Economics of Using Calcium Chloride vs. Sodium Chloride for Deicing/Anti-Icing, TR-488, 2008

The use of chemicals is a critical part of a pro-active winter maintenance program. However, ensuring that the correct chemicals are used is a challenge. On the one hand, budgets are limited, and thus price of chemicals is a major concern. On the other, performance of chemicals, especially at lower pavement temperatures, is not always assured. Two chemicals that are used extensively by the Iowa Department of Transportation (Iowa DOT) are sodium chloride (or salt) and calcium chloride. While calcium chloride can be effective at much lower temperatures than salt, it is also considerably more expensive. Costs for a gallon of salt brine are typically in the range of $0.05 to $0.10, whereas calcium chloride brine may cost in the range of $1.00 or more per gallon. These costs are of course subject to market forces and will thus change from year to year. The idea of mixing different winter maintenance chemicals is by no means new, and in general discussions it appears that many winter maintenance personnel have from time to time mixed up a jar of chemicals and done some work around the yard to see whether or not their new mix “works.” There are many stories about the mixture turning to “mayonnaise” (or, more colorfully, to “snot”) suggesting that mixing chemicals may give rise to some problems most likely due to precipitation. Further, the question of what constitutes a mixture “working” in this context is a topic of considerable discussion. In this study, mixtures of salt brine and calcium chloride brine were examined to determine their ice melting capability and their freezing point. Using the results from these tests, a linear interpolation model of the ice melting capability of mixtures of the two brines has been developed. Using a criterion based upon the ability of the mixture to melt a certain thickness of ice or snow (expressed as a thickness of melt-water equivalent), the model was extended to develop a material cost per lane mile for the full range of possible mixtures as a function of temperature. This allowed for a comparison of the performance of the various mixtures. From the point of view of melting capacity, mixing calcium chloride brine with salt brine appears to be effective only at very low temperatures (around 0° F and below). However, the approach described herein only considers the material costs, and does not consider application costs or other aspects of the mixture performance than melting capacity. While a unit quantity of calcium chloride is considerably more expensive than a unit quantity of sodium chloride, it also melts considerably more ice. In other words, to achieve the same result, much less calcium chloride brine is required than sodium chloride brine. This is important in considering application costs, because it means that a single application vehicle (for example, a brine dispensing trailer towed behind a snowplow) can cover many more lane miles with calcium chloride brine than with salt brine before needing to refill. Calculating exactly how much could be saved in application costs requires an optimization of routes used in the application of liquids in anti-icing, which is beyond the scope of the current study. However, this may be an area that agencies wish to pursue for future investigation. In discussion with winter maintenance personnel who use mixtures of sodium chloride and calcium chloride, it is evident that one reason for this is because the mixture is much more persistent (i.e. it stays longer on the road surface) than straight salt brine. Operationally this persistence is very valuable, but at present there are not any established methods to measure the persistence of a chemical on a pavement. In conclusion, the study presents a method that allows an agency to determine the material costs of using various mixtures of salt brine and calcium chloride brine. The method is based upon the requirement of melting a certain quantity of snow or ice at the ice-pavement interface, and on how much of a chemical or of a mixture of chemicals is required to do that.

Protease modulation of the activity of the epithelial sodium channel expressed in Xenopus oocytes.

