320 ± 49 to 117 ± 14%, whereas in the presence of benzamil this inhibitory effect was not observed (335 ± 74%). Immunoprecipitation of [35S]methionine-labeled β-rabbit epithelial Na+ channel (rbENaC) revealed that the effects of modulation of apical Na+ entry on transepithelial Na+ transport are exactly mirrored by β-rbENaC protein levels, because short-circuiting the monolayers decreased aldosterone-induced β-rbENaC protein synthesis from 310 ± 51 to 56 ± 17%. Exposure to benzamil doubled the β-rbENaC protein level to 281 ± 68% in control cells but had no significant effect on aldosterone-stimulated β-rbENaC levels (282 ± 68%). In conclusion, stimulation of apical Na+ entry suppresses the aldosterone-induced increase in transepithelial Na+transport. This negative-feedback inhibition is reflected in a decrease in β-rbENaC synthesis or in an increase in β-rbENaC degradation.

Basolateral Na(+)-independent Cl(-)-HCO3- exchange in primary cultures of rat IMCD cells

1992 ◽  
Vol 263 (3) ◽  
pp. F401-F410 ◽  
Author(s):  
J. A. Kraut ◽  
D. Hart ◽  
E. P. Nord
Keyword(s):  
Steady State ◽  
Cell Surface ◽  
Primary Culture ◽  
Anion Exchange ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Disulfonic Acid ◽  
Absolute Requirement ◽  
Ethanesulfonic Acid ◽  
Base Load

The role of anion exchange in the regulation of intracellular pH (pHi) under base load and steady-state conditions was investigated in confluent monolayers of rat inner medullary collecting duct (IMCD) cells in primary culture using the pH-sensitive fluoroprobe 2,7-bis(carboxyethyl)-5(6')-carboxyfluorescein (BCECF). Recovery of pHi after imposition of a base load induced either by replacement of HCO3-/CO2 by N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) at the same extracellular pH (pHo) or deletion of Cl- from a HCO3-/CO2-buffered solution had an absolute requirement for Cl-, was Na+ independent, and was inhibited approximately 90% by 50 microM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). When pHo was decreased by lowering HCO3- concentration in the constant presence of 5% CO2, the rate of decrement in pHi was significantly blunted in the absence of Cl-. Imposition of a positive or negative diffusion potential of equal but opposite magnitude did not modify the anion exchange rate, confirming the electroneutrality of the process. Under steady-state conditions, pHi of cells bathed in a HCO3-/CO2-buffered solution was 7.33 +/- 0.06, significantly lower than that of cells bathed in a nominally HCO3-/CO2-free buffer (7.50 +/- 0.04), indicating that under physiological conditions the pathway functions as a base extruder. In studies performed on cells grown on permeable supports, the anion exchange pathway was found to be confined exclusively to the basolateral-equivalent cell surface. In summary, confluent monolayers of rat IMCD cells in primary culture possess a Na(+)-independent, DIDS-inhibitable electroneutral Cl(-)-HCO3- exchange pathway that is confined to the basolateral cell surface. The transporter is an important determinant of steady-state pHi and is the predominant mechanism whereby the cell recovers from imposed elevations in pHi.

Characteristics of papillary collecting duct cells in primary culture

1988 ◽  
Vol 255 (6) ◽  
pp. F1160-F1169 ◽  
Author(s):  
R. F. Husted ◽  
M. Hayashi ◽  
J. B. Stokes
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Calf Serum ◽  
Morphological Characteristics ◽  
Short Circuit Current ◽  
Papillary Collecting Duct ◽  
Collecting Duct Cells ◽  
Na Uptake

We examined the electrophysiological and Na+ transport characteristics of rat papillary collecting duct (PCD) cells grown in primary cultures. Grown as monolayers on polycarbonate filters, the cells displayed similar morphological characteristics to native epithelia. They also bound Dolichus biflorus lectin, a property shared by native cells. Monolayers developed a peak electrical resistance of 100-200 omega.cm2 and a transmonolayer voltage of less than 2 mV. Similar values were measured in the perfused, native PCD of the same species as well as PCD cells cultured from rabbit and bovine kidneys. Hamster cells did not readily develop confluent monolayers under the same conditions. Exposure of the cultured cells to 10% fetal calf serum for 24 h caused the Na+ uptake across the apical membrane to double, an effect not reproduced by indomethacin, insulin, vasopressin, aldosterone, dexamethasone, or hexamethylene bisacetamide (an inducer of differentiation). Amiloride (1 mM) inhibited Na+ uptake by 50-80%. The measured short-circuit current did not correlate with Na+ uptake and was clearly dissociated by exposure to serum. The results suggest that there is more than one mechanism of ion transport by the rat PCD.

