Regulation of sodium transport in the cortical collecting duct : physiological role of GILZ in vitro and in vivo : characterisation of Na+ transport in the mCCDcl1 cell line

SUMMARY Regulation of sodium excretion by the kidney is a key mechanism in the long term regulation of blood pressure, and when altered it constitutes a risk factor for the appearance of arterial hypertension. Aldosterone, which secretion depends upon salt intake in the diet, is a steroid hormone that regulates sodium reabsorption in the distal part of the nephron (functional unit of the kidney) by modulating gene transcription. It has been shown that it can act synergistically with the peptidic hormone insulin through the interaction of their signalisation pathways. Our work consisted of two distinct parts: 1) the in vitro and in vivo characterisation of Glucocorticoid-Induced Leucine Zipper (GILZ) (an aldosterone-induced gene) mechanism of action; 2) the in vitro characterisation of insulin mechanism of action and its interaction with aldosterone. GILZ mRNA, coded by the TSC22D3 gene, is strongly induced by aldosterone in the cell line of principal cells of the cortical collecting duct (CCD) mpkCCDc14, suggesting that GILZ is a mediator of aldosterone response. Co-expression of GILZ and the amiloride-sensitive epithelial sodium channel ENaC in vitro in the Xenopus oocyte expression system showed that GILZ has no direct effect on the ENaC-mediated Na+ current in basal conditions. To define the role of GILZ in the kidney and in other organs (colon, heart, skin, etc.), a conditional knock-out mouse is being produced and will allow the in vivo study of its role. Previous data showed that insulin induced a transepithelial sodium transport at supraphysiological concentrations. Insulin and the insulin-like growth factor 1 (IGF-1) are able to bind to each other receptor with an affinity 50 to 100 times lower than to their cognate receptor. Our starting hypothesis was that the insulin effect observed at these supraphysiological concentrations is actually mediated by the IGF receptor type 1 (IGF-1R). In a new cell line that presents all the characteristics of the principal cells of the CCD (mCCDc11) we have shown that both insulin and IGF-1 induce a physiologically significant increase of Na+ transport through the activation of IGF-1R. Aldosterone and insulin/IGF-1 have an additive effect on Na+ transport, through the activation of the PI3-kinase (PI3-K) pathway and the phosphorylation of the serum- and glucocorticoid-induced kinase 1 (Sgk1) by the IGF-1R, and the induction of Sgk1 expression by aldosterone. Thus, Sgk1 integrates IGF-1/insulin and aldosterone effects. We suggest that IGF-1 is physiologically relevant in the modulation of sodium balance, while insulin can only regulate Na+ transport at supraphysiological conditions. Both hormones would bind to the IGF-1R and induce Na+ transport by activating the PI3-K PDK1/2 - Sgk1 pathway. We have shown for the first time that Sgk1 is expressed and phosphorylated in principal cells of the CCD in basal conditions, although the mechanism that maintains Sgk1 phosphorylation is not known. This new role for IGF-1 suggests that it could be a salt susceptibility gene. In effect, IGF-1 stimulates Na+ and water transport in the kidney in vivo. Moreover, 35 % of the acromegalic patients (overproduction of growth hormone and IGF-1) are hypertensives (higher proportion than in normal population), and genetic analysis suggest a link between the IGF-1 gene locus and blood pressure. RÉSUMÉ La régulation de l'excrétion rénale de sodium (Na+) joue un rôle principal dans le contrôle à long terme de la pression sanguine, et ses altérations constituent un facteur de risque de l'apparition d'une hypertension artérielle. L'aldosterone, dont la sécrétion dépend de l'apport en sel dans la diète, est une hormone stéroïdienne qui régule la réabsorption de Na+ dans la partie distale du nephron (unité fonctionnelle du rein) en contrôlant la transcription de gènes. Elle peut agir de façon synergistique avec l'hormone peptidique insuline, probablement via l'interaction de leurs voies de signalisation cellulaire. Le but de notre travail comportait deux volets: 1) caractériser in vitro et in vivo le mécanisme d'action du Glucocorticoid Induced Leucine Zipper (GILZ) (un gène induit par l'aldosterone); 2) caractériser in vitro le mécanisme d'action de l'insuline et son interaction avec l'aldosterone. L'ARNm de GILZ, codé par le gène TSC22D3, est induit par l'aldosterone dans la lignée cellulaire de cellules principales du tubule collecteur cortical (CCD) mpkCCDc14, suggérant que GILZ est un médiateur potentiel de la réponse à l'aldosterone. La co-expression in vitro de GILZ et du canal à Na+ sensible à l'amiloride ENaC dans le système d'expression de l'oocyte de Xénope a montré que GILZ n'a pas d'effet sur les courants sodiques véhiculées par ENaC en conditions basales. Une souris knock-out conditionnelle de GILZ est en train d'être produite et permettra l'étude in vivo de son rôle dans le rein et d'autres organes. Des expériences préliminaires ont montré que l'insuline induit un transport transépithelial de Na+ à des concentrations supraphysiologiques. L'insuline et l'insulin-like growth factor 1 (IGF-1) peuvent se lier à leurs récepteurs réciproques avec une affinité 