Effects of neuropeptide Y on intrarenal hemodynamics, plasma renin activity and urinary sodium excretion in rats.

Neuropeptide Y (NPY) is a vasoconstrictor peptide possibly involved in the regulation of renal sodium handling and renin release. This investigation was undertaken to assess in conscious normotensive rats the acute effects of a non-pressor dose of NPY on renal plasma flow, glomerular filtration rate, sodium excretion and plasma renin activity. Experiments were also performed during concomitant beta-adrenoceptor stimulation with isoproterenol. NPY per se had no effect on the studied parameters. Renal plasma flow was increased by isoproterenol and was significantly higher when the beta-adrenoceptor stimulant was infused alone (13.4 +/- 2.1 ml/min, p < 0.05, mean +/- SEM) that when administered together with NPY (7.2 +/- 2.0 ml/min). This was also true for glomerular filtration rate (3.3 +/- 0.3 vs. 1.8 +/- 0.3 ml/min, p < 0.01) and plasma renin activity (6.3 +/- 1.7 vs. 2.1 +/- 0.4 ng Ang I/ml/h, p < 0.05). Our data however do not allow to deduce whether the inhibitory effect of NPY on isoproterenol-induced renin release is mediated by changes in intrarenal hemodynamics or a direct effect on juxtaglomerular cells.

Copper, selenium, zinc, and thiamine balances during continuous venovenous hemodiafiltration in critically ill patients.

BACKGROUND: Acute renal failure is a serious complication in critically ill patients and frequently requires renal replacement therapy, which alters trace element and vitamin metabolism. OBJECTIVE: The objective was to study trace element balances during continuous renal replacement therapy (CRRT) in intensive care patients. DESIGN: In a prospective randomized crossover trial, patients with acute renal failure received CRRT with either sodium bicarbonate (Bic) or sodium lactate (Lac) as a buffering agent over 2 consecutive 24-h periods. Copper, selenium, zinc, and thiamine were measured with highly sensitive analytic methods in plasma, replacement solutions, and effluent during 8-h periods. Balances were calculated as the difference between fluids administered and effluent losses and were compared with the recommended intakes (RI) from parenteral nutrition. RESULTS: Nineteen sessions were conducted in 11 patients aged 65 +/- 10 y. Baseline plasma concentrations of copper were normal, whereas those of selenium and zinc were below reference ranges; glutathione peroxidase was in the lower range of normal. The replacement solutions contained no detectable copper, 0.01 micromol Se/L (Bic and Lac), and 1.42 (Bic) and 0.85 (Lac) micromol Zn/L. Micronutrients were detectable in all effluents, and losses were stable in each patient; no significant differences were found between the Bic and Lac groups. The 24-h balances were negative for selenium (-0.97 micromol, or 2 times the daily RI), copper (-6.54 micromol, or 0.3 times the daily RI), and thiamine (-4.12 mg, or 1.5 times the RI) and modestly positive for zinc (20.7 micromol, or 0.2 times the RI). CONCLUSIONS: CRRT results in significant losses and negative balances of selenium, copper, and thiamine, which contribute to low plasma concentrations. Prolonged CRRT is likely to result in selenium and thiamine depletion despite supplementation at recommended amounts.

Effect of Sodium Loading/Depletion on Renal Oxygenation in Young Normo-and Hypertensive Men Measured with BOLD-MRI