CFTR disruption impairs cAMP-dependent Cl−secretion in primary cultures of mouse cortical collecting ducts

2001 ◽  
Vol 281 (3) ◽  
pp. F434-F442 ◽  
Author(s):  
Marcelle Bens ◽  
Jean-Paul Duong Van Huyen ◽  
Françoise Cluzeaud ◽  
Jacques Teulon ◽  
Alain Vandewalle
Keyword(s):  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Similar Degree ◽  
Transmembrane Conductance Regulator ◽  
Cortical Collecting Duct ◽  
Transmembrane Conductance ◽  
Nacl Transport ◽  
Cystic Fibrosis Transmembrane

The role of the cystic fibrosis transmembrane conductance regulator (CFTR) in the renal cortical collecting duct (CCD) has not yet been fully elucidated. Here, we investigated the effects of deamino-8-d-arginine vasopressin (dDAVP) and isoproterenol (ISO) on NaCl transport in primary cultured CCDs microdissected from normal [CFTR(+/+)] and CFTR-knockout [CFTR(−/−)] mice. dDAVP stimulated the benzamyl amiloride (BAm)-sensitive transport of Na+ assessed by the short-circuit current ( I sc) method in both CFTR(+/+) and CFTR(−/−) CCDs to a very similar degree. Apical addition of 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) or glibenclamide partially inhibited the rise in I sc induced by dDAVP and ISO in BAm-treated CFTR(+/+) CCDs, whereas dDAVP, ISO, and NPPB did not alter I sc in BAm-treated CFTR(−/−) CCDs. dDAVP stimulated the apical-to-basal flux and, to a lesser extent, the basal-to-apical flux of 36Cl− in CFTR(+/+) CCDs. dDAVP also increased the apical-to-basal36Cl− flux in CFTR(−/−) CCDs but not the basal-to-apical 36Cl− flux. These results demonstrate that CFTR mediates the cAMP-stimulated component of secreted Cl− in mouse CCD.

AVP stimulates Na+ transport in primary cultures of rabbit cortical collecting duct cells

1992 ◽  
Vol 262 (3) ◽  
pp. F454-F461 ◽  
Author(s):  
C. M. Canessa ◽  
J. A. Schafer
Keyword(s):  
Cultured Cells ◽  
Collecting Duct ◽  
Short Circuit ◽  
Primary Cultures ◽  
Short Circuit Current ◽  
Cortical Collecting Duct ◽  
Na Transport ◽  
Permeable Membrane ◽  
Ussing Chambers ◽  
Principal And Intercalated Cells

Immunodissected rabbit cortical collecting duct (CCD) cells were grown in primary culture on permeable membrane supports. Transepithelial voltage, Na+, K+, and H+ gradients developed as expected for a mixed population of principal and intercalated cells. The amiloride-sensitive short-circuit current (Isc) was measured in Ussing chambers as an index of Na+ transport via apical membrane Na+ channels. Treatment of the cells in culture with 10 nM aldosterone for 48 h increased Isc from 7.4 +/- 1.4 to 19.3 +/- 3.2 microA/cm2. In contrast to the native rabbit CCD, 220 pM arginine vasopressin (AVP) produced a rapid and stable (greater than 60 min) increase in Isc to 15.8 +/- 2.0 and 29.0 +/- 3.8 microA/cm2 in untreated and aldosterone-treated cultures, respectively. Although prostaglandin E2 (PGE2) inhibits Na+ transport in the native rabbit CCD, it did not in the cultured cells, and it has previously been shown that PGE2 inhibition of AVP-dependent adenosine 3',5'-cyclic monophosphate production is lost in culture (W. K. Sonnenburg and W. L. Smith, J. Biol. Chem. 263: 6155-6160, 1988). We conclude that the development of a stable stimulation of Na+ transport by AVP is linked to the loss of the inhibitory effects of PGE2.

