Identification of SPLUNC1's ENaC-inhibitory domain yields novel strategies to treat sodium hyperabsorption in cystic fibrosis airways.

The epithelial sodium channel (ENaC) is responsible for Na+ and fluid absorption across colon, kidney, and airway epithelia. We have previously identified SPLUNC1 as an autocrine inhibitor of ENaC. We have now located the ENaC inhibitory domain of SPLUNC1 to SPLUNC1's N terminus, and a peptide corresponding to this domain, G22-A39, inhibited ENaC activity to a similar degree as full-length SPLUNC1 (∼2.5 fold). However, G22-A39 had no effect on the structurally related acid-sensing ion channels, indicating specificity for ENaC. G22-A39 preferentially bound to the β-ENaC subunit in a glycosylation-dependent manner. ENaC hyperactivity is contributory to cystic fibrosis (CF) lung disease. Addition of G22-A39 to CF human bronchial epithelial cultures (HBECs) resulted in an increase in airway surface liquid height from 4.2±0.6 to 7.9±0.6 μm, comparable to heights seen in normal HBECs, even in the presence of neutrophil elastase. Our data also indicate that the ENaC inhibitory domain of SPLUNC1 may be cleaved away from the main molecule by neutrophil elastase, which suggests that it may still be active during inflammation or neutrophilia. Furthermore, the robust inhibition of ENaC by the G22-A39 peptide suggests that this peptide may be suitable for treating CF lung disease.

Hemodynamic effects of sodium bicarbonate in critically ill neonates.

OBJECTIVE: To analyze the cardiovascular effects of sodium bicarbonate in neonates with metabolic acidosis. DESIGN: Prospective, open, non-randomized, before-after intervention study with hemodynamic measurements performed before and 1, 5, 10, 20, and 30 min after bicarbonate administration. SETTING: Neonatal intensive care unit, tertiary care center. PATIENTS: Sequential sample of 16 paralysed and mechanically ventilated newborn infants with a metabolic acidosis (pH < 7.25 in premature and < 7.30 in term infants, base deficit > -8). INTERVENTION: An 8.4% sodium bicarbonate solution diluted 1:1 with water (final osmolality of 1000 mOsm/l) was administered in two equal portions at a rate of 0.5 mmol/min. The dose in mmol was calculated using the formula "base deficit x body weight (kg) x 1/3 x 1/2". MEASUREMENTS AND RESULTS: Sodium bicarbonate induced a significant but transient rise in pulsed Doppler cardiac output (CO) (+27.7%), aortic blood flow velocity (+15.3%), systolic blood pressure (BP) (+9.3%), (+14.6%), transcutaneous carbon dioxide pressure (PtcCO2) (+11.8%), and transcutaneous oxygen pressure (PtcO2) (+8%). In spite of the PaCO2 elevation, pH significantly improved (from a mean of 7.24 to 7.30), and the base deficit decreased (-39.3%). Calculated systemic vascular resistance (SVR) (-10.7%) and diastolic BP (-11.7%) decreased significantly, while PaO2 and heart rate (HR) did not change. Central venous pressure (CVP) (+6.5%) increased only slightly. By 30 min after bicarbonate administration all hemodynamic parameters, with the exception of the diastolic BP, had returned to baseline. CONCLUSION: Sodium bicarbonate in neonates with metabolic acidosis induces an increase in contractility and a reduction in afterload.

Astrocyte-neuron lactate transport is required for long-term memory formation.

We report that, in the rat hippocampus, learning leads to a significant increase in extracellular lactate levels that derive from glycogen, an energy reserve selectively localized in astrocytes. Astrocytic glycogen breakdown and lactate release are essential for long-term but not short-term memory formation, and for the maintenance of long-term potentiation (LTP) of synaptic strength elicited in vivo. Disrupting the expression of the astrocytic lactate transporters monocarboxylate transporter 4 (MCT4) or MCT1 causes amnesia, which, like LTP impairment, is rescued by L-lactate but not equicaloric glucose. Disrupting the expression of the neuronal lactate transporter MCT2 also leads to amnesia that is unaffected by either L-lactate or glucose, suggesting that lactate import into neurons is necessary for long-term memory. Glycogenolysis and astrocytic lactate transporters are also critical for the induction of molecular changes required for memory formation, including the induction of phospho-CREB, Arc, and phospho-cofilin. We conclude that astrocyte-neuron lactate transport is required for long-term memory formation.

