Stimulation of the epithelial sodium channel (ENaC) by the serum- and glucocorticoid-inducible kinase (Sgk) involves the PY motifs of the channel but is independent of sodium feedback inhibition

The epithelial sodium channel (ENaC) is the major mediator of sodium transport across the apical membranes of the distal nephron, the distal colon, the respiratory tract and the ducts of exocrine glands. It is subject to feedback inhibition by increased intracellular Na⁺, a regulatory system wherein the ubiquitin protein ligases, Nedd4 and Nedd4-2, bind to conserved PY motifs in the C-termini of ENaC and inactivate the channel. It has been proposed recently that the kinase Sgk activates the channel as a consequence of phosphorylating Nedd4-2, thus preventing it from inhibiting the channels. This proposal predicts that Sgk should interfere with Na⁺ feedback regulation of ENaC. We have tested this prediction in Xenopus laevis oocytes and in mouse salivary duct cells and found that in neither system did increased activity of Sgk interrupt Na⁺ feedback inhibition of ENaC. We found, however, that Sgk stimulation was largely abolished in oocytes expressing ENaC channels with C-terminal truncations or mutated PY motifs. We were also unable to confirm that Sgk directly interacts with Nedd4-2 in vitro. We conclude that the stimulatory effect of Sgk on ENaC requires the presence of the channel’s PY motifs, but it is not due to the interruption of Na⁺ feedback regulation.Robert Rauh and Anuwat Dinudom cotributed equally to this work.     

Firewall function of the endothelial glycocalyx in the regulation of sodium homeostasis

Plasma sodium, slightly above normal and in presence of aldosterone, stiffens vascular endothelium and reduces nitric oxide release with the consequence of endothelial dysfunction. This process is mediated by epithelial sodium channels (ENaC) and, most likely, the endothelial Na⁺/K⁺-ATPase. Both, ENaC and Na⁺/K⁺-ATPase, are located in the plasma membrane of endothelial cells and embedded in the endothelial glycocalyx (eGC). This negatively charged biopolymer is directly exposed to the blood stream and selectively buffers sodium ions. We hypothesize that the glycocalyx could interfere with endothelial sodium transport when extracellular sodium varies in the physiological range. Therefore, we modeled the endothelial cell as a pump–leak system measuring changes of intracellular sodium in cultured human endothelial cells. Experiments were performed under low/high extracellular sodium conditions before and after enzymatic eGC removal, and with inhibition of Na⁺/K⁺-ATPase and ENaC, respectively. Three major observations were made: (1) eGC removal by heparinase treatment facilitates sodium to enter/exit the endothelial cells. (2) The direction of net sodium movement across the endothelial plasma membrane depends on the concentration of extracellular sodium which regulates both the Na⁺/K⁺-ATPase and ENaC activity. (3) Removal of eGC and inhibition of sodium transport modify the electrical resistance of endothelial cells. We conclude that the eGC serves as a potential “firewall” preventing uncontrolled access of sodium to the pump–leak system of the endothelial cell. After eGC removal, sodium access to the system is facilitated. Thus the pump–leak system could be regulated by ambient sodium and control vascular permeability in pathophysiological conditions.     

Regulation of Na+ excretion and arterial blood pressure by purinergic signalling intrinsic to the distal nephron: consequences and mechanisms

Discretionary control of Na⁺ excretion is a key component of the regulation of arterial blood pressure in mammals. Sodium excretion is fine‐tuned in the aldosterone‐sensitive distal nephron by the activity of the epithelial Na⁺ channel (ENaC). Here, ENaC functions as a final effector of the renin–angiotensin–aldosterone system (RAAS) during negative feedback control of blood pressure. Mutations affecting ENaC activity and abnormal regulation of this channel affect blood pressure through pathological changes to Na⁺ excretion. Recent evidence demonstrates that powerful signalling pathways function in parallel with the RAAS to modulate ENaC activity and blood pressure. An inclusive paradigm is emerging with respect to regulation of blood pressure where ENaC serves as a critical point of convergence for several important signalling systems that affect renal Na⁺ excretion. A robust inhibitory purinergic signalling system intrinsic to the distal nephron dynamically regulates ENaC through paracrine ATP signalling via the metabotropic P2Y₂ purinergic receptor to properly match urinary Na⁺ excretion to dietary Na⁺ intake. This enables blood pressure to be maintained within a normal range despite broad changes in dietary Na⁺ consumption. Loss of purinergic inhibition of ENaC increases blood pressure by causing inappropriate Na⁺ excretion. In contrast, stimulation of the P2Y₂ receptor promotes natriuresis and a decrease in blood pressure. Such observations identify purinergic signalling in the distal nephron as possibly causative, when dysfunctional, for certain forms of elevated blood pressure, and as a possible therapeutic target for the treatment of elevated blood pressure particularly that associated with salt sensitivity.     