We have investigated the effect of extracellular proteases on the amiloride-sensitive Na+ current (INa) in Xenopus oocytes expressing the three subunits alpha, beta, and gamma of the rat or Xenopus epithelial Na+ channel (ENaC). Low concentrations of trypsin (2 microg/ml) induced a large increase of INa within a few minutes, an effect that was fully prevented by soybean trypsin inhibitor, but not by amiloride. A similar effect was observed with chymotrypsin, but not with kallikrein. The trypsin-induced increase of INa was observed with Xenopus and rat ENaC, and was very large (approximately 20-fold) with the channel obtained by coexpression of the alpha subunit of Xenopus ENaC with the beta and gamma subunits of rat ENaC. The effect of trypsin was selective for ENaC, as shown by the absence of effect on the current due to expression of the K+ channel ROMK2. The effect of trypsin was not prevented by intracellular injection of EGTA nor by pretreatment with GTP-gammaS, suggesting that this effect was not mediated by G proteins. Measurement of the channel protein expression at the oocyte surface by antibody binding to a FLAG epitope showed that the effect of trypsin was not accompanied by an increase in the channel protein density, indicating that proteolysis modified the activity of the channel present at the oocyte surface rather than the cell surface expression. At the single channel level, in the cell-attached mode, more active channels were observed in the patch when trypsin was present in the pipette, while no change in channel activity could be detected when trypsin was added to the bath solution around the patch pipette. We conclude that extracellular proteases are able to increase the open probability of the epithelial sodium channel by an effect that does not occur through activation of a G protein-coupled receptor, but rather through proteolysis of a protein that is either a constitutive part of the channel itself or closely associated with it.

Revealing a subclinical salt-losing phenotype in heterozygous carriers of the novel S562P mutation in the alpha subunit of the epithelial sodium channel.

OBJECTIVE: Pseudohypoaldosteronism type I (PHA1) is a rare inborn disease causing severe salt loss. Mutations in the three coding genes of the epithelial sodium channel (ENaC) are responsible for the systemic autosomal recessive form. So far, no phenotype has been reported in heterozygous carriers. PATIENTS: A consanguineous family from Somalia giving birth to a neonate suffering from PHA1 was studied including clinical and hormonal characteristics of the family, mutational analysis of the SCNN1A, SCNN1B, SCNN1G and CFTR genes and in vitro analysis of the functional consequences of a mutant ENaC channel. RESULTS: CFTR mutations have been excluded. SCNN1A gene analysis revealed a novel homozygous c.1684T > C mutation resulting in a S562P substitution in the alphaENaC protein of the patient. Functional analysis showed a significantly reduced S562P channel function compared to ENaC wild type. Protein synthesis and channel subunit assembly were not altered by the S562P mutation. Co-expression of mutant and wild-type channels revealed a dominant negative effect. In heterozygote carriers, sweat sodium and chloride concentrations were increased without additional hormonal or clinical phenotypes. CONCLUSION: Hence, the novel mutation S562P is causing systemic PHA1 in the homozygous state. A thorough clinical investigation of the heterozygote SCNN1A mutation carriers revealed increased sweat sodium and chloride levels consistent with a dominant effect of the mutant S562P allele. Whether this subclinical phenotype is of any consequence for the otherwise asymptomatic heterozygous carriers has to be elucidated.