Proximal sodium reabsorption: An independent determinant of blood pressure response to salt.

The purpose of this study was to evaluate the contribution of renal sodium handling by the proximal tubule as an independent determinant of blood pressure responsiveness to salt in hypertension. We measured blood pressure (BP), renal hemodynamics, and segmental renal sodium handling (with lithium used as a marker of proximal sodium reabsorption) in 38 hypertensive patients and 27 normotensive subjects (15 young and 12 age-matched) on a high and low sodium diet. In control subjects, changing the diet from a low to a high sodium content resulted in no change in BP and increases in glomerular filtration rate (P<0.05), renal plasma flow (P<0.05), and fractional excretion of lithium (FE(Li), P<0.01). In hypertensive patients, comparable variations of sodium intake induced an increase in BP with no change in renal hemodynamics and proximal sodium reabsorption. When analyzed by tertiles of their BP response to salt, salt-insensitive hypertensive patients of the first tertile disclosed a pattern of adaptation of proximal sodium reabsorption comparable to that of control subjects, whereas the most salt-sensitive patients of the third tertile had an inverse pattern with a high FE(Li) on low salt and a lower FE(Li) on high salt, suggesting an inappropriate modulation of proximal sodium reabsorption. The BP response to salt correlated positively with age (r=0.34, P=0.036) and negatively with the changes in FE(Li) (r=-0.37, P=0.029). In a multivariate analysis, the changes in FE(Li) were significantly and independently associated with the salt-induced changes in BP. These results suggest that proximal sodium reabsorption is an independent determinant of the BP response to salt in hypertension.

Nedd4-2 modulates renal Na+-Cl- cotransporter via the aldosterone-SGK1-Nedd4-2 pathway.

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.

Activity-dependent phosphorylation of SNAP-25 in hippocampal organotypic cultures.

Synaptosomal-associated protein of 25 kDa (SNAP-25) is thought to play a key role in vesicle exocytosis and in the control of transmitter release. However, the precise mechanisms of action as well as the regulation of SNAP-25 remain unclear. Here we show by immunoprecipitation that activation of protein kinase C (PKC) by phorbol esters results in an increase in SNAP-25 phosphorylation. In addition, immunochemical analysis of two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels shows that SNAP-25 focuses as three or four distinct spots in the expected range of molecular weight and isoelectric point. Changing the phosphorylation level of the protein by incubating the slices in the presence of either a PKC agonist (phorbol 12,13-dibutyrate) or antagonist (chelerythrine) modified the distribution of SNAP-25 among these spots. Phorbol 12,13-dibutyrate increased the intensity of the spots with higher molecular weight and lower isoelectric point, whereas chelerythrine produced the opposite effect. This effect was specific for regulators of PKC, as agonists of other kinases did not produce similar changes. Induction of long-term potentiation, a property involved in learning mechanisms, and production of seizures with a GABA(A) receptor antagonist also increased the intensity of the spots with higher molecular weight and lower isoelectric point. This effect was prevented by the PKC inhibitor chelerythrine. We conclude that SNAP-25 can be phosphorylated in situ by PKC in an activity-dependent manner.

A comparative study between crosslinked sodium hyaluronate (healaflow glaucoma implant) and high molecular weight sodium hyaluronate (healon 5) as subconjunctival spacers injected into failed filtration blebs during needling with MMC