50 à 100 fois moindre qu'à leur propre récepteur. Nous avons donc proposé que l'effet de l'insuline soit médié par le récepteur à l'IGF type 1 (IGF-1R). Dans une nouvelle lignée cellulaire qui présente toutes les caractéristiques des cellules principales du CCD (mCCDc11) nous avons montré que les deux hormones induisent une augmentation physiologiquement significative du transport du Na+ par l'activation des IGF-1 R. Aldosterone et insuline/IGF-1 ont un effet additif sur le transport de Na+, via l'activation de la voie de la PI3-kinase et la phosphorylation de la serum- and glucocorticoid-induced kinase 1 (Sgk1) par l'IGF-1R, dont l'expression est induite par l'aldosterone. Sgk1 intègre les effets de l'insuline et l'aldosterone. Nous proposons que l'IGF-1 joue un rôle dans la modulation physiologique de la balance sodique, tandis que l'insuline régule le transport de Na+ à des concentrations supraphysiologiques. Les deux hormones agissent en se liant à l'IGF-1R et induisent le transport de Na+ en activant la cascade de signalisation PI3-K - PDK1/2 - Sgk1. Nous avons montré pour la première fois que Sgk1 est exprimée et phosphorylée dans des conditions basales dans les cellules principales du CCD, mais le mécanisme qui maintient sa phosphorylation n'est pas connu. Ce nouveau rôle pour l'IGF-1 suggère qu'il pourrait être un gène impliqué de susceptibilité au sel. Aussi, l'IGF-1 stimule le transport rénal de Na+ in vivo. De plus, 35 % des patients atteints d'acromégalie (surproduction d'hormone de croissance et d'IGF-1) sont hypertensifs (prévalence plus élevée que la population normale), et des analyses génétiques suggèrent un lien entre le locus du gène de l'IGF-1 et la pression sanguine. RÉSUMÉ GRAND PUBLIC Nos ancêtres se sont génétiquement adaptés pendant des centaines de millénaires à un environnement pauvre en sel (chlorure de sodium) dans la savane équatoriale, où ils consommaient moins de 0,1 gramme de sel par jour. On a commencé à ajouter du sel aux aliments avec l'apparition de l'agriculture (il y a 5000 à 10000 années), et aujourd'hui une diète omnivore, qui inclut des plats préparés, contient plusieurs fois la quantité de sodium nécessaire pour notre fonction physiologique normale (environ 10 grammes par jour). Le corps garde sa concentration constante dans le sang en s'adaptant à une consommation très variable de sel. Pour ceci, il module son excrétion soit directement, soit en sécrétant des hormones régulatrices. Le rein joue un rôle principal dans cette régulation puisque l'excrétion urinaire de sel change selon la diète et peut aller d'une quantité dérisoire à plus de 36 grammes par jour. L'attention qu'on prête au sel est liée à sa relation avec l'hypertension essentielle. Ainsi, le contrôle rénal de l'excrétion de sodium et d'eau est le principal mécanisme dans la régulation de la pression sanguine, et une ingestion excessive de sel pourrait être l'un des facteurs-clé déclenchant l'apparition d'un phénotype hypertensif. L'hormone aldosterone diminue l'excrétion de sodium par le rein en modulant l'expression de gènes qui pourraient être impliqués dans la sensibilité au sel. Dans une lignée cellulaire de rein l'expression du gène TSC22D3, qui se traduit en la protéine Glucocorticoid Induced Leucine Zipper (GILZ), est fortement induite par l'aldosterone. Ceci suggère que GILZ est un médiateur potentiel de l'effet de l'aldosterone, et pourrait être impliqué dans la sensibilité au sel. Pour analyser la fonction de GILZ dans le rein plusieurs approches ont été utilisées. Par exemple, une souris dans laquelle GILZ est spécifiquement inactivé dans le rein est en train d'être produite et permettra l'étude du rôle de GILZ dans l'organisme. De plus, on a montré que GILZ, en conditions basales, n'a pas d'effet direct sur la protéine transportant le sodium à travers la membrane des cellules, le canal sodique épithélial ENaC. On a aussi essayé de trouver des protéines qui interagissent directement avec GILZ utilisant une technique appelée du « double-hybride dans la levure », mais aucun candidat n'a émergé. Des études ont montré que, à de hautes concentrations, l'insuline peut aussi diminuer l'excrétion de sodium. A ces concentrations, elle peut activer son récepteur spécifique, mais aussi le récepteur d'une autre hormone, l'Insulin-Like Growth Factor 1 (IGF-1). En plus, l'infusion d'IGF-1 augmente la rétention rénale de sodium et d'eau, et des mutations du gène codant pour l'IGF-1 sont liées aux différents niveaux de pression sanguine. On a utilisé une nouvelle lignée cellulaire de rein développée dans notre laboratoire, appelée mCCDc11, pour analyser l'importance relative des deux hormones dans l'induction du transport de sodium. On a montré que les deux hormones induisent une augmentation significative du transport de sodium par l'activation de récepteurs à l'IGF-1 et non du récepteur à l'insuline. On a montré qu'à l'intérieur de la cellule leur activation induit une augmentation du transport sodique par le biais du canal ENaC en modifiant la quantité de phosphates fixés sur la protéine Serumand Glucocorticoid-induced Kinase 1 (Sgk1). On a finalement montré que l'IGF-1 et l'aldosterone ont un effet additif sur le transport de sodium en agissant toutes les deux sur Sgk1, qui intègre leurs effets dans le contrôle du transport de sodium dans le rein.

