The Epithelial Sodium Channel α subunit (α ENaC) alternatively spliced form "b" in Dahl rats: What's next?

<p>Abstract</p> <p>Background</p> <p>The amiloride-sensitive Epithelial Sodium Channel (ENaC) is critical in maintaining Na⁺balance, extracellular fluid volume and long term blood pressure control. ENaC is composed of three main subunits α, β, & γ. While α ENaC is critical for channel functionality, β & γ ENaC maximize channel function. To date, there are four alternatively spliced forms of the α subunit of ENaC (α ENaC-a, -b, -c, & -d) that have been published in rats, in addition to the major α ENaC transcript. While α ENaC-a, -c & -d transcripts are low abundance transcripts compared to full-length α ENaC, α ENaC-b is a higher abundance and salt-sensitive transcript compared to full-length α ENaC.</p> <p>Presentation of the hypothesis</p> <p>α ENaC-b protein, which is preferentially produced in Dahl R rats, to a greater extent on high salt diet, exerts a dominant negative effect on full-length α ENaC subunit by physically binding to and trapping full-length α ENaC subunit in the endoplasmic reticulum, and finally accelerating full-length α ENaC proteolytic degradation in a dose-dependent manner.</p> <p>Testing the hypothesis</p> <p>1) To examine the mRNA and protein abundance of α ENaC-b relative to α ENaC full-length in kidney, lung, and taste tissues of Dahl rats. 2) To compare the expression (mRNA and protein) of α ENaC-b in kidneys of Dahl S and R rats on regular and high salt diet. 3) To examine the putative binding of α ENaC-b proteins to full-length α ENaC <it>in vitro </it>and to determine the impact of such binding on full-length α ENaC expression <it>in vitro</it>.</p> <p>Implications of the hypothesis</p> <p>Our studies will be the first to demonstrate the over-expression of salt-sensitive α ENaC-b spliced form in kidney tissues of Dahl R rats at the expense of full-length α ENaC. The current proposal will provide highly novel insights into the putative mechanisms leading to ENaC hypoactivity in high-salt-fed Dahl R rats. Finally, findings from the present proposal will uncover a new mechanism by which alternative splicing may control the regulation of ENaC expression/function.</p

Regulation of the epithelial sodium channel [ENaC] in kidneys of salt-sensitive Dahl rats: Insights on alternative splicing

The epithelial sodium channel [ENaC] is critical for the maintenance of sodium balance, extracellular fluid volume and long term blood pressure control. Monogenic disorders causing ENaC hyperactivity have led to a severe form of hereditary hypertension in humans, known as Liddle's syndrome. Similarly, in animal models, ENaC hyperactivity has been well documented in kidneys of salt-sensitive [S] Dahl rats [a genetic model of salt-sensitive hypertension] versus their normotensive control [Dahl salt-resistant [R] rats]. The purpose of the present review is to highlight the differential regulation of ENaC in kidneys of Dahl S versus R rats. A systematic overview of the putative role of alternative splicing of the main α subunit of ENaC [α ENaC] in modulating ENaC expression in kidneys of Dahl rats will be discussed. Finally, a better understanding of the meaningful contribution of ENaC in the pathogenesis of salt-sensitive hypertension will be achieved upon completion of this review

Characterization of the epithelial sodium channel α subunit coding and non-coding transcripts and their corresponding mRNA expression levels in Dahl R versus S rat kidney cortex on normal and high salt diet