Purpose: To compare the efficacy and safety of a slowly resorbable glaucoma implant, Healaflow (HF) and Healon 5 (H5) as spacers injected into failing filtration blebs at the time of bleb needlings, augmented with MMC Methods: Eighty-four glaucoma patients with filtration bleb failure following glaucoma surgery (deep sclerectomy/ trabeculectomy) underwent bleb needling using either HF (n=44) or H5 (n=40) as an intrableb viscoelastic spacer. All needlings were augmented by MMC and performed in the operating room, by a single surgeon. The choice between HF and H5 was randomised. Postoperative data for IOP, number of glaucoma medications (GMs), antimetabolite injections (AMIs), bleb morphology and all complications were recorded at day 1 (D1), weeks 1 (W1), 4 (W4), month 6 (M6) and last visit. Success was defined as IOP≤21mmHg and 20% reduction in IOP from baseline Results: Age was comparable between groups (HF vs H5; 66 vs 72 years; p=0.13*). There were no differences in mean postoperative IOP or GMs at any time point between groups (mean IOP = HF vs H5; prior to needling=19.8mmHg vs 18.7mmHg, p=0.48*; D1=10.4mmHg vs 10.2mmHg, p=0.85*; W1=12.3mmHg vs 13.8mmHg, p=0.33*; W4=15.7mmHg vs 15.1mmHg, p=0.69*; M6=14.3mmHg vs 13.8mmHg, p=0.69*. Mean GMs= HF vs H5; prior to needling=1.7 vs 1.7, p=0.96*; M6=0.9 vs 1.1, p=0.66*). Success rates were comparable between HF and H5 groups (74% vs 71%). Requirements for AMIs were similar between groups; post filtration surgery and prior to needling (mean AMIs 1.54 vs 1.65 p=0.82*) and within the first six months following needling ( 0.77 vs 0.65 p=0.70*). Complication rates were infrequent in both groups (9%, HF; 3%, H5) * two-sample t-test Conclusions: The early to mid-term success rates of needlings using intrableb spacers were high. However there were no differences observed between the use of crosslinked and high molecular weight sodium hyaluronate

Sodium reduction in cooked meat products by using commercial potassium phoshate mixtures

Selostus: Natriumpitoisuuden pienentäminen lihavalmisteissa korvaamalla natriumfosfaatti kaliumfosfaatilla

Role of the epithelial sodium channel (ENaC) in the epidermal barrier function of the skin