CARDIAC AND MUSCLE CHANNELOPATHIES: ROLES AND REGULATION OF VOLTAGE-GATED SODIUM CHANNELS

Abstract :The contraction of the heart or skeletal muscles is mainly due to the propagation, through excitable cells, of an electrical influx called action potential (AP). The AP results from the sequential opening of ion channels that generate inward or outward currents through the cell membrane. Among all the channels involved, the voltage-gated sodium channel is responsible for the rising phase of the action potential. Ten genes encode the different isoforms of these channels (from Nav1.1 to Nav1.9 and an atypical channel named NavX). Nav1.4 and Nav1.5 are the main skeletal muscle and cardiac sodium channels respectively. Their importance for muscle and heart function has been highlighted by the description of mutations in their encoding genes SCN4A and SCNSA. They lead respectively to neuromuscular disorders such as myotonia or paralysis (for Nav1.4), and to cardiac arrhythmias that can deteriorate into sudden cardiac death (for Nav1.5).The general aim of my PhD work has been to study diseases linked with channels dysfunction, also called channelopathies. In that purpose, I investigated the function and the regulation of the muscle and cardiac voltage-gated sodium channels. During the two first studies, I characterized the effects of two mutations affecting Nav1.4 and Nav1.5 function. I used the HEK293 model cells to express wild-type or mutant channels and then studied their biophysical properties with the patch-clamp technique, in whole cell configuration. We found that the SCN4A mutation produced complex alterations of the muscle sodium channel function, that could explain the myotonic phenotype described in patients carrying the mutation. In the second study, the index case was an heterozygous carrier of a SCNSA mutation that leads to a "loss of function" of the channel. The decreased sodium current measured with mutated Nay 1.5 channels, at physiological temperature, was a one of the factors that could explain the observed Brugada syndrome. The last project aimed at identifying a new potential protein interacting with the cardiac sodium channel. We found that the protein SAP97 binds the three last amino-acids of the C-terminus of Na,, 1.5. Our results also indicated that silencing the expression of SAP97 in HEK293 cells decreased the sodium current. Sodium channels lacking their three last residues also produced a reduced INa. These preliminary results suggest that SAP97 is implicated in the regulation of sodium channel. Whether this effect is direct or imply the action of an adaptor protein remains to be investigated. Moreover, our group has previously shown that Nav1.5 channels are localized to lateral membranes of cardiomyocytes by the dystrophin multiprotein complex (DMC). This suggests that sodium channels are distributed in, at least, two different pools: one targeted at lateral membranes by DMC and the other at intercalated discs by another protein such as SAP97.These studies reveal that cardiac and muscle diseases may result from ion channel mutations but also from regulatory proteins affecting their regulation.Résumé :La contraction des muscles et du coeur est principalement due à la propagation, à travers les cellules excitables, d'un stimulus électrique appelé potentiel d'action (PA). C'est l'ouverture séquentielle de plusieurs canaux ioniques transmembranaires, permettant l'entrée ou la sortie d'ions dans la cellule, qui est à l'origine de ce PA. Parmi tous les canaux ioniques impliqués dans ce processus, les canaux sodiques dépendant du voltage sont responsables de la première phase du potentiel d'action. Les différentes isoformes de ces canaux (de Nav1.1 à Nav1.9 et NavX) sont codées par dix gènes distincts. Nav1.4 et Nav1.5 sont les principaux variants exprimés respectivement dans le muscle et le coeur. Plusieurs mutations ont été décrites dans les gènes qui codent pour ces deux canaux: SCN4A (pour Nav1.4) et SCNSA (pour Nav1.5). Elles sont impliquées dans des pathologies neuromusculaires telles que des paralysies ou myotonies (SCN4A) ou des arythmies cardiaques pouvant conduire à la mort subite cardiaque (SCNSA).Mon travail de thèse a consisté à étudier les maladies liées aux dysfonctionnements de ces canaux, aussi appelées canalopathies. J'ai ainsi analysé la fonction et la régulation des canaux sodiques dépendant du voltage dans le muscle squelettique et le coeur. A travers les deux premières études, j'ai ainsi pu examiner les conséquences de deux mutations affectant respectivement les canaux Nav1.4 et Nav1.5. Les canaux sauvages ou mutants ont été exprimés dans des cellules HEK293 afin de caractériser leurs propriétés biophysiques par la technique du patch clamp en configuration cellule entière. Nous avons pu déterminer que la mutation trouvée dans le gène SCN4A engendrait des modifications importantes de la fonction du canal musculaire. Ces altérations fournissent des indications nous permettant d'expliquer certains aspects de la myotonie observée chez les membres de la famille étudiée. Le patient présenté dans la deuxième étude était hétérozygote pour la mutation identifiée dans le gène SCNSA. La perte de fonction des canaux Nav1.5 ainsi engendrée, a été observée lors d'analyses à températures physiologiques. Elle représente l'un des éléments pouvant potentiellement expliquer le syndrome de Brugada du patient. La dernière étude a consisté à identifier une nouvelle protéine impliquée dans la régulation du canal sodique cardiaque. Nos expériences ont démontré que les trois derniers acides aminés de la partie C-terminale de Nav1.5 pouvaient interagir avec la protéine SAP97. Lorsque que l'expression de la SAP97 est réduite dans les cellules HEK293, cela induit une baisse importante du courant sodique. De même, les canaux tronqués de leurs trois derniers acides aminés génèrent un flux ionique réduit. Ces résultats préliminaires suggèrent que SAP97 est peut-être impliquée dans la régulation du canal Na,,1.5. Des expériences complémentaires permettront de déterminer si ces deux protéines interagissent directement ou si une protéine adaptatrice est nécessaire. De plus, nous avons préalablement montré que les canaux Nav1.5 étaient localisés au niveau de la membrane latérale des cardiomyocytes par le complexe multiprotéique de la dystrophine (DMC). Ceci suggère que les canaux sodiques peuvent être distribués dans un minimum de deux pools, l'un ciblé aux membranes latérales pax le DMC et l'autre dirigé vers les disques intercalaires par des protéines telles que SAP97.L'ensemble de ces études met en évidence que certaines maladies musculaires et cardiaques peuvent être la conséquence directe de mutations de canaux ioniques, mais que l'action de protéines auxiliaires peut aussi affecter leur fonction.