Objective: To measure renal tissue oxygenation in young normo-and hypertensive volunteers under conditions of salt loading and depletion using blood oxygen level dependent magnetic resonance imaging (BOLD-MRI). Design and Methods: Ten normotensive (NT) male volunteers (age 26.5_7.4 y) and eight non-treated, hypertensive (HT) male volunteers (age 28.8_5.7 y) were studied after one week on a high salt (HS) regimen (6g of salt/day added to their normal regimen) and again after one week of a low sodium diet (LS). On the 8th day, BOLD-MRI was performed under standard hydration conditions. Four coronal slices were selected in each kidney, and combination sequence was used to acquire T2* weighted images. The mean R2* (1/T2*) was measured to determine cortical and medullar oxygenation. Results: Baseline characteristics and their changes are shown in the table. The mean cortical R2* was not different under conditions of HS or LS (17.8_1.3 vs. 18.2_0.6 respectively in NT group, p_0.27; 17.4_0.6 vs 17.8_0.9 in HT group, p_0.16). However, the mean medullary R2* was significantly lower under LS conditions in both groups (31.3_0.6 vs 28.1_0.8 in NT group, p_0.05; 30.3_0.8 vs 27.9_1.5 in HT group, p_0.05), corresponding to higher medullary oxygenation as compared to HS conditions, without significant changes in hemoglobin or hematocrit values. The salt induced changes in medullary oxygenation were comparable in the two groups (ANOVA, p_0.1). Conclusion: Dietary sodium restriction leads to increased renal medullary oxygenation compared to high sodium intake in normo-and hypertensive subjects. This observation may in part explain the potential renal benefits of a low sodium intake.

Mutational analysis of cysteine-rich domains of the epithelium sodium channel (ENaC). Identification of cysteines essential for channel expression at the cell surface.

One of the characteristic features of the structure of the epithelial sodium channel family (ENaC) is the presence of two highly conserved cysteine-rich domains (CRD1 and CRD2) in the large extracellular loops of the proteins. We have studied the role of CRDs in the functional expression of rat alphabetagamma ENaC subunits by systematically mutating cysteine residues (singly or in combinations) into either serine or alanine. In the Xenopus oocyte expression system, mutations of two cysteines in CRD1 of alpha, beta, or gamma ENaC subunits led to a temperature-dependent inactivation of the channel. In CRD1, one of the cysteines of the rat alphaENaC subunit (Cys158) is homologous to Cys133 of the corresponding human subunit causing, when mutated to tyrosine (C133Y), pseudohypoaldosteronism type 1, a severe salt-loosing syndrome in neonates. In CRD2, mutation of two cysteines in alpha and beta but not in the gamma subunit also produced a temperature-dependent inactivation of the channel. The main features of the mutant cysteine channels are: (i) a decrease in cell surface expression of channel molecules that parallels the decrease in channel activity and (ii) a normal assembly or rate of degradation as assessed by nondenaturing co-immunoprecipitation of [35S]methionine-labeled channel protein. These data indicate that the two cysteines in CRD1 and CRD2 are not a prerequisite for subunit assembly and/or intrinsic channel activity. We propose that they play an essential role in the efficient transport of assembled channels to the plasma membrane.

Colitis induced in mice with dextran sulfate sodium (DSS) is mediated by the NLRP3 inflammasome.

BACKGROUND: The proinflammatory cytokines interleukin 1beta (IL-1beta) and IL-18 are central players in the pathogenesis of inflammatory bowel disease (IBD). In response to a variety of microbial components and crystalline substances, both cytokines are processed via the caspase-1-activating multiprotein complex, the NLRP3 inflammasome. Here, the role of the NLRP3 inflammasome in experimental colitis induced by dextran sodium sulfate (DSS) was examined. METHODS: IL-1beta production in response to DSS was studied in macrophages of wild-type, caspase-1(-/-), NLRP3(-/-), ASC(-/-), cathepsin B(-/-) or cathepsin L(-/-) mice. Colitis was induced in C57BL/6 and NLRP3(-/-) mice by oral DSS administration. A clinical disease activity score was evaluated daily. Histological colitis severity and expression of cytokines were determined in colonic tissue. RESULTS: Macrophages incubated with DSS in vitro secreted high levels of IL-1beta in a caspase-1-dependent manner. IL-1beta secretion was abrogated in macrophages lacking NLRP3, ASC or caspase-1, indicating that DSS activates caspase-1 via the NLRP3 inflammasome. Moreover, IL-1beta secretion was dependent on phagocytosis, lysosomal maturation, cathepsin B and L, and reactive oxygen species (ROS). After oral administration of DSS, NLRP3(-/-) mice developed a less severe colitis than wild-type mice and produced lower levels of proinflammatory cytokines in colonic tissue. Pharmacological inhibition of caspase-1 with pralnacasan achieved a level of mucosal protection comparable with NLRP3 deficiency. CONCLUSIONS: The NLRP3 inflammasome was identified as a critical mechanism of intestinal inflammation in the DSS colitis model. The NLRP3 inflammasome may serve as a potential target for the development of novel therapeutics for patients with IBD.