Decreased amiloride-sensitive Na+ absorption in collecting duct principal cells isolated from BPK ARPKD mice

2004 ◽  
Vol 286 (2) ◽  
pp. F244-F254 ◽  
Author(s):  
Elias I. Veizis ◽  
Cathleen R. Carlin ◽  
Calvin U. Cotton
Keyword(s):  
Fluorescent Protein ◽  
Kidney Diseases ◽  
Collecting Duct ◽  
Short Circuit ◽  
Expression Patterns ◽  
Renal Cysts ◽  
Short Circuit Current ◽  
Sodium Absorption ◽  
Principal Cells ◽  
Green Fluorescent

The main feature of polycystic kidney diseases (PKD) is formation and progressive enlargement of renal cysts. Alterations in epithelial cell proliferation, extracellular matrix, and ion transport are thought to contribute to cyst enlargement and loss of renal function. Abnormal Cl- secretion is implicated in cyst enlargement in autosomal dominant PKD (ADPKD), but little is known about transport abnormalities in autosomal recessive PKD (ARPKD). We developed a method to isolate collecting duct (CD) principal cells (site of the lesion in ARPKD) from normal and ARPKD mice. A transgenic mouse (Hoxb7/GFP) in which enhanced green fluorescent protein (GFP) is expressed in CDs was bred with an ARPKD mouse (BPK), and GFP-positive cells from normal and cystic mice were selected by fluorescence-activated cell sorting. GFP-positive CD cells (>95 ± 3%) obtained from either normal or cystic mice formed high-resistance, polarized epithelial monolayers. Expression patterns for marker proteins and the presence of a central cilium confirmed that the monolayers are composed of principal cells. Under basal conditions, the Cl- secretory responses elicited by elevation of cAMP or calcium were not significantly different between normal and cystic monolayers. In contrast, the amiloride-sensitive short-circuit current was significantly reduced in monolayers of cells isolated from cystic mice (12.9 ± 1.6 μA/cm2; n = 10) compared with monolayers of cells isolated from normal mice (27.3 ± 3.4 μA/cm2; n = 12). The results of these studies suggest that epithelial sodium channel-mediated sodium absorption is decreased in principal cells of ARPKD CD cysts and that the reduction in sodium absorption may contribute to the accumulation of luminal fluid.

scholarly journals Epithelial Na+ channel mutants causing Liddle's syndrome retain ability to respond to aldosterone and vasopressin

2003 ◽  
Vol 285 (3) ◽  
pp. F459-F471 ◽  
Author(s):  
Muriel Auberson ◽  
Nicole Hoffmann-Pochon ◽  
A. Vandewalle ◽  
Stephan Kellenberger ◽  
Laurent Schild
Keyword(s):  
Cell Surface ◽  
Collecting Duct ◽  
Short Circuit ◽  
Critical Role ◽  
Na Channel ◽  
Cortical Collecting Duct ◽  
Liddle’S Syndrome ◽  
Liddle's Syndrome ◽  
Py Motif ◽  
In Cells

Liddle's syndrome is a monogenic form of hypertension caused by mutations in the PY motif of the COOH terminus of β- and γ-epithelial Na+ channel (ENaC) subunits. These mutations lead to retention of active channels at the cell surface. Because of the critical role of this PY motif in the stability of ENaCs at the cell surface, we have investigated its contribution to the ENaC response to aldosterone and vasopressin. Mutants of the PY motif in β- and γ-ENaC subunits (β-Y618A, β-P616L, β-R564stop, and γ-K570stop) were stably expressed by retroviral gene transfer in a renal cortical collecting duct cell line (mpkCCDcl4), and transepithelial Na+ transport was assessed by measurements of the benzamil-sensitive short-circuit current ( Isc). Cells that express ENaC mutants of the PY motif showed a five- to sixfold higher basal Isc compared with control cells and responded to stimulation by aldosterone (10-6 M) or vasopressin (10-9 M) with a further increase in Isc. The rates of the initial increases in Isc after aldosterone or vasopressin stimulation were comparable in cells transduced with wild-type and mutant ENaCs, but reversal of the effects of aldosterone and vasopressin was slower in cells that expressed the ENaC mutants. The conserved sensitivity of ENaC mutants to stimulation by aldosterone and vasopressin together with the prolonged activity at the cell surface likely contribute to the increased Na+ absorption in the distal nephron of patients with Liddle's syndrome.