The epidthelial sodium channel ENaC and its regulators in the epidermal permeability barrier function

The highly amiloride-sensitive epithelial sodium channel ENaC is well known to be involved in controlling whole body sodium homeostasis and lung liquid clearance. ENaC expression has also been detected in the skin of amphibians and mammals. Mice lacking ENaC expression lose rapidly weight associated with an epidermal barrier defect that develops following birth. This dehydration is accompanied with a highly abnormal lipid matrix composition and an impaired skin surface acidification. This strongly suggests a role of ENaC in the maturation of barrier function rather than in the prenatal generation of the barrier, and may be as such an important modulator for skin hydration. In parallel, gene targeting experiments of regulators of ENaC activity, membrane serine proteases, also termed channel activating proteases, like CAP1/Prss8 and matriptase/MT-SP1 by themselves have been shown to be crucial for the epidermal barrier function. In our review, we mainly focus on the role of ENaC and its regulators in the skin and discuss their importance in the epidermal permeability barrier function.

Transcriptome of a mouse kidney cortical collecting duct cell line: effects of aldosterone and vasopressin.

Aldosterone and vasopressin are responsible for the final adjustment of sodium and water reabsorption in the kidney. In principal cells of the kidney cortical collecting duct (CCD), the integral response to aldosterone and the long-term functional effects of vasopressin depend on transcription. In this study, we analyzed the transcriptome of a highly differentiated mouse clonal CCD principal cell line (mpkCCD(cl4)) and the changes in the transcriptome induced by aldosterone and vasopressin. Serial analysis of gene expression (SAGE) was performed on untreated cells and on cells treated with either aldosterone or vasopressin for 4 h. The transcriptomes in these three experimental conditions were determined by sequencing 169,721 transcript tags from the corresponding SAGE libraries. Limiting the analysis to tags that occurred twice or more in the data set, 14,654 different transcripts were identified, 3,642 of which do not match known mouse sequences. Statistical comparison (at P < 0.05 level) of the three SAGE libraries revealed 34 AITs (aldosterone-induced transcripts), 29 ARTs (aldosterone-repressed transcripts), 48 VITs (vasopressin-induced transcripts) and 11 VRTs (vasopressin-repressed transcripts). A selection of the differentially-expressed, hormone-specific transcripts (5 VITs, 2 AITs and 1 ART) has been validated in the mpkCCD(cl4) cell line either by Northern blot hybridization or reverse transcription-PCR. The hepatocyte nuclear transcription factor HNF-3-alpha (VIT39), the receptor activity modifying protein RAMP3 (VIT48), and the glucocorticoid-induced leucine zipper protein (GILZ) (AIT28) are candidate proteins playing a role in physiological responses of this cell line to vasopressin and aldosterone.

Dysregulation of voltage-gated sodium channels by ubiquitin ligase NEDD4-2 in neuropathic pain.