<p>Abstract</p> <p>Aims/hypothesis</p> <p>The α subunit of the amiloride-sensitive epithelial sodium channel (α ENaC) is critical for the expression of functional channels. In humans and rats, non functional alternatively spliced forms of α ENaC have been proposed to act as negative regulatory components for ENaC. The purpose of this study was to examine the presence and consequently investigate the mRNA expression levels of alternatively spliced forms of α ENaC in kidney cortex of Dahl salt-resistant rats (R) <it>versus </it>Dahl salt-sensitive rats (S) on high salt and normal diets.</p> <p>Methods</p> <p>Using quantitative RT-PCR strategy, we examined the mRNA expression levels of previously reported α ENaC-a and -b alternatively spliced forms in kidney cortex of Dahl S and R rats on normal and four-week high salt diet and compared their corresponding abundance to wildtype α ENaC mRNA levels. We identified 2 novel non-coding C-terminus spliced forms and examined their mRNA expression in Dahl R <it>versus </it>S rat kidney cortex. We also tested the presence of five previously reported lung-specific α ENaC spliced forms in Dahl rat kidney cortex (CK479583, CK475461, CK364785, CK475819, and CB690980).</p> <p>Results</p> <p>Previously reported α ENaC-a and -b alternatively spliced forms are present in Dahl rat kidney cortex and are significantly higher in Dahl R <it>versus </it>S rats (P < 0.05). Four-week high salt diet significantly increases α ENaC-b (P < 0.05), but not α ENaC-a transcript abundance in Dahl R, but not S rats. Two non-coding α ENaC spliced forms -c and -d are newly identified in the present study, whose levels are comparable in Dahl R and S rats. Compared to α ENaC-wt, α ENaC-a, -c and -d are low abundance transcripts (4 +/- 2, 110 +/- 20, and 10 +/- 2 fold less respectively), in contrast to α ENaC-b abundance that exceeds α ENaC-wt by 32 +/- 3 fold. We could not identify any of the five previously reported lung-specific α ENaC spliced forms (CK479583, CK475461, CK364785, CK475819, and CB690980) in Dahl rat kidney cortex.</p> <p>Conclusion/interpretation</p> <p>α ENaC alternative splicing might regulate α ENaC by the formation of coding RNA species (α ENaC-a and -b) and non-coding RNA species (α ENaC-c and -d). α ENaC-a and -b mRNA levels are significantly higher in Dahl R <it>versus </it>S rats. Additionally, α ENaC-b is a salt-sensitive transcript whose levels are significantly higher 4-weeks post high salt diet compared to normal salt diet in Dahl R rats. Among the four α ENaC transcripts (-a, -b, -c and -d), α ENaC-b is a predominant transcript that exceeds α ENaC-wt abundance by ~32 fold. α ENaC-a and -b spliced forms, particularly, α ENaC-b, might potentially act as dominant negative proteins for ENaC activity, thereby rescuing Dahl R rats from developing salt-sensitive hypertension on high salt diet. On the other hand, non-coding α ENaC-c and -d might assist alternative splicing, facilitate RNA processing, or regulate α ENaC as well as each other.</p

Important genetic checkpoints for insulin resistance in salt-sensitive (S) Dahl rats

Despite the marked advances in research on insulin resistance (IR) in humans and animal models of insulin resistance, the mechanisms underlying high salt-induced insulin resistance remain unclear. Insulin resistance is a multifactorial disease with both genetic and environmental factors (such as high salt) involved in its pathogenesis. High salt triggers insulin resistance in genetically susceptible patients and animal models of insulin resistance. One of the mechanisms by which high salt might precipitate insulin resistance is through its ability to enhance an oxidative stress-induced inflammatory response that disrupts the insulin signaling pathway. The aim of this hypothesis is to discuss two complementary approaches to find out how high salt might interact with genetic defects along the insulin signaling and inflammatory pathways to predispose to insulin resistance in a genetically susceptible model of insulin resistance. The first approach will consist of examining variations in genes involved in the insulin signaling pathway in the Dahl S rat (an animal model of insulin resistance and salt-sensitivity) and the Dahl R rat (an animal model of insulin sensitivity and salt-resistance), and the putative cellular mechanisms responsible for the development of insulin resistance. The second approach will consist of studying the over-expressed genes along the inflammatory pathway whose respective activation might be predictive of high salt-induced insulin resistance in Dahl S rats

A Proposed Study on How Far Anger Contributes to Initiating Essential Hypertension in Canadian Caucasian Adults with A Family History of Hypertension

The goal of the present correlational study is to test the hypothesis that anger initiates essential hypertension in Canadian Caucasian adults with a family history of hypertension. The study population will include a cohort of 100 men and women aged 25 to 45 years at enrollment recruited from the University of Ottawa General and Civic Hospitals. Participants who are normotensives will be included in the study given that they have a family history of hypertension. The Spielberger State Trait Anger Expression inventory (STAXI) scales will assess the three main dimensions of anger: State, Trait and Anger Expression. Using a semiautomated blood pressure machine, blood pressure measurements will be conducted by the attending nurse. It is hypothesized that during the three year study, participants with higher STAXI scores will more likely develop hypertension. Results obtained from the present study are expected to highlight the significant contribution of anger as a modifiable behavioral risk factor in the pathogenesis of hypertension