Hyperosmolality stimulates Na-K-ATPase gene expression in inner medullary collecting duct cells

1996 ◽  
Vol 270 (5) ◽  
pp. F728-F738 ◽  
Author(s):  
A. Ohtaka ◽  
S. Muto ◽  
J. Nemoto ◽  
K. Kawakami ◽  
K. Nagano ◽  
...  
Keyword(s):  
Atpase Activity ◽  
Collecting Duct ◽  
Primary Cultures ◽  
Protein Synthesis Inhibitor ◽  
Dependent Manner ◽  
Mrna Accumulation ◽  
Inner Medullary Collecting Duct ◽  
Subunit Mrna ◽  
Atpase Gene ◽  
Medullary Collecting Duct

Primary cultures of inner medullary collecting duct (IMCD) cells of rats were incubated in hyperosmotic media to determine the effects on Na-K-ATPase alpha 1- and beta 1-subunit mRNA expression. Osmolality of the incubation media was raised from 300 up to 500 mosmol/kgH2O by adding NaCl, mannitol, raffinose, or urea. Hyperosmotic media supplemented with NaCl, mannitol, or raffinose caused two- to fourfold increases in the alpha 1-subunit mRNA accumulation and five- to eightfold increases in the beta 1-subunit mRNA accumulation, with peak elevations of both subunits at 12 h after addition. In sharp contrast, hyperosmolar urea medium had no effect at any time. When NaCl or mannitol was added to the media in amounts ranging from 300 to 600 mosmol/kgH2O, the maximal effects on both alpha 1- and beta 1-subunit mRNA accumulation occurred at 500 mosmol/kgH2O. In urea-supplemented medium, however, there was no significant change at any level of osmolality. The upregulation of alpha 1- and beta 1-subunit mRNA induced by hyperosmotic mannitol- or raffinose-supplemented media was markedly inhibited by removal of Na from the culture medium. Furthermore, pretreatment with a protein synthesis inhibitor cycloheximide partially inhibited the upregulation of alpha 1- and beta 1-subunit mRNA in IMCD cells exposed to hyperosmotic media treated with NaCl or mannitol. When IMCD cells were incubated with hyperosmotic media (500 mosmol/kgH2O) supplemented with NaCl or mannitol for 24 h, Na-K-ATPase activity increased by 78.6 and 82.8%, respectively. In contrast, hyperosmolar urea medium had no significant effect on Na-K-ATPase activity. These results demonstrate that 1) hyperosmolality induced by the poorly permeating solutes (NaCl, mannitol, and raffinose) but not the rapidly permeating solute (urea) stimulates both alpha 1- and beta 1-subunit mRNA accumulations in IMCD cells in a time- and an osmolality-dependent manner, 2) the hyperosmolality-induced upregulation of alpha 1- and beta 1-subunit mRNA leads to an increase in Na- K -ATPase activity; and 3) the above upregulation of alpha1- and beta 1-subunit mRNA in response to hyperosmotic media requires, at least in part, the presence of Na in the extracellular medium and the de novo synthesis of intermediate proteins.