Peripheral neuropathic pain is a disabling condition resulting from nerve injury. It is characterized by the dysregulation of voltage-gated sodium channels (Navs) expressed in dorsal root ganglion (DRG) sensory neurons. The mechanisms underlying the altered expression of Navs remain unknown. This study investigated the role of the E3 ubiquitin ligase NEDD4-2, which is known to ubiquitylate Navs, in the pathogenesis of neuropathic pain in mice. The spared nerve injury (SNI) model of traumatic nerve injury-induced neuropathic pain was used, and an Nav1.7-specific inhibitor, ProTxII, allowed the isolation of Nav1.7-mediated currents. SNI decreased NEDD4-2 expression in DRG cells and increased the amplitude of Nav1.7 and Nav1.8 currents. The redistribution of Nav1.7 channels toward peripheral axons was also observed. Similar changes were observed in the nociceptive DRG neurons of Nedd4L knockout mice (SNS-Nedd4L-/-). SNS-Nedd4L-/- mice exhibited thermal hypersensitivity and an enhanced second pain phase after formalin injection. Restoration of NEDD4-2 expression in DRG neurons using recombinant adenoassociated virus (rAAV2/6) not only reduced Nav1.7 and Nav1.8 current amplitudes, but also alleviated SNI-induced mechanical allodynia. These findings demonstrate that NEDD4-2 is a potent posttranslational regulator of Navs and that downregulation of NEDD4-2 leads to the hyperexcitability of DRG neurons and contributes to the genesis of pathological pain.

Sodium/hydrogen exchanger NHA2 in osteoclasts: subcellular localization and role in vitro and in vivo.

NHA2 was recently identified as a novel sodium/hydrogen exchanger which is strongly upregulated during RANKL-induced osteoclast differentiation. Previous in vitro studies suggested that NHA2 is a mitochondrial transporter required for osteoclast differentiation and bone resorption. Due to the lack of suitable antibodies, NHA2 was studied only on RNA level thus far. To define the protein's role in osteoclasts in vitro and in vivo, we generated NHA2-deficient mice and raised several specific NHA2 antibodies. By confocal microscopy and subcellular fractionation studies, NHA2 was found to co-localize with the late endosomal and lysosomal marker LAMP1 and the V-ATPase a3 subunit, but not with mitochondrial markers. Immunofluorescence studies and surface biotinylation experiments further revealed that NHA2 was highly enriched in the plasma membrane of osteoclasts, localizing to the basolateral membrane of polarized osteoclasts. Despite strong upregulation of NHA2 during RANKL-induced osteoclast differentiation, however, structural parameters of bone, quantified by high-resolution microcomputed tomography, were not different in NHA2-deficient mice compared to wild-type littermates. In addition, in vitro RANKL stimulation of bone marrow cells isolated from wild-type and NHA2-deficient mice yielded no differences in osteoclast development and activity. Taken together, we show that NHA2 is a RANKL-induced plasmalemmal sodium/hydrogen exchanger in osteoclasts. However, our data from NHA2-deficient mice suggest that NHA2 is dispensable for osteoclast differentiation and bone resorption both in vitro and in vivo.

Functional analysis of a missense mutation in the serine protease inhibitor SPINT2 associated with congenital sodium diarrhea.

Membrane-bound serine proteases play important roles in different biological processes. Their regulation by endogenous inhibitors is poorly understood. A Y163C mutation in the SPINT2 gene encoding the serine protease inhibitor Hepatocyte Growth Factor Inhibitor HAI-2 is associated with a congenital sodium diarrhea. The functional consequences of this mutation on HAI-2 activity and its physiological targets are unknown. We established a cellular assay in Xenopus laevis oocytes to study functional interactions between HAI-2 and candidate membrane-bound serine proteases expressed in the gastro-intestinal tract. We found that the wild-type form of HAI-2 is a potent inhibitor of nine gastro-intestinal serine proteases. The Y163C mutation in the second Kunitz domain of HAI-2 resulted in a complete loss of inhibitory activity on two intestinal proteases, prostasin and tmprss13. The effect of the mutation of the homologous Y68C in the first Kunitz domain of HAI-2 is consistent with a differential contribution of the two Kunitz domains of HAI-2 in the inhibition of serine proteases. By contrast to the Tyr to Cys, the Tyr to Ser substitution did not change the inhibitory potency of HAI-2, indicating that the thiol-group of the cysteine rather than the Tyr deletion is responsible for the HAI-2 loss of function. Our functional assay allowed us to identify membrane-bound serine proteases as cellular target for inhibition by HAI-2 wild type and mutants, and to better define the role of the Tyr in the second Kunitz domain in the inhibitory activity of HAI-2.