Physiological analysis of leaf senescence of two rice cultivars with different yield potential

The objective of this work was to evaluate the physiological changes that occur in different leaves during the early and late grain-filling stages of two rice genotypes (Oryza sativa subsp. indica , BRS Pelota cultivar, and O. sativa subsp. japonica , BRS Firmeza cultivar), which present differences in grain yield potential. The plants were cultivated in greenhouse. Pigment content, chlorophyll fluorescence, electron transport and oxygen evolution rate were determined in the grain-filling stage, from the first to the forth leaf (top to bottom). Pigment content, photochemical efficiency of photosystem II and electron transport decreased significantly according to the position of leaves in 'BRS Pelota'. The BRS Firmeza cultivar shows higher pigment content and higher activity of the photosynthetic apparatus in comparison to 'BRS Pelota' during the grain-filling stage.

Physiological requirements in triathlon

This article aims to present the current knowledge on physiological requirements in Olympic distance and Ironman triathlon. Showing the data available from a "traditional point of view" (aerobic power, anaerobic threshold, heart rate, running economy) and from a "contemporary" point of view (V̇O2 kinetics), it emphasises where we are currently and the areas that remain unknown.

Fruit flies learn to avoid odours associated with virulent infection.

While learning to avoid toxic food is common in mammals and occurs in some insects, learning to avoid cues associated with infectious pathogens has received little attention. We demonstrate that Drosophila melanogaster show olfactory learning in response to infection with their virulent intestinal pathogen Pseudomonas entomophila. This pathogen was not aversive to taste when added to food. Nonetheless, flies exposed for 3 h to food laced with P. entomophila, and scented with an odorant, became subsequently less likely to choose this odorant than flies exposed to pathogen-laced food scented with another odorant. No such effect occurred after an otherwise identical treatment with an avirulent mutant of P. entomophila, indicating that the response is mediated by pathogen virulence. These results demonstrate that a virulent pathogen infection can act as an aversive unconditioned stimulus which flies can associate with food odours, and thus become less attracted to pathogen-contaminated food.