Peptide YY inhibits vasopressin-stimulated chloride secretion in inner medullary collecting duct cells

1998 ◽  
Vol 275 (3) ◽  
pp. F452-F457
Author(s):  
Christopher M. Breen ◽  
Peter J. Mannon ◽  
Bruce A. Benjamin
Keyword(s):  
Peptide Yy ◽  
Collecting Duct ◽  
Short Circuit ◽  
Chloride Secretion ◽  
Chloride Current ◽  
Sodium Absorption ◽  
Receptor Subtype ◽  
Apical Surface ◽  
Inner Medullary Collecting Duct ◽  
Medullary Collecting Duct

mIMCD-k2 cells are derived from the inner medullary collecting duct of a mouse and exhibit electrogenic sodium absorption and cAMP- and vasopressin (AVP)-stimulated electrogenic chloride secretion [N. L. Kizer, B. Lewis, and B. A. Stanton. Am. J. Physiol. 268 ( Renal Fluid Electrolyte Physiol. 37): F347–F355, 1995; and N. L. Kizer, D. Vandorpe, B. Lewis, B. Bunting, J. Russell, and B. A. Stanton. Am. J. Physiol. 268 ( Renal Fluid Electrolyte Physiol. 37): F854–F861, 1995]. The purpose of the present study was to determine how peptide YY (PYY) affects electrogenic Na+ and Cl− current in mIMCD-k2 cells. Short-circuit currents ( I sc) were measured across monolayers of mIMCD-k2 cells mounted in Ussing-type chambers. PYY did not alter baseline I sc, nor did it alter I sc in chloride-free conditions, indicating no effect on electrogenic sodium transport. Baseline chloride current in these cells is low; therefore, chloride short-circuit current[Formula: see text] was stimulated with AVP (10 nM) added to the basolateral surface and 10 μM amiloride added to the apical surface. Although apical applications of PYY had no effect, basolateral application of PYY caused attenuation of[Formula: see text], with the maximal inhibitory dose (100 nM) causing 52 ± 1.3% inhibition (IC50 = 0.11 nM). Inhibition by PYY of [Formula: see text] is mediated through the Y2 receptor subtype, as PYY-(3–36) was the only PYY analog tested that caused inhibition and was equipotent to PYY. Inhibition by PYY of[Formula: see text] was abolished following incubation with pertussis toxin. We also show that PYY inhibits AVP-stimulated cAMP accumulation, with a maximal inhibitory dose (100 nM) causing a 38% ± 6% inhibition (IC50 = 0.16 nM), comparable to inhibition by PYY of [Formula: see text]. We conclude that PYY acts through either Gi or Go to inhibit adenylate cyclase activity, leading to a decrease in AVP-stimulated chloride current.

scholarly journals A primary culture of mouse proximal tubular cells, established on collagen-coated membranes

2007 ◽  
Vol 293 (2) ◽  
pp. F476-F485 ◽  
Author(s):  
Sara Terryn ◽  
François Jouret ◽  
Frank Vandenabeele ◽  
Inge Smolders ◽  
Marjan Moreels ◽  
...  
Keyword(s):  
Proximal Tubule ◽  
Short Circuit ◽  
Primary Cultures ◽  
Endocytic Pathway ◽  
Simple Method ◽  
Proximal Tubule Cells ◽  
Apical Side ◽  
Microscopic Evaluation ◽  
Tubule Cells ◽  
Glutamyl Transferase

A simple method is described to establish primary cultures of kidney proximal tubule cells (PTC) on membranes. The permeable membranes represent a unique culture surface, allowing a high degree of differentiation since both apical and basolateral membranes are accessible for medium. Proximal tubule (PT) segments from collagenase-digested mouse renal cortices were grown for 7 days, by which time cells were organized as a confluent monolayer. Electron microscopic evaluation revealed structurally polarized epithelial cells with numerous microvilli, basolateral invaginations, and apical tight junctions. Immunoblotting for markers of distinct parts of the nephron demonstrated that these primary cultures only expressed PT-specific proteins. Moreover immunodetection of distinct components of the receptor-mediated endocytic pathway and uptake of FITC-albumin indicated that these cells expressed a functional endocytotic apparatus. In addition, primary cultures possessed the PT brush-border enzymes, alkaline phosphatase, and γ-glutamyl-transferase, and a phloridzin-sensitive sodium-dependent glucose transport at their apical side. Electrophysiological measurements show that the primary cultured cells have a low transepithelial resistance and high short-circuit current that was completely carried by Na+ similar to a leaky epithelium like proximal tubule cells. This novel method established well-differentiated PTC cultures.

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