Structure function relationships of ENaC and its role in sodium handling.

The epithelial sodium channel (ENaC) in the apical membrane of polarized epithelial cells is the rate-limiting step for Na entry into the cell; in series with the basolateral Na pump, it allows the vectorial transepithelial transport of Na ions. ENaC is expressed in different epithelia like the distal nephron or colon, and the airways epithelium. In the lung ENaC controls the composition and the amount of pulmonary fluid, whereas in the distal nephron ENaC under the control of aldosterone and vasopressin, is essential to adapt the amount of Na+ reabsorbed with the daily sodium intake. Activating mutations of ENaC cause severe disturbances of Na+ homeostasis leading to hypertension in human and in mouse models. Functional expression of ENaC in different cell systems allowed the identification of structural domains of the protein that are essential for channel function and/or modulation of channel activity. Site-directed mutations in specific domains of the channel protein lead to channel hyperactivity or channel loss of function. Knowledge about ENaC structure-function relationships opens new opportunities for development of pharmacological tools for controlling ENaC activity, such as channel activators of potential benefit in the treatment of pulmonary edema, or highly potent ENaC blockers with natriuretic effects.

L'approche de l'homéostasie du sodium par le dosage du lithium trace

Le rein participe directement ou indirectement à de nombreux processus pathologiques s'accompagnant d'une rétention hydrosodée. L'étude des mécanismes impliqués et de leur localisation intrarénale est un élément important pour l'élaboration d'un diagnostic et d'une thérapeutique rationnelle. Des outils sont nécessaires à cette fin. Il y a 25 ans, Thomsen et Schou ont proposé la clairance du lithium comme marqueur de la réabsorption de fluide et de sodium au niveau du tubule rénal proximal. L'administration de lithium exogène semble cependant perturber l'homéostasie électrolytique rénale en entraînant une natriurèse transitoire. Depuis peu, la possibilité existe de quantifier le lithium présent en trace dans le corps humain et de déterminer ainsi sa clairance rénale. Cette nouvelle approche évite toute altération de l'homéostasie du sodium et ouvre un vaste champ d'étude. Il permet de préciser certains problèmes diagnostiques, d'éclairer des mécanismes physiopathologiques, et mène ainsi à des thérapies plus judicieuses.

Metabolic attributes, yield and stability of milk in Jersey cows fed diets containing sodium citrate and sodium bicarbonate

The objective of this work was to evaluate the inclusion of sodium citrate and sodium bicarbonate in the diet of lactating Jersey cows, and its effects on the metabolic attributes, productivity and stability of milk. We evaluated urinary pH, levels of glucose and urea in blood, body weight, body condition score, milk yield, milk stability (ethanol test), and milk physicochemical properties of 17 cows fed diets containing sodium citrate (100 g per cow per day), sodium bicarbonate (40 g per cow per day) or no additives. Assessments were made at the 28th and 44th days. Supply of sodium citrate or bicarbonate has no influence on the metabolic attributes, productivity, body weight, and body condition score of the cows, neither on the composition and stability of milk.

Intracellular thiol-mediated modulation of epithelial sodium channel activity.

The epithelial sodium channel ENaC is physiologically important in the kidney for the regulation of the extracellular fluid volume, and in the lungs for the maintenance of the appropriate airway surface liquid volume that lines the pulmonary epithelium. Besides the regulation of ENaC by hormones, intracellular factors such as Na(+) ions, pH, or Ca(2+) are responsible for fast adaptive responses of ENaC activity to changes in the intracellular milieu. In this study, we show that ENaC is rapidly and reversibly inhibited by internal sulfhydryl-reactive molecules such as methanethiosulfonate derivatives of different sizes, the metal cations Cd(2+) and Zn(2+), or copper(II) phenanthroline, a mild oxidizing agent that promotes the formation of disulfide bonds. At the single channel level, these agents applied intracellularly induce the appearance of long channel closures, suggesting an effect on ENaC gating. The intracellular reducing agent dithiothreitol fully reverses the rundown of ENaC activity in inside-out patches. Our observations suggest that changes in intracellular redox potential modulate ENaC activity and may regulate ENaC-mediated Na(+) transport in epithelia. Finally, substitution experiments reveal that multiple cysteine residues in the amino and carboxyl termini of ENaC subunits are responsible for this thiol-mediated inhibition of ENaC.