Regulation of NaCl transport in the renal collecting duct: lessons from cultured cells

The fine control of NaCl absorption regulated by hormones takes place in the distal nephron of the kidney. In collecting duct principal cells, the epithelial sodium channel (ENaC) mediates the apical entry of Na⁺, which is extruded by the basolateral Na⁺,K⁺-ATPase. Simian virus 40-transformed and “transimmortalized” collecting duct cell lines, derived from transgenic mice carrying a constitutive, conditionally, or tissue-specific promoter-regulated large T antigen, have been proven to be valuable tools for studying the mechanisms controlling the cell surface expression and trafficking of ENaC and Na⁺,K⁺-ATPase. These cell lines have made it possible to identify sets of aldosterone- and vasopressin-stimulated proteins, and have provided new insights into the concerted mechanism of action of serum- and glucocorticoid-inducible kinase 1 (Sgk1), ubiquitin ligase Nedd4-2 (neural precursor cell-expressed, developmentally down-regulated protein 4-2), and 14-3-3 regulatory proteins in modulating ENaC-mediated Na⁺ currents. Epidermal growth factor and induced leucine zipper protein have also been shown to repress and stimulate ENaC-dependent Na⁺ absorption, respectively, by activating or repressing the mitogen-activated protein kinase externally regulated kinase_1/2. Overall, these findings have provided evidence suggesting that multiple pathways are involved in regulating NaCl absorption in the distal nephron.     

The second sodium pump: from the function to the gene

Transepithelial Na⁺ transport is mediated by passive Na⁺ entry across the luminal membrane and exit through the basolateral membrane by two active mechanisms: the Na⁺/K⁺ pump and the second sodium pump. These processes are associated with the ouabain-sensitive Na⁺/K⁺-ATPase and the ouabain-insensitive, furosemide-inhibitable Na⁺-ATPase, respectively. Over the last 40?years, the second sodium pump has not been successfully associated with any particular membrane protein. Recently, however, purification and cloning of intestinal α-subunit of the Na⁺-ATPase from guinea pig allowed us to define it as a unique biochemical and molecular entity. The Na⁺- and Na⁺/K⁺-ATPase genes are at the same locus, atp1a1, but have independent promoters and some different exons. Herein, we spotlight the functional characteristics of the second sodium pump, and the associated Na⁺-ATPase, in the context of its role in transepithelial transport and its response to a variety of physiological and pathophysiological conditions. Identification of the Na⁺-ATPase gene (atna) allowed us, using a bioinformatics approach, to explore the tertiary structure of the protein in relation to other P-type ATPases and to predict regulatory sites in the promoter region. Potential regulatory sites linked to inflammation and cellular stress were identified in the atna gene. In addition, a human atna ortholog was recognized. Finally, experimental data obtained using spontaneously hypertensive rats suggest that the Na⁺-ATPase could play a role in the pathogenesis of essential hypertension. Thus, the participation of the second sodium pump in transepithelial Na⁺ transport and cellular Na⁺ homeostasis leads us to reconsider its role in health and disease.     