Physiological indicators of stress in gestating sows under different cooling systems

The objective of this work was to determine physiological stress markers, neutrophil:lymphocyte ratio (N/L) and corticoid concentrations, in gestating sows under different cooling systems. A sprinkling cooling system (SS) and a system based on fan-assisted evaporative cellulose pad (PS) were used. SS showed higher N/L ratio (1.095) than PS (0.850). Corticoid concentrations showed high variability. Corticosteroids are more efficient short-term stress indicators while N/L ratio is a good medium and long-term stress indicator. According to N/L ratio, gestating sows under PS benefit from a higher level of welfare.

Postharvest mechanical damage affects fruit quality of 'Montenegrina' and 'Rainha' tangerines

The objective of this work was to evaluate the visual and chemical quality of tangerines after mechanical damage by impacts. The tangerine cultivars Montenegrina and Rainha were submitted to different degrees of impact and evaluated for decay and oleocellosis, loss of fresh weight, total soluble solids, total titratable acidity and ascorbic acid degradation, as well as for epicarp color changes. Experiments with three replicates and experimental units of six fruit for each cultivar were done in a completely randomized design. Impact produced qualitative internal and minor external changes on tangerines. The main modifications produced by impact on the fruit were losses of citric acid and soluble solids, which increased the solid:acid ratio, and losses of ascorbic acid. 'Montenegrina' tangerines are more susceptible to internal quality damage than 'Rainha'.

Hot spots for diversity of Magnaporthe oryzae physiological races in irrigated rice fields in Brazil

The objective of this work was to evaluate the Magnaporthe oryzae pathotype diversity in new commercial irrigated rice fields in the Araguaia River Valley, state of Tocantins, Brazil. The causal agent of rice blast has heavily affected rice production in the region. Despite the efforts of breeding programs, blast resistance breakdown has been recorded shortly after the release of new resistant cultivars developed for the region. Among the causes of resistance breakage is the capacity of the fungus to rapidly develop new pathotypes. A sample of 479 M. oryzae monosporic isolates was obtained and tested using the international rice blast differential set. Isolate collections were made in small areas designed as trap nurseries and in scattered sites in their vicinity. Analysis of 250 M. oryzae isolates from three trap nurseries indicated the presence of 45 international M. oryzae races belonging to seven pathotype groups (IA-IG). In the isolates tested, 61 M. oryzae pathotypes belonging to all but the IH group were detected. The new areas of irrigated rice in the Araguaia River Valley have the highest diversity of M. oryzae pathotypes reported so far in Brazil.

On an asymptotic formula for the maximum voltage drop in a on-chip power distribution network

We present a new asymptotic formula for the maximum static voltage in a simplified model for on-chip power distribution networks of array bonded integrated circuits. In this model the voltage is the solution of a Poisson equation in an infinite planar domain whose boundary is an array of circular pads of radius ", and we deal with the singular limit Ɛ → 0 case. In comparison with approximations that appear in the electronic engineering literature, our formula is more complete since we have obtained terms up to order Ɛ15. A procedure will be presented to compute all the successive terms, which can be interpreted as using multipole solutions of equations involving spatial derivatives of functions. To deduce the formula we use the method of matched asymptotic expansions. Our results are completely analytical and we make an extensive use of special functions and of the Gauss constant G

Development and fruit yield of strawberry plants as affected by crown diameter and plantlet growing period

The objective of this work was to evaluate the influence of crown diameter and growing period of runner tips in 128 cell-trays on growth and yield of strawberry in the field. Treatments consisted of three classes of runner tip crown diameters, between 2.0 to 3.9 mm; 4.0 to 5.5 mm and 5.6 to 7.0 mm, respectively, and four growing periods in trays, 24; 39; 54 and 69 days, respectively. Higher shoot dry mass of transplants at planting and earlier yield of plants in the field were obtained in transplants grown for 69 days in trays. Larger runner tips lead to more vigorous transplants at planting and plants with higher vegetative growth in the field, with minor impact on yield. Increasing the growing time of runner tips in trays improved early fruit yield and decreased plant vegetative growth in the field.