Abstract In humans, the skin is the largest organ of the body, covering up to 2m2 and weighing up to 4kg in an average adult. Its function is to preserve the body from external insults and also to retain water inside. This barrier function termed epidermal permeability barrier (EPB) is localized in the functional part of the skin: the epidermis. For this, evolution has built a complex structure of cells and lipids sealing the surface, the stratum corneum. The formation of this structure is finely tuned since it is not only formed once at birth, but renewed all life long. This active process gives a high plasticity and reactivity to skin, but also leads to various pathologies. ENaC is a sodium channel extensively studied in organs like kidney and lung due to its importance in regulating sodium homeostasis and fluid volume. It is composed of three subunits α, ß and r which are forming sodium selective channel through the cell membrane. Its presence in the skin has been demonstrated, but little is known about its physiological role. Previous work has shown that αENaC knockout mice displayed an abnormal epidermis, suggesting a role in differentiation processes that might be implicated in the EPB. The principal aim of this thesis has been to study the consequences for EPB function in mice deficient for αENaC by molecular and physiological means and to investigate the underlying molecular mechanisms. Here, the barrier function of αENaC knockout pups is impaired. Apparently not immediately after birth (permeability test) but 24h later, when evident water loss differences appeared compared to wildtypes. Neither the structural proteins of the epithelium nor the tights junctions showed any obvious alterations. In contrary, stratum corneum lipid disorders are most likely responsible for the barrier defect, accompanied by an impairment of skin surface acidification. To analyze in details this EPB defect, several hypotheses have been proposed: reduced sensibility to calcium which is the key activator far epidermal formation, or modification of ENaC-mediated ion fluxes/currents inside the epidermis. The cellular localization of ENaC and the action in the skin of CAPl, a positive regulator of ENaC, have been also studied in details. In summary, this study clearly demonstrates that ENaC is a key player in the EPB maintenance, because αENaC knockout pups are not able to adapt to the new environment (ex utero) as efficiently as the wildtypes, most likely due to impaired of sodium handling inside the epidermis. Résumé Chez l'homme, la peau est le plus grand organe, couvrant presque 2m2 et pesant près de 4kg chez l'adulte. Sa fonction principale est de protéger l'organisme des agressions extérieures mais également de conserver l'eau à l'intérieur du corps. Cette fonction nommée barrière épithéliale est localisée dans la partie fonctionnelle de la peau : l'épiderme. A cette fin, l'évolution s'est dotée d'une structure complexe composée de cellules et de lipides recouvrant la surface, la couche cornée. Sa formation est finement régulée, car elle n'est pas seulement produite à la naissance mais constamment renouvelée tout au long de la vie, ce qui lui confère une grande plasticité mais ce qui est également la cause de nombreuses pathologies. ENaC est un canal sodique très étudié dans le rein et le poumon pour son importance dans la régulation de l'homéostasie sodique et la régulation du volume du milieu intérieur. Il est composé de 3 sous unités, α, ß et y qui forment un pore sélectif pour le sodium dans les membranes. Ce canal est présent dans la peau mais sa fonction n'y est pas connue. Des travaux précédents ont pu montrer que les souris dont le gène codant pour αENaC a été invalidé présentent un épiderme pathologique, suggérant un rôle dans la différentiation et pourrait même être impliqué dans la barrière épithéliale. Le but de cette thèse fut l'étude de la barrière dans ces souris knockouts avec des méthodes moléculaires et physiologiques et la caractérisation des mécanismes moléculaire impliqués. Dans ce travail, il a été montré que les souris mutantes présentaient un défaut de la barrière. Ce défaut n'est pas visible immédiatement à la naissance (test de perméabilité), mais 24h plus tard, lorsque les tests de perte d'eau transépithéliale montrent une différence évidente avec les animaux contrôles. Ni les protéines de structures ni les jonctions serrées de l'épiderme ne présentaient d'imperfections majeures. A l'inverse, les lipides de la couche cornée présentaient un problème de maturation (expliquant le phénotype de la barrière), certainement consécutif au défaut d'acidification à la surface de la peau que nous avons observé. D'autres mécanismes ont été explorées afin d'investiguer cette anomalie de la barrière, comme la réduction de sensibilité au calcium qui est le principal activateur de la formation de l'épiderme, ou la modification des flux d'ions entre les couches de l'épiderme. La localisation cellulaire d'ENaC, et l'action de son activateur CAPl ont également été étudiés en détails. En résumé, cette étude démontre clairement qu'ENaC est un acteur important dans la formation de la barrière épithéliale, car la peau des knockouts ne s'adapte pas aussi bien que celle des sauvages au nouvel environnement ex utero à cause de la fonction d'ENaC dans les mouvements de sodium au sein même de l'épiderme. Résumé tout public Chez l'homme, la peau est le plus grand organe, couvrant presque 2m2 et pesant près de 4kg chez l'adulte. Sa fonction principale est de protéger l'organisme des agressions extérieures mais également de conserver l'eau à l'intérieur du corps. Cette fonction nommée barrière épithéliale est localisée dans la partie fonctionnelle de la peau : l'épiderme. A cette fin, l'évolution s'est dotée d'une structure complexe composée de cellules et de lipides recouvrant la surface, la couche cornée. Sa formation est finement régulée, car elle n'est pas seulement produite à la naissance mais constamment renouvelée tout au long de la vie, ce qui lui confère une grande plasticité mais ce qui est également la cause de nombreuses maladies. ENaC est une protéine formant un canal qui permet le passage sélectif de l'ion sodium à travers la paroi des cellules. Il est très étudié dans le rein pour son importance dans la récupération du sel lors de la concentration de l'urine. Ce canal est présent dans la peau mais sa fonction n'y est pas connue. Des travaux précédents ont pu montrer que les souris où le gène codant pour αENaC a été invalidé présentent un épiderme pathologique, suggérant un rôle dans la peau et plus particulièrement la fonction de barrière de l'épiderme. Le but de cette thèse fut l'étude de la fonction de barrière dans ces souris mutantes, au niveau tissulaire et cellulaire. Dans ce travail, il a été montré que les souris mutantes présentaient une peau plus perméable que celle des animaux contrôles, grâce à une machine mesurant la perte d'eau à travers la peau. Ce défaut n'est visible que 24h après la naissance, mais nous avons pu montrer que les animaux mutants perdaient quasiment 2 fois plus d'eau que les contrôles. Au niveau moléculaire, nous avons pu montrer que ce défaut provenait d'un problème de maturation des lipides qui composent la barrière de la peau. Cette maturation est incomplète vraisemblablement à cause d'un défaut de mouvement des ions dans les couches les plus superficielles de l'épiderme, et cela à cause de l'absence du canal ENaC. En résumé, cette étude démontre clairement qu'ENaC est un acteur important dans la formation de la barrière épithéliale, car la peau des mutants ne s'adapte pas aussi bien que celle des sauvages au nouvel environnement ex utero à cause de la fonction d'ENaC dans les mouvements de sodium au sein même de l'épiderme.