Amiloride-sensitive sodium transport of the rat distal colon during early postnatal development

To evaluate developmental changes in colonic sodium transport, the sensitivity of the transepithelial potential and short-circuit current to amiloride was investigated. The amiloride-sensitive short-circuit current (I _sc ^Na ), which represents the electrogenic sodium transport through Na⁺ channels, rose significantly from day 5, reached a peak on day 10, and entirely disappeared after weaning. The maximum rate of electrogenic, amiloride-sensitive sodium transport was 12.0 Eq/cm² · h. TheI _sc ^Na was suppressed by adrenalectomy and/or premature weaning but not by a mineralocorticoid antagonist, spironolactone. On the contrary, treatments which increase aldosterone levels in vivo (low-sodium diet, furosemide-induced natriuresis, high dietary intake of potassium) stimulated theI _sc ^Na . The effect of adrenalectomy increased during postnatal development. The sensitivity ofI _sc ^Na to aldosterone was highest at the end of the weaning period. High-sodium diet, which causes a decrease in circulating aldosterone, was associated with a partial inhibition ofI _sc ^Na (P<0.016). These data suggest that the distal colon of neonatal rats can transport sodium via an electrogenic, amiloride-sensitive mechanism and that adrenocortical hormones exert the main regulatory control of this pathway.Parts of this study have been presented at the 6th Symposium on Developmental Pharmacology, Schloss Reinhardsbrunn 1986     

Cellular heterogeneity of the distal nephron and its relation to function

Summary The distal segments beyond the macula densa — distal convoluted tubule, connecting tubule, cortical collecting duct — display cellular heterogeneity. The four different cell types, namely the DCT cell, CNT cell, the principal cell and intercalated cell differ mainly by the pattern of membrane amplification and they reveal also qualitative differences as to some cytoplasmic proteins.Each of the four cell types adapts to chronic changes in electrolyte metabolism with structural alteration, concerning essentially the membrane area over which the active transport step of the cell proceeds, in DCT-, CNT- and P-cells the basolateral cell membrane with the Na-K-ATPase, in intercalated cells the luminal cell membrane with a H⁺ ATPase. Since each cell type responds only to specific conditions with changes in membrane area and associated transcellular transport activity, morphological studies can help to determine the specific role of each cell type in the regulation of renal electrolyte excretion. Such investigations demonstrated that besides mineralocorticoid hormones the transport capacity of certain cells should depend on the solute composition of tubular fluid. Thus, changes in the transport pattern specifically induced in only one segment alters also the transport patterns of segments downstream. Cellular heterogeneity seems to guarantee the optimal regulation of renal electrolyte excretion.     

Extracellular Na+ removal attenuates rundown of the epithelial Na+-channel (ENaC) by reducing the rate of channel retrieval

Regulation of the epithelial sodium channel (ENaC) is important for the long-term control of arterial blood pressure as evidenced by gain of function mutations of ENaC causing Liddles syndrome, a rare form of hereditary arterial hypertension. In Xenopus laevis oocytes expressing ENaC a spontaneous decline of ENaC currents over time, so-called rundown, is commonly observed. Mechanisms involved in rundown may be physiologically relevant and may be related to feedback regulation of ENaC by intra- or extracellular Na⁺. We tested the effect of extracellular Na⁺ removal on ENaC rundown. Spontaneous rundown of ENaC was largely prevented by extracellular Na⁺ removal and was partially prevented by primaquine suggesting that it is due to endocytic channel retrieval. Liddles syndrome mutation caused a reduced rate of rundown, and in oocytes expressing the mutated channel extracellular Na⁺ removal not only prevented rundown but even increased the ENaC currents (runup). Acute exposure to high extracellular Na⁺ drastically reduced whole-cell currents and surface expression of wild-type ENaC, while these effects were much smaller in ENaC with Liddles syndrome mutation consistent with a stabilization of the mutated channel in the plasma membrane. Interestingly, the apparent intracellular Na⁺ concentration [Na⁺]_i-app was high (>60 mM) in ENaC-expressing oocytes but rundown was not associated with a further increase in [Na⁺]_i-app. We conclude that the inhibitory effect of extracellular Na⁺ removal on rundown is due to an inhibition of endocytic ENaC retrieval.     