Physiological studies on heavy metals and blue-green algae

Mutants of Anacystis nidulans tolerant to high levels of Co, Ni,Cu, Zn and Cd were obtained by repeated subculturing at strongly inhibitory levels of metal. For instance, the level of Zn at which-1strong inhibition occurred was raised from 1.45 to 16.5 mg 1(^-1) Zn after 75 subcultures. Isolates resistant to 5.0 mg 1(^-1) Zn and 12.0 mg 1(^-1) Zn maintained their resistance for at least 72 cell generations in the absence of Zn, though there was subsequently an increased lag during the first subculture back to high Zn levels. This and plating experiment sindicate that the strains are mutants. Assays of cross-resistance of each of the five types of mutant were made to the other four metals. Inmost cases changes in cross-resistance were only slight, with about equal numbers of examples of increased and decreased resistance. Examples of marked changes were increased Co-resistance of a Cd-tolerant strain and decreased Cd-resistance of a Ni-tolerant strain. The environmental factors influencing toxicity were investigated for Cu, Zn and Cd. Increases in Ca, Mn, Fe and P reduced Zn toxicity to both wild-type and Zn-tolerant strains, but the two differed in their response to pH. Effects on morphology were evident at high metal levels with all strains. In most cases increased levels of metal led to the formation of filaments, but with Cu subspherical structures sometimes made up most of the-1population. Uptake from media enriched 0.1 and 1.0 mg 1(^-1) Zn was similar in both wild-type and Zn-tl2.0, whether judged by total Zn accumulated or by that remaining after EDTA washes. Isolates from high Zn sites were found in general to tolerate considerably higher levels of Zn than laboratory research strains(presumably isolated from environments not enriched with Zn). A comparison of the influence of Zn on nitrogen fixation by a strain from low zinc site{Anabaena cylindrica) and one from a high Zn site (Calothrix D184) showed only a slight difference when Zn was first added, but a pronounced effect after 24 h

Sequence analysis of coding and 3' and 5' flanking regions of the epithelial sodium channel α, β, and γ genes in Dahl S versus R rats

<p>Abstract</p> <p>Background</p> <p>To test whether epithelial sodium channel (ENaC) genes' variants contribute to salt sensitive hypertension in Dahl rats, we screened ENaC α, β, and γ genes entire coding regions, intron-exon junctions, and the 3' and 5' flanking regions in Dahl S, R and Wistar rats using both Denaturing High Performance Liquid Chromatography (DHPLC) and sequencing.</p> <p>Results</p> <p>Our analysis revealed no sequence variability in the three genes encoding ENaC in Dahl S <it>versus </it>R rats. One homozygous sequence variation predicted to result in a D75E substitution was identified in Dahl and Wistar rat ENaC α compared to Brown Norway. Six and two previously reported polymorphic sites in Brown Norway sequences were lost in Dahl and Wistar rats, respectively. In the 5' flanking regions, we found a deletion of 5GCTs in Dahl and Wistar rat ENaC α gene, five new polymorphic sites in ENaC β and γ genes, one homozygous sequence variation in Dahl and Wistar rat ENaC γ gene, as well as one Dahl rat specific homozygous insertion of -1118CCCCCA in ENaC γ gene. This insertion created additional binding sites for Sp1 and Oct-1. Five and three Brown Norway polymorphic sites were lost in Dahl and Wistar rats, respectively. No sequence variability in ENaC 3' flanking regions was identified in Dahl compared to Brown Norway rats.</p> <p>Conclusion</p> <p>The first comprehensive sequence analysis of ENaC genes did not reveal any differences between Dahl S and R rats that were isogenic in the regions screened. Mutations in ENaC genes intronic sequence or in ENaC-regulatory genes might possibly account for increased ENaC activity in Dahl S <it>versus </it>R rats.</p

How to Capture Reciprocal Communication Dynamics: Comparing Longitudinal Statistical Approaches in Order to Analyze Within- and Between-Person Effects

Choosing an appropriate statistical model to analyze reciprocal relations between individuals’ attitudes, beliefs, or behaviors over time can be challenging. Often, decisions for or against specific models are rather implicit and it remains unclear whether the statistical approach fits the theory of interest. For longitudinal models, this is problematic since within- and between-person processes can be confounded leading to wrong conclusions. Taking the perspective of the reinforcing spirals model (RSM) focusing on media effects and selection, we compare six statistical models that were recently used to analyze the RSM and show their ability to separate within- and between-person components. Using empirical data capturing respondents’ development during adolescence, we show that results vary across statistical models. Further, Monte Carlo simulations indicate that some approaches might lead to wrong conclusions if specific communication dynamics are present. In sum, we recommend using approaches that explicitly model and clearly separate within- and between-person effects

Development of two reference materials for all trans-retinol, retinyl palmitate, α- and γ-tocopherol in milk powder and infant formula

AbstractVitamins are important food constituents that can be present in almost every foodstuff. Food quality and safety depends on food surveillance by reliable quantitative analysis enabled by appropriate quality control. Certified matrix reference materials are versatile tools to support quality assurance and control. However, in the case of vitamins, which are important in various foods, there is a lack of matrix reference materials. Two certified reference materials for the determination of all–trans-retinol, retinyl palmitate, and α- and γ-tocopherol in milk powder and infant formula have been developed by the National Institute of Standards, Egypt. This article presents the preparation, characterization, homogeneity, and stability testing as well as statistical treatment of data and certified value assignment. The assignment of the certified values and associated uncertainties in the prepared natural-matrix reference materials were based on the widely used approach of combining data from independent and reliable analytical methods