Anesthesia of Biomphalaria spp. (Mollusca, Gastropoda): sodium pentobarbital is the drug of choice

The anesthetic effect of some water-soluble anesthesic or narcotic drugs currently used in mice was tested in molluscs of the Biomphalaria genus. Sodium thiopental was very toxic to the snails resulting in high rates of mortality in all the treatment schedules tested. Cetamine base, at concentration of 0.25 mg/ml of water, resulted in partial snail anesthesia (40% of snails were anesthetized) only after 20 h of exposition. The association of Cetamine base with Tiazine chloridrate did not improve the anesthesic effect, and higher concentrations of these drugs were toxic to the snails. Sodium pentobarbital at 0.4 mg/ml in water for 8 h was the best treatment schedule to anesthetize Biomphalaria snails. In this schedule, the snails were anesthetized without any toxic effect. The procedure provides a powerful tool for in vivo studies that demande a complete state of snail anesthesia.

A methodology to event reconstruction from trace images

The widespread use of digital imaging devices for surveillance (CCTV) and entertainment (e.g., mobile phones, compact cameras) has increased the number of images recorded and opportunities to consider the images as traces or documentation of criminal activity. The forensic science literature focuses almost exclusively on technical issues and evidence assessment [1]. Earlier steps in the investigation phase have been neglected and must be considered. This article is the first comprehensive description of a methodology to event reconstruction using images. This formal methodology was conceptualised from practical experiences and applied to different contexts and case studies to test and refine it. Based on this practical analysis, we propose a systematic approach that includes a preliminary analysis followed by four main steps. These steps form a sequence for which the results from each step rely on the previous step. However, the methodology is not linear, but it is a cyclic, iterative progression for obtaining knowledge about an event. The preliminary analysis is a pre-evaluation phase, wherein potential relevance of images is assessed. In the first step, images are detected and collected as pertinent trace material; the second step involves organising and assessing their quality and informative potential. The third step includes reconstruction using clues about space, time and actions. Finally, in the fourth step, the images are evaluated and selected as evidence. These steps are described and illustrated using practical examples. The paper outlines how images elicit information about persons, objects, space, time and actions throughout the investigation process to reconstruct an event step by step. We emphasise the hypothetico-deductive reasoning framework, which demonstrates the contribution of images to generating, refining or eliminating propositions or hypotheses. This methodology provides a sound basis for extending image use as evidence and, more generally, as clues in investigation and crime reconstruction processes.

Characterization of Sm14 related components in different helminths by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis

Sm14 was the first fatty acid-binding protein homologue identified in helminths. Thereafter, members of the same family were identified in several helminth species, with high aminoacid sequence homology between them. In addition, immune crossprotection was also reported against Fasciola hepatica infection, in animals previously immunized with the Schistosoma mansoni vaccine candidate, r-Sm14. In the present study, data on preliminary sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting analysis of nine different helminth extracts focusing the identification of Sm14 related proteins, is reported. Out of these, three extracts - Ascaris suum (males and females), Echinostoma paraensei, and Taenia saginata - presented components that comigrated with Sm14 in SDS-PAGE, and that were recognized by anti-rSm14 policlonal serum, in Western blotting tests.