The effects of adenosine on transepithelial resistance and sodium uptake in the inner medullary collecting duct

It has been previously demonstrated that adenosine induces natriuresis when administered directly into the renal circulation of the rat. It was postulated that the mechanism was inhibition of tubule Na⁺ reabsorption. In the current study, the hypothesis was tested that adenosine inhibits ion reabsorption across the inner medullary collecting duct (IMCD), a tubule segment which is rich in adenosine receptors. IMCD epithelium from rat kidney was grown in primary culture as a confluent monolayer on Costar filters, allowing selective access to the basolateral and apical surfaces of the cells. Transepithelial resistance was taken as a measure of epithelial permeability and ion conductance. Na⁺ uptake was studied using ²²Na⁺ and used to determine the permeability of the epithelial monolayer specifically to Na⁺. Exposure of the basolateral aspect of the monolayer to adenosine (10⁻⁸–10⁻⁷ M) increased transepithelial resistance in a dose- and time-dependent manner; in parallel, adenosine (10⁻⁷–10⁻⁶ M) reduced apical Na⁺ uptake from 20±5 to 10±2 nmol/cm². 1,3-Dipropyl-8-(2-amino-4-chlorophenyl)-xanthine (PACPX, 5×10⁻⁹ M), an adenosine antagonist with selectivity for the A₁ receptor, inhibited the rise in transepithelial resistance and the decrease in Na⁺ uptake following the addition of adenosine. The effects of adenosine on transepithelial resistance were reproduced with the A₁ receptor selective adenosine analogue N ⁶-cyclohexyladenosine (CHA, 10⁻⁸ –10⁻⁷ M) but not with the A₂ selective analogues, 5′-N-ethylcarboxamidoadenosine (NECA) or CGS 21680. CHA (10⁻⁷ M) inhibited apical Na⁺ uptake by 50%, an effect abolished by PACPX. The effects of adenosine on transepithelial resistance and Na⁺ uptake were inhibited, but only in part, by amiloride. These data suggest that adenosine inhibits ion movement, specifically apical Na⁺ uptake, across the IMCD epithelium and that this effect is mediated by A₁ receptors from the basolateral aspect of the cell. The results are consistent with the hypothesis that adenosine inhibits Na⁺ reabsorption across the IMCD.     

PPARγ agonists do not directly enhance basal or insulin-stimulated Na+ transport via the epithelial Na+ channel

Selective agonists of peroxisome proliferator-activated receptor gamma (PPAR) are anti-diabetic drugs that enhance cellular responsiveness to insulin. However, in some patients, fluid retention, plasma volume expansion, and edema have been observed. It is well established that insulin regulates Na⁺ reabsorption via the epithelial sodium channel (ENaC) located in the distal tubule. Therefore, we hypothesized that these agonists may positively modulate insulin-stimulated ENaC activity leading to increased Na⁺ reabsorption and fluid retention. Using electrophysiological techniques, dose–response curves for insulin-mediated Na⁺ transport in the A6, M-1, and mpkCCD_cl4 cell lines were performed. Each line demonstrated hormone efficacy within physiological concentration ranges and, therefore, can be used to monitor clinically relevant effects of pharmacological agents which may affect electrolyte transport. Immunodetection and quantitative PCR analyses showed that each cell line expresses viable and functional PPAR receptors. Despite this finding, two PPAR agonists, pioglitazone and GW7845 did not directly enhance basal or insulin-stimulated Na⁺ flux via ENaC, as shown by electrophysiological methodologies. These studies provide important results, which eliminate insulin-mediated ENaC activation as a candidate mechanism underlying the fluid retention observed with PPAR agonist use.     

SGK1 increases Na,K-ATP cell-surface expression and function in Xenopus laevis oocytes

The Na⁺-retaining hormone aldosterone increases the cell-surface expression of the luminal epithelial sodium channel (ENaC) and the basolateral Na⁺ pump (Na,K-ATPase) in aldosterone-sensitive distal nephron cells in a coordinated fashion. To address the question of whether aldosterone-induced serum and glucocorticoid-regulated kinase-1 (SGK1) might be involved in mediating this regulation of Na,K-ATPase subcellular localization, similar to that of the epithelial Na⁺ channel (ENaC), we co-expressed the Na,K-ATPase (rat 1- and Xenopus laevis 1-subunits) and Xenopus SGK1 in Xenopus oocytes. Measurements of the Na⁺ pump current showed that wild-type SGK1 increases the function of exogenous Na,K-ATPase at the surface of Xenopus oocytes. This appeared to be secondary to an increase in Na,K-ATPase cell-surface expression as visualized by Western blotting of surface-biotinylated proteins. In contrast, the functional surface expression of two other exogenous transporters, the heterodimeric amino acid transporter LAT1-4F2hc and the Na⁺/phosphate cotransporter NaPi-IIa, was not increased by SGK1 co-expression. The total pool of exogenous Na,K-ATPase was increased by the co-expression of SGK1, and similarly also by ENaC co-expression. This latter effect depended on the [Na⁺] of the buffer and was not additive to that of SGK1. When the total Na,K-ATPase was increased by ENaC co-expression, SGK1 still increased Na,K-ATPase cell-surface expression. These observations in Xenopus oocytes suggest the possibility that SGK1 induction and/or activation could participate in the coordinated regulation of Na,K-ATPase and ENaC cell-surface expression in the aldosterone-sensitive distal nephron.     