Leaf morphological and physiological responses of Quercus aquifolioides along an altitudinal gradient

Abstract

Traffic Control in Work Zones with Edge Drop-Offs, 2002

Pavement and shoulder edge drop-offs commonly occur in work zones as the result of overlays, pavement replacement, or shoulder construction. The depth of these elevation differentials can vary from approximately one inch when a flexible pavement overlay is applied to several feet where major reconstruction is undertaken. The potential hazards associated with pavement edge differentials depend on several factors including depth of the drop-off, shape of the pavement edge, distance from traveled way, vehicle speed, traffic mix, volume, and other factors. This research was undertaken to review current practices in other states for temporary traffic control strategies addressing lane edge differentials and to analyze crash data and resultant litigation related to edge drop-offs. An objective was to identify cost-effective practices that would minimize the potential for and impacts of edge drop crashes in work zones. Considerable variation in addressing temporary traffic control in work zones with edge drop-off exposure was found among the states surveyed. Crashes related to pavement edge drop-offs in work zones do not commonly occur in the state of Iowa, but some have resulted in significant tort claims and settlements. The use of benefit/cost analysis may provide guidance in selection of an appropriate mitigation and protection of edge drop-off conditions. Development and adoption of guidelines for design of appropriate traffic control for work zones that include edge drop-off exposure, particularly identifying effective use of temporary barrier rail, may be beneficial in Iowa.

'Royal Gala' apple quality stored under ultralow oxygen concentration and low temperature conditions

The objective of this work was to evaluate the interaction of ultralow oxygen concentrations (ULO) with storage temperatures and carbon dioxide partial pressures and its influence on fruit quality preservation and on the occurrence of physiological disorders in 'Royal Gala' apples. The experiment was carried out in a completely randomized design, with four replicates 25-fruit. ULO conditions (1.0 kPa O2 + 2.0 kPa CO2; 0.8 kPa O2 + 1.5 kPa CO2; 0.8 kPa O2 + 1.0 kPa CO2; 0.6 kPa O2 + 1.5 kPa CO2; and 0.6 kPa O2 + 1.0 kPa CO2) were tested at 0, 0.5 and 1.0°C, in a 5x3 factorial arrangement. Fruit quality and ripening analyses were performed after eight-month storage plus seven days of shelf-life at 20°C. Oxygen partial pressures below 0.8 kPa increased the occurrence of internal breakdown and mealiness. The best ULO condition was 1.0 kPa O2 + plus 2.0 kPa CO2 at 1.0°C. The interaction of ULO conditions and storage temperatures shows the need of increasing O2 partial pressure at higher storage temperatures.

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.

Sensitivity of yellow passion fruit to ultraviolet-B radiation

The objective of this work was to evaluate the effects of UV-B radiation on the vegetative growth and on the gas exchange characteristics of passion fruit plants (Passiflora edulis) grown in greenhouse. The average unweighted UV-B radiation near the apex of the plants was 8 W m-2 for the UV-B treatment (high UV-B), and 0.8 W m-2 for the control plants (low UV-B). Plants were irradiated with UV-B for 7 hours per day, centered on solar noon, during 16 days. High UV-B radiation resulted in lower shoot dry matter accumulation per plant. The content of UV-B absorbing compounds and anthocyanins was increased in the plants exposed to high UV-B radiation, when compared with the control. UV-B radiation did not affect stomatal conductance or transpiration rate, but reduced photosynthesis and instantaneous water-use efficiency, and increased intercellular CO2 concentration. The accumulation of UV-B-absorbing compounds and anthocyanins did not effectively shield plants from supplementary UV-B radiation, since the growth and photosynthetic processes were significantly reduced.

Growth of champa fruit under agroecological conditions of Miraflores, Boyacá, Colombia

The objective of this work was to analyze the growth of champa fruit (Campomanesia lineatifolia) as a function of growing-degree days (GDD) in the municipality of Miraflores, in Boyacá, Colombia. Thirty trees were selected at random, and 100 flowers in full bloom were marked in each tree. From the 26th day after flowering until harvest, 10 samples were taken every 15 days to determine the fruit parameters and growth rate. Temperature was recorded to calculate the GDD. From flowering until harvest, 1,489.1 GDD were accumulated over 145 days. Dry and fresh matter mass of pulp, seed, and total fruit were fitted to a logistic growth model, and three growth stages (S1, S2 and S3) were defined. In the S1, growth was slow, and the relative growth remained nearly stable, whereas the absolute growth rate (AGR) increased slowly. In the S2, maximum growth was observed. In the S3, which corresponds to maturation, dry mass increased gradually, and the AGR decreased, while the fresh pulp and total mass did not cease to increase. The polar and equatorial diameters increased linearly, while the volume followed an exponential model. Champa fruit show a simple sigmoid growth curve.