Trace element differentiation in ferruginous accumulation soil patterns under tropical rainforest of southern Cameroon, the role of climatic change

Regions under tropical rainforest cover, such as central Africa and Brazil are characterised by degradation and dismantling of old ferricrete structures. In southern Cameroon, these processes are relayed by present-day ferruginous accumulation soil facies, situated on the middle and the lower part of hill slopes. These facies become progressively harder towards the surface, containing from bottom to top, mainly kaolinite, kaolinite-goethite and Al-rich goethite-hematite, and are discontinuous to the relictic hematite-dominated ferricrete that exist in the upper part of the hill slope. These features were investigated in terms of geochemical differentiation of trace elements. It appears that, in contrast to the old ferricrete facies, the current ferruginous accumulations are enriched in transitional trace elements (V, Cr, Co, Y, Sc) and Ph, while alkali-earth elements are less differentiated. This recent chemical accumulation is controlled both by intense weathering of the granodiorite bedrock and by mobilisation of elements previously accumulated in the old ferricrete. The observed processes are clearly linked to the present-day humid climate with rising groundwater tables. They slowly replace the old ferricretes formed during Cretaceous time under more seasonal climatic conditions, representing an instructive case of continuos global change. (C) 2002 Elsevier Science B.V. All rights reserved.

NF-kappaB inhibits sodium transport via down-regulation of SGK1 in renal collecting duct principal cells.

Tubulointerstitial inflammation is a common feature of renal diseases. We have investigated the relationship between inflammation and Na(+) transport in the collecting duct (CD) using the mCCD(cl1) and mpkCDD(cl4) principal cell models. Lipopolysaccharide (LPS) decreased basal and aldosterone-stimulated amiloride-sensitive transepithelial current in a time-dependent manner. This effect was associated with a decrease in serum and glucocorticoid-regulated kinase 1 (SGK1) mRNA and protein levels followed by a decrease in epithelial sodium channel (ENaC) alpha-subunit mRNA levels. The LPS-induced decrease in SGK1 expression was confirmed in isolated rat CD. This decreased expression of either SGK1 or the ENaC alpha-subunit was not due to enhanced degradation of mRNA. In contrast, LPS inhibited transcriptional activity of the SGK1 promoter measured by luciferase-reporter gene assay. The effect of LPS was not mediated by inhibition of mineralocorticoid or glucocorticoid receptor, because expression of both receptors was unchanged and blockade of either receptor by spironolactone or RU486, respectively, did not prevent the down-regulation of SGK1. The effect of LPS was mediated by the canonical NF-kappaB pathway, as overexpression of a constitutively active mutant, IKKbeta (inhibitor of nuclear factor kappaB kinase-beta) decreased SGK1 mRNA levels, and knockdown of p65 NF-kappaB subunit by small interfering RNA increased SGK1 mRNA levels. Chromatin immunoprecipitation showed that LPS increased p65 binding to two NF-kappaB sites along the SGK1 promoter. In conclusion, we show that activation of the NF-kappaB pathway down-regulates SGK1 expression, which might lead to decreased ENaC alpha-subunit expression, ultimately resulting in decreased Na(+) transport.

The heterotetrameric architecture of the epithelial sodium channel (ENaC).

The epithelial sodium channel (ENaC) is a key element for the maintenance of sodium balance and the regulation of blood pressure. Three homologous ENaC subunits (alpha, beta and gamma) assemble to form a highly Na+-selective channel. However, the subunit stoichiometry of ENaC has not yet been solved. Quantitative analysis of cell surface expression of ENaC alpha, beta and gamma subunits shows that they assemble according to a fixed stoichiometry, with alpha ENaC as the most abundant subunit. Functional assays based on differential sensitivities to channel blockers elicited by mutations tagging each alpha, beta and gamma subunit are consistent with a four subunit stoichiometry composed of two alpha, one beta and one gamma. Expression of concatameric cDNA constructs made of different combinations of ENaC subunits confirmed the four subunit channel stoichiometry and showed that the arrangement of the subunits around the channel pore consists of two alpha subunits separated by beta and gamma subunits.