EP3 receptors inhibit antidiuretic-hormone-dependent sodium transport across frog skin epithelium

 We examined the effect of prostaglandin E₂ (PGE₂) on antidiuretic hormone (ADH)-dependent Na⁺ transport and cAMP production in isolated frog skin epithelium. ADH caused an increase in transepithelial Na⁺ transport and a decrease in cellular potential, indicating an increase in apical Na⁺ permeability. Subsequent addition of PGE₂ decreased Na⁺ transport and repolarised the cells. The PGE₂ receptor EP_1/3-selective analogue sulprostone and the PGE₂ receptor EP_2/3-selective analogue misoprostol were able to mimic the effect of PGE₂. ADH increased cellular cAMP levels, whereas PGE₂, sulprostone and misoprostol were able to reduce the ADH-dependent cAMP production. Measurements of intracellular Ca²⁺ concentration ([Ca²⁺]_i) revealed that it was unaffected by both PGE₂ and sulprostone. The inhibitory effect of PGE₂ on ADH-dependent Na⁺ transport was also observed in Ca²⁺-depleted epithelia. We conclude that ADH stimulates transepithelial Na⁺ transport by increasing cellular cAMP levels, whereas PGE₂ inhibits ADH-dependent Na⁺ transport by activating EP₃-type receptors, which decrease cellular cAMP levels. We have found no evidence that [Ca²⁺]_i is involved in the regulation of ADH-dependent Na⁺ transport by PGE₂. Received: 27 May 1998 / Received after revision: 18 August 1998 / Accepted: 1 September 1998     

The role of mitochondria-rich cells in sodium transport across amphibian skin

 The possible participation of mitochondria-rich cells in transepithelial Na⁺ transport across frog skin under ”physiological conditions” (low apical [Na⁺], open circuited) was analysed by recording electrophysiological parameters from principal cells with intracellular microelectrodes and using measurement of Rb⁺ uptake into the epithelial cells from the serosal side via the Na⁺/K⁺-ATPase. It was observed that transport perturbation with amiloride induced changes in the apical potential difference and fractional apical resistance in principal cells, observations which are compatible with the notion that the essential fraction of transcellular current flow occurs across these cells. Amiloride-inhibitable uptake of Rb⁺ was also restricted to principal cells, the amount being about equivalent to the predicted rate of K⁺ recycling via the Na⁺/K⁺-ATPase. The results indicate that principal cells are responsible for transepithelial Na⁺ transport irrespective of the experimental conditions. Flow of Na⁺ across mitochondria-rich cells appears to be negligible. Received: 29 February 1996 / Received after revision: 23 June 1996 / Accepted 9 September 1996     

Regulatory volume decrease in a renal distal tubular cell line (A6) II. Effect of Na+ transport rate