Studies on Soil-Aggregate-Sodium Chloride Stabilized Roads in Franklin County, Iowa, HR-33

This investigation was conducted to study the performance characteristics of low cost roadway surfaces of soil-aggregate-sodium chloride mixtures. Many roads have been successfully stabilized with sodium chloride. However, little information is available on either the properties of the road materials or the effects of sodium chloride on the materials. The performance of some of the sodium chloride stabilized roads in Franklin County, Iowa, and the performance of some near-by nonchemically treated roads has been studied. The study of sodium chloride stabilized roads was restricted to the roads in which the binder soil used in construction came from the same source. The effects of sodium chloride on some of the engineering properties of the soil and soil-aggregate mixtures used were studied in the laboratory.

Scnn1 sodium channel gene family in genetically engineered mice.

The amiloride-sensitive epithelial sodium channel is the limiting step in salt absorption. In mice, this channel is composed of three subunits (alpha, beta, and gamma), which are encoded by different genes (Scnn1a, Scnn1b, and Scnn1c, respectively). The functions of these genes were recently investigated in transgenic (knockout) experiments, and the absence of any subunit led to perinatal lethality. More defined phenotypes have been obtained by introducing specific mutations or using transgenic rescue experiments. In this report, these approaches are summarized and a current gene-targeting strategy to obtain conditional inactivation of the channel is illustrated. This latter approach will be indispensable for the investigation of channel function in a wide variety of organ systems.

Role of reactive hyperreninemia in blood pressure changes induced by sodium depletion in patients with refractory hypertension

Sixteen patients with refractory hypertension were submitted to vigorous sodium depletion while cardiovascular homeostasis was monitored with measurements of hormonal and hemodynamic parameters and repeat saralasin tests. This regimen resulted in a negative sodium balance by an average of 300 mEq. The loss of sodium closely correlated to the decrease of body weight (r = 0.70, p less than 0.005). Blood pressure (BP) decreased from 176/166 +/- 8/3 to 155/109 +/-6/3 mm Hg. There was a significant correlation between percent increments in plasma renin activity (PRA) and the rise in plasma norepinephrine (r = 0.68, p less than 0.05) and a close negative correlation between percent increase in PRA and the ratio of fall in mean blood pressure (MAP) per unit of weight loss (r = -0.73, p less than 0.005). Thus, patients with the least percent increase in PRA demonstrated the greatest fall in BP per unit of weight loss, indicating that relative rather than absolute elevation of renin may be the factor limiting antihypertensive efficacy of sodium depletion. Sodium depletion induced increase in peripheral resistance and decrease in cardiac output, both mostly attributable to relative hyperreninemia. Indeed, the adverse hemodynamic changes were reversed by angiotensin inhibition, during which BP normalized. It is concluded that vigorous sodium depletion complemented by angiotensin blockade or suppression with sympatholytic agents improves management of otherwise refractory hypertension.

Remaining phosphorus and sodium fluoride pH in soils with different clay contents and clay mineralogies

The remaining phosphorus (Prem) has been used for estimating the phosphorus buffer capacity (PBC) of soils of some Brazilian regions. Furthermore, the remaining phosphorus can also be used for estimating P, S and Zn soil critical levels determined with PBC-sensible extractants and for defining P and S levels to be used not only in P and S adsorption studies but also for the establishment of P and S response curves. The objective of this work was to evaluate the effects of soil clay content and clay mineralogy on Prem and its relationship with pH values measured in saturated NaF solution (pH NaF). Ammonium-oxalate-extractable aluminum exerts the major impacts on both Prem and pH NaF, which, in turn, are less dependent on soil clay content. Although Prem and pH NaF have consistent correlation, the former has a soil-PBC discriminatory capacity much greater than pH NaF.