Short-term increase in particulate matter blunts nocturnal blood pressure dipping and daytime urinary sodium excretion

Short-term exposure to ambient particulate matter with aerodynamic diameters<10 µm were found to be positively associated with blood pressure. Yet, little information exists regarding the association between particles and circadian rhythm of blood pressure. Hence, we analyzed the association of exposure to particulate matter with aerodynamic diameters<10 µm on the day of examination and ≤7 days before with ambulatory blood pressure and with sodium excretion in 359 adults from the general population using multiple linear regression. After controlling for potential confounders, a 10-µg/m3 increase in particulate matter with aerodynamic diameters<10 µm levels was associated with nighttime systolic blood pressure (β=1.32 mm Hg 95% CI, 0.06-2.58 mm Hg at lag 0; P=0.04), nighttime diastolic blood pressure (0.72 mm Hg 95% CI, 0.03-1.42 mm Hg at lag 2; P=0.04), nocturnal systolic blood pressure dipping (-0.96 mm Hg 95% CI, -1.89 to -0.03 mm Hg at lag 0; P=0.044), and daytime urinary sodium excretion (-0.05 log-mmol/min 95% CI, -0.10 to -0.01 log-mmol/min at lag 0; P=0.027) but not with nighttime sodium excretion. The associations with blood pressure rapidly diminished with increasing lag days, and the associations with daytime sodium excretion were maximal with particulate matter with aerodynamic diameters<10 µm in exposures 2 to 5 days before. The associations of short-term increases in particulate matter with aerodynamic diameters<10 µm with higher nighttime blood pressure and blunted systolic blood pressure dipping were preceded by associations with reduced ability of the kidney to excrete sodium during daytime. The underlying mechanism linking air pollution to increased cardiovascular risk may include disturbed circadian rhythms of renal sodium handling and blood pressure.

Use of constant denaturant capillary electrophoresis of pooled blood samples to identify single-nucleotide polymorphisms in the genes (Scnn1a and Scnn1b) encoding the alpha and beta subunits of the epithelial sodium channel.

BACKGROUND: The epithelial sodium channel (ENaC) is composed of three homologous subunits: alpha, beta, and gamma. Mutations in the Scnn1b and Scnn1g genes, which encode the beta and the gamma subunits of ENaC, cause a severe form of hypertension (Liddle syndrome). The contribution of genetic variants within the Scnn1a gene, which codes for the alpha subunit, has not been investigated. METHODS: We screened for mutations in the COOH termini of the alpha and beta subunits of ENaC. Blood from 184 individuals from 31 families participating in a study on the genetics of hypertension were analyzed. Exons 13 of Scnn1a and Scnn1b, which encode the second transmembrane segment and the COOH termini of alpha- and beta-ENaC, respectively, were amplified from pooled DNA samples of members of each family by PCR. Constant denaturant capillary electrophoresis (CDCE) was used to detect mutations in PCR products of the pooled DNA samples. RESULTS: The detection limit of CDCE for ENaC variants was 1%, indicating that all members of any family or up to 100 individuals can be analyzed in one CDCE run. CDCE profiles of the COOH terminus of alpha-ENaC in pooled family members showed that the 31 families belonged to four groups and identified families with genetic variants. Using this approach, we analyzed 31 rather than 184 samples. Individual CDCE analysis of members from families with different pooled CDCE profiles revealed five genotypes containing 1853G-->T and 1987A-->G polymorphisms. The presence of the mutations was confirmed by DNA sequencing. For the COOH terminus of beta-ENaC, only one family showed a different CDCE profile. Two members of this family (n = 5) were heterozygous at 1781C-->T (T594M). CONCLUSION: CDCE rapidly detects point mutations in these candidate disease genes.