A6 epithelia, a cell line originating from the distal tubular part of the kidney ofXenopus laevis, were cultured on permeable supports and mounted in an Ussing-type chamber. Cell thickness (T _c), short-circuit current (I _sc) and transepithelial conductance (G _t) were recorded while tissues were bilaterally incubated in NaCl solutions and the transepithelial potential was clamped to zero. Effects of inhibition and stimulation of transepithelial Na⁺ transport on cell volume and on its regulation during a hyposmotic challenge were investigated. Under control conditions a slow spontaneous decrease ofT _c described by a linear baseline was recorded. The reduction of the apical osmolality from 260 to 140 mosmol/kg did not alter cell volume significantly, demonstrating a negligible water permeability of the apical barrier. The inhibition of Na⁺ uptake by replacing apical Na⁺ byN-methyl-d-glucamine (NMDG⁺) did not affect cell volume under isotonic conditions. An increase ofT _c by 12.1% above the control baseline was recorded after blocking active transport with ouabain for 60 min. The activation of Na⁺ transport with insulin or oxytocin, which is known to activate the apical water permeability in other epithelia, did not alter cell volume significantly. The insensitivity of cell volume to alterations in apical Na⁺ uptake or Na⁺ pump rate confirms the close coupling between apical and basolateral transport processes. The blockage of basolateral K⁺ channels by 5 mM Ba²⁺ elicited a significant increase inT _c of 16.3% above control. Quinine, a potent blocker of volume-activated K⁺ channels, did not changeT _c significantly. Basolateral hypotonicity elicited a rapid rise inT _c followed by a regulatory volume decrease (RVD). An RVD was also recorded after blocking apical Na⁺ uptake as well as after stimulating apical Na⁺ uptake with oxytocin or insulin. Inhibition of active transport with ouabain as well as blocking K⁺ efflux at the basolateral side with Ba²⁺ or quinine abolished the RVD. The inhibition of the RVD by ouabain seems to be caused by a depletion of cellular K⁺, whereas the effects of Ba²⁺ and quinine are most likely due to the blockage of the basolateral K⁺ pathway.     

Activity of (Na+K+)-stimulated adenosintriphosphatase in the rat nephron

Summary In 17 male Wistar rats in antidiuresis 10 different nephron segments and arteries are identified with the aid of Lowry's technique, dissected and total-and (Mg⁺⁺)-adenosintriphosphatase (=ATPase) determined. (Na⁺K⁺)-activated ATPase in the distal tubule is four to five times (max. eight times) more active than in the proximal segment. This difference of activity may speak for a high pump mechanism mediated by the way of a (Na⁺K⁺)-activated enzyme system in the distal nephron and for a partially passive reabsorption of sodium from the proximal convolution.With the support of the Schweiz. Nationalfonds zur Förderung der wissenschaftlichen Forschung (Nr. 4256 and Nr. 4809.3)     

The key role of the mitochondria-rich cell in Na+ and H+ transport across the frog skin epithelium

We have investigated the possibility that the mitochondria-rich (MR) cells participate in sodium and proton transport, when the frog skin epithelium is bathed on its apical side with solutions of low Na⁺ concentration, by comparing transport rates with morphological observations (MR cell number and MR cell pit surface area). Frogs were adapted to various salinities or the isolated skins were treated with the following hormones, deoxycorticosterone acetate (DOCA), arginine vasotocin (AVT) and oxytocin in order to modify the transport of sodium and hydrogen ions. Adaptation of the frogs (either 3–4 days or 7–10 days) to distilled water, NaCl (50 mmol/l), KCl (50 mmol/l) or Na₂SO₄ (25 mmol/l) solutions modified the Na⁺ transport rate and the morphology of the epithelium. The highest Na⁺ transport rates were found for the animals adapted to the Na⁺ free solutions and were correlated with an increase in the total MR cell pit-surface area (number of MR cells x individual cell pit-surface area). The KCl adaptated group showed the largest increase in sodium and proton transport and also presented a metabolic acidosis as reflected by plasma acidification (pCO₂ increase and HCO ₃ ^– decrease). Proton secretion and sodium absorption were also found to be stimulated by either serosal DOCA addition (10^–6 M) or during acidification of the epithelium by serosally applied CO₂. Na⁺ transport was enhanced by AVT (10^–6 M) or oxytocin (100mU/ml) when the skin was bathed on its apical side with a high Na⁺ containing solution (115 mmol/l), whereas these hormones did not exert any effect on Na⁺ transport when the apical solution was low in Na⁺ (0.5mmol/l). It is concluded that MR cells play a key role in Na⁺ and H⁺ transport through the frog skin epithelium when bathed on its apical side with a low Na⁺ containing solution. Distinct pathways for sodium transport through two cell types (MR cells and granular cells) are proposed depending on the Na⁺ concentration of the solution bathing the apical side of the epithelium.     

Sodium conductance changes by aldosterone in the rat kidney

Summary To asses passive permeability properties of distal, and proximal tubules of the rat kidney the tubular lumen was perfused with solutions of 1.5 and 150 mM Na/l while transtubular potential differences were recorded. Sodium transport numbers (T _Na) were calculated.T _Na in the distal tubule of adrenalectomized rats was acutely increased from 0.21 to 0.27 by aldosterone (5 g/100 g B.W.). This effect of aldosterone could not be reduced by concomitant injection of cycloheximide (100 g/100 g B.W.). Aldosterone was also effective in control rats. In the proximal tubule similar data were obtained. However, the aldosterone-induced increase of conductance was slightly reduced with cycloheximide.These measurements of transepithelial sodium conductance indicate that aldosterone, in addition to the already known stimulation of active sodium transport, increases overall permeability of the tubular wall to sodium. In the distal tubule this effect indicates an increase of the luminal membrane permeability whereas in the proximal tubule aldosterone may facilitate the diffusion of sodium through the intercellular shunt path and/or the luminal membrane. The passive components of transepithelial electrolyte transfer seem to be less sensitive to inhibition of protein synthesis than the active transport components.Supported by Deutsche Forschungemeinschaft.     

Sodium transport-related proteins in the mammalian distal nephron – distribution, ontogeny and functional aspects

The mammalian distal nephron plays a pivotal role in adjusting urinary sodium excretion. Successive portions of the renal tubule are formed to adapt to this function, and an axial heterogeneity of the distal segments has been defined. The specific transport properties of these epithelia are accomplished by the expression of proteins (cotransporters, exchangers, channels) governing the movement of ions on either cell side. Molecular cloning of these proteins has had a marked impact on the study of their localization and function in the healthy and diseased kidney. Electroneutral cation-chloride cotransporters [Na(K)CC] have been localized to the thick ascending limb and the distal convoluted tubule using specific probes. Proteins implicated in the function of aldosterone target cells, such as the epithelial Na⁺ channel (ENaC), the mineralocorticoid receptor (MR) and 11β-hydroxysteroid dehydrogenase type 2 (11HSD2), an enzyme that confers mineralocorticoid specificity, have been found in the terminal portion of the nephron and the collecting duct. A mineralocorticoid-sensitive component of thiazide-sensitive NaCl transport has been identified in the distal convoluted tubule. Analysis of the ontogeny of these proteins in the maturing kidney has provided a detailed picture of epithelial differentiation and morphological specialization of the renal tubule. The study of mutations of the proteins related with NaCl transport has led to the identification of the molecular causes of inherited human diseases associated with hypo- or hypertension, and the respective sites of an impaired ion transport could be mapped to the renal tubule. Accepted: 13 April 1999     

AMPK controls epithelial Na+ channels through Nedd4-2 and causes an epithelial phenotype when mutated

The metabolic sensor adenosine-monophosphate-activated kinase (AMPK) detects the cellular energy status and adjusts metabolic activity according to the cytosolic AMP to ATP ratio. Na⁺ absorption by epithelial Na⁺ channels (ENaC) is a highly energy-consuming process that is inhibited by AMPK. We show that the catalytic subunit α1 of AMPK inhibits ENaC in epithelial tissues from airways, kidney, and colon and that AMPK regulation of ENaC is absent in AMPKα1−/− mice. These mice demonstrate enhanced electrogenic Na⁺ absorption that leads to subtle changes in intestinal and renal function and may also affect Na⁺ absorption and mucociliary clearance in the airways. We demonstrate that AMPK uses the ubiquitin ligase Nedd4-2 to inhibit ENaC by increasing ubiquitination and endocytosis of ENaC. Thus, enhanced expression of epithelial Na⁺ channels was detected in colon, airways, and kidney of AMPKα1−/− mice. Therefore, AMPKα1 is a physiologically important regulator of electrogenic Na⁺ absorption and may provide a novel pharmacological target for controlling epithelial Na⁺ transport. Categories: Membranes and Transport; bioenergetics, anabolic/catabolic processes studied at